Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene

USDA-ARS?s Scientific Manuscript database

The widespread use of atrazine and other s-triazine herbicides to control weeds in agricultural production fields has impacted surface and ground water in the United States and elsewhere. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradat...

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Control of the Biofilms Formed by Curli- and Cellulose-Expressing Shiga Toxin-Producing Escherichia coli Using Treatments with Organic Acids and Commercial Sanitizers.

PubMed

Park, Yoen Ju; Chen, Jinru

2015-05-01

Biofilms are a mixture of bacteria and extracellular products secreted by bacterial cells and are of great concern to the food industry because they offer physical, mechanical, and biological protection to bacterial cells. This study was conducted to quantify biofilms formed by different Shiga toxin-producing Escherichia coli (STEC) strains on polystyrene and stainless steel surfaces and to determine the effectiveness of sanitizing treatments in control of these biofilms. STEC producing various amounts of cellulose (n = 6) or curli (n = 6) were allowed to develop biofilms on polystyrene and stainless steel surfaces at 28°C for 7 days. The biofilms were treated with 2% acetic or lactic acid and manufacturer-recommended concentrations of acidic or alkaline sanitizers, and residual biofilms were quantified. Treatments with the acidic and alkaline sanitizers were more effective than those with the organic acids for removing the biofilms. Compared with their counterparts, cells expressing a greater amount of cellulose or curli formed more biofilm mass and had greater residual mass after sanitizing treatments on polystyrene than on stainless steel. Research suggests that the organic acids and sanitizers used in the present study differed in their ability to control biofilms. Bacterial surface components and cell contact surfaces can influence both biofilm formation and the efficacy of sanitizing treatments. These results provide additional information on control of biofilms formed by STEC.

Compositions and Methods for the Treatment of Pierce's Disease

DOEpatents

Gupta, Goutam

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

MRP8/14 Enhances Corneal Susceptibility to Pseudomonas aeruginosa Infection by Amplifying Inflammatory Responses

PubMed Central

Deng, Qiuchan; Sun, Mingxia; Yang, Kun; Zhu, Min; Chen, Kang; Yuan, Jin; Wu, Minhao; Huang, Xi

2013-01-01

Purpose. We explored the role of myeloid-related protein 8 and 14 (MRP8/14) in Pseudomonas aeruginosa (PA) keratitis. Methods. MRP8/14 mRNA levels in human corneal scrapes and mouse corneas infected by PA were tested using real-time PCR. MRP8/14 protein expression in C57BL/6 (B6) corneas was confirmed using Western blot assay and immunohistochemistry. B6 mice were injected subconjunctivally with siRNA for MRP8/14, and then infected with PA. Bacterial plate counts and myeloperoxidase assays were used to determine the bacterial load and polymorphonuclear neutrophil (PMN) infiltration in infected B6 corneas. Pro-inflammatory cytokine levels in vivo and in vitro were examined with PCR and ELISA. In murine macrophage-like RAW264.7 cells, phagocytosis and bacterial killing were assessed using plate count assays, and reactive oxygen species (ROS) and nitric oxide (NO) levels were tested with flow cytometry and Griess assay, respectively. Results. MRP8/14 expression levels were increased significantly in human corneal scrapes and B6 corneas after PA infection. Silencing of MRP8/14 in B6 corneas significantly reduced the severity of corneal disease, bacterial clearance, PMN infiltration, and pro-inflammatory cytokine expression after PA infection. In vitro studies demonstrated further that silencing of MRP8/14 suppressed pro-inflammatory cytokine production, bacterial killing, and ROS production, but not phagocytosis or NO production. Conclusions. Our study demonstrated a dual role for MRP8/14 in bacterial keratitis. Although MRP8/14 promotes bacterial clearance by enhancing ROS production, it functions more importantly as an inflammatory amplifier at the ocular surface by enhancing pro-inflammatory cytokine expression, thus contributing to the corneal susceptibility. PMID:23299480

Surface physicochemical properties at the micro and nano length scales: role on bacterial adhesion and Xylella fastidiosa biofilm development.

PubMed

Lorite, Gabriela S; Janissen, Richard; Clerici, João H; Rodrigues, Carolina M; Tomaz, Juarez P; Mizaikoff, Boris; Kranz, Christine; de Souza, Alessandra A; Cotta, Mônica A

2013-01-01

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.

Surface Physicochemical Properties at the Micro and Nano Length Scales: Role on Bacterial Adhesion and Xylella fastidiosa Biofilm Development

PubMed Central

Lorite, Gabriela S.; Janissen, Richard; Clerici, João H.; Rodrigues, Carolina M.; Tomaz, Juarez P.; Mizaikoff, Boris; Kranz, Christine; de Souza, Alessandra A.; Cotta, Mônica A.

2013-01-01

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant. PMID:24073256

Expression of β-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

PubMed

Cheng, C-M; Chen, F M; Lu, Y-L; Tzou, S-C; Wang, J-Y; Kao, C-H; Liao, K-W; Cheng, T-C; Chuang, C-H; Chen, B-M; Roffler, S; Cheng, T-L

2013-05-01

Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

Bacteria-surface interactions.

PubMed

Tuson, Hannah H; Weibel, Douglas B

2013-05-14

The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.

Impact of cranberry on Escherichia coli cellular surface characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Brandy J.; Lin Baochuan; Dinderman, Michael A.

2008-12-19

The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae.more » The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.« less

Adhesion of Porphyromonas gingivalis and Tannerella forsythia to dentin and titanium with sandblasted and acid etched surface coated with serum and serum proteins - An in vitro study.

PubMed

Eick, Sigrun; Kindblom, Christian; Mizgalska, Danuta; Magdoń, Anna; Jurczyk, Karolina; Sculean, Anton; Stavropoulos, Andreas

2017-03-01

To evaluate the adhesion of selected bacterial strains incl. expression of important virulence factors at dentin and titanium SLA surfaces coated with layers of serum proteins. Dentin- and moderately rough SLA titanium-discs were coated overnight with human serum, or IgG, or human serum albumin (HSA). Thereafter, Porphyromonas gingivalis, Tannerella forsythia, or a six-species mixture were added for 4h and 24h. The number of adhered bacteria (colony forming units; CFU) was determined. Arg-gingipain activity of P. gingivalis and mRNA expressions of P. gingivalis and T. forsythia proteases and T. forsythia protease inhibitor were measured. Coating specimens never resulted in differences exceeding 1.1 log10 CFU, comparing to controls, irrespective the substrate. Counts of T. forsythia were statistically significantly higher at titanium than dentin, the difference was up to 3.7 log10 CFU after 24h (p=0.002). No statistically significant variation regarding adhesion of the mixed culture was detected between surfaces or among coatings. Arg-gingipain activity of P. gingivalis was associated with log10 CFU but not with the surface or the coating. Titanium negatively influenced mRNA expression of T. forsythia protease inhibitor at 24h (p=0.026 uncoated, p=0.009 with serum). The present findings indicate that: a) single bacterial species (T. forsythia) can adhere more readily to titanium SLA than to dentin, b) low expression of T. forsythia protease inhibitor may influence the virulence of the species on titanium SLA surfaces in comparison with teeth, and c) surface properties (e.g. material and/or protein layers) do not appear to significantly influence multi-species adhesion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arsenic alters transcriptional responses to Pseudomonas aeruginosa infection and decreases antimicrobial defense of human airway epithelial cells.

PubMed

Goodale, Britton C; Rayack, Erica J; Stanton, Bruce A

2017-09-15

Arsenic contamination of drinking water and food threatens the health of hundreds of millions of people worldwide by increasing the risk of numerous diseases. Arsenic exposure has been associated with infectious lung disease in epidemiological studies, but it is not yet understood how ingestion of low levels of arsenic increases susceptibility to bacterial infection. Accordingly, the goal of this study was to examine the effect of arsenic on gene expression in primary human bronchial epithelial (HBE) cells and to determine if arsenic altered epithelial cell responses to Pseudomonas aeruginosa, an opportunistic pathogen. Bronchial epithelial cells line the airway surface, providing a physical barrier and serving critical roles in antimicrobial defense and signaling to professional immune cells. We used RNA-seq to define the transcriptional response of HBE cells to Pseudomonas aeruginosa, and investigated how arsenic affected HBE gene networks in the presence and absence of the bacterial challenge. Environmentally relevant levels of arsenic significantly changed the expression of genes involved in cellular redox homeostasis and host defense to bacterial infection, and decreased genes that code for secreted antimicrobial factors such as lysozyme. Using pathway analysis, we identified Sox4 and Nrf2-regulated gene networks that are predicted to mediate the arsenic-induced decrease in lysozyme secretion. In addition, we demonstrated that arsenic decreased lysozyme in the airway surface liquid, resulting in reduced lysis of Microccocus luteus. Thus, arsenic alters the expression of genes and proteins in innate host defense pathways, thereby decreasing the ability of the lung epithelium to fight bacterial infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells.

PubMed

Cruz, Rui; Pereira-Castro, Isabel; Almeida, Maria T; Moreira, Alexandra; Cabanes, Didier; Sousa, Sandra

2018-01-01

The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes . We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin β1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.

Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces.

PubMed

Cavalcanti, Yuri Wanderley; Wilson, Melanie; Lewis, Michael; Del-Bel-Cury, Altair Antoninha; da Silva, Wander José; Williams, David W

2016-01-01

Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.

Adhesive Fiber Stratification in Uropathogenic Escherichia coli Biofilms Unveils Oxygen-Mediated Control of Type 1 Pili

PubMed Central

Floyd, Kyle A.; Moore, Jessica L.; Eberly, Allison R.; Good, James A. D.; Shaffer, Carrie L.; Zaver, Himesh; Almqvist, Fredrik; Skaar, Eric P.; Caprioli, Richard M.; Hadjifrangiskou, Maria

2015-01-01

Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the “OFF” orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were up-regulated under anoxic conditions. Tethering the fim promoter in the “ON” orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms. PMID:25738819

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

PubMed

Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

2015-01-01

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Bacterial Modulation of Plant Ethylene Levels

PubMed Central

Gamalero, Elisa; Glick, Bernard R.

2015-01-01

A focus on the mechanisms by which ACC deaminase-containing bacteria facilitate plant growth.Bacteria that produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, when present either on the surface of plant roots (rhizospheric) or within plant tissues (endophytic), play an active role in modulating ethylene levels in plants. This enzyme activity facilitates plant growth especially in the presence of various environmental stresses. Thus, plant growth-promoting bacteria that express ACC deaminase activity protect plants from growth inhibition by flooding and anoxia, drought, high salt, the presence of fungal and bacterial pathogens, nematodes, and the presence of metals and organic contaminants. Bacteria that express ACC deaminase activity also decrease the rate of flower wilting, promote the rooting of cuttings, and facilitate the nodulation of legumes. Here, the mechanisms behind bacterial ACC deaminase facilitation of plant growth and development are discussed, and numerous examples of the use of bacteria with this activity are summarized. PMID:25897004

Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

PubMed

Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

2015-01-01

Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response▿ †

PubMed Central

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J.

2006-01-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway. PMID:16980424

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response.

PubMed

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J

2006-11-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation.

PubMed

Bruna, Roberto E; Molino, María Victoria; Lazzaro, Martina; Mariscotti, Javier F; García Véscovi, Eleonora

2018-04-15

PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescen s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescens IMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the prtA gene. Moreover, we reveal that PrtA expression favors the ability of S. marcescens to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of S. marcescens on abiotic surfaces and in bacterial host colonization capacity. Copyright © 2018 American Society for Microbiology.

N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

PubMed Central

Nothaft, Harald; Zheng, Jing

2013-01-01

Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

PubMed Central

Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

2016-01-01

ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chunmao; Yu, You; Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn

2015-10-23

Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. Wemore » speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.« less

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

PubMed Central

Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

2016-01-01

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169

Extract of corn silk (stigma of Zea mays) inhibits the tumour necrosis factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1 expression.

PubMed

Habtemariam, S

1998-05-01

Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking.

Effect of respiratory syncytial virus (RSV) infection on the adherence of pathogenic bacteria to human epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faden, H.; Hong, J.J.; Ogra, P.L.

1986-03-01

The effect of RSV infection on the adherence of Streptococcus pneumoniae (SP), Haemophilus influenzae (HI) and Staphylococcus aureus (SA) to human epithelial cells was determined. RSV-infected Hep-2 cell cultures at different stages of expression of surface viral antigens and bacteria labeled with /sup 3/H-thymidine were employed to examine the kinetics of bacterial adherence to virus-infected cells. RSV infection did not alter the magnitude of adherence of HI or SA to HEp-2 cells. However, adherence of SP to HEp-2 cells was significantly (P < 0.01) enhanced by prior RSV infection. The degree of adherence was directly related to the amount ofmore » viral antigen expressed on the cell surface. The adherence was temperature dependent, with maximal adherence observed at 37/sup 0/C. Heat-inactivation of SP did not alter adherence characteristics. These data suggest that RSV infection increases adherence of SP to the surface of epithelial cells in vitro. Since attachment of bacteria to mucosal surfaces is the first step in many infections, it is suggested that viral infections of epithelial cells render them more susceptible to bacterial adherence. Thus, RSV infection in vivo may predispose children to SP infections, such as in otitis media, by increasing colonization with SP.« less

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

PubMed

Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

2015-05-01

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. Copyright © 2015 Elsevier GmbH. All rights reserved.

Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans.

PubMed

Spinola, Stanley M; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Zwickl, Beth; Katz, Barry P; Munson, Robert S

2012-02-01

Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.

Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yun

The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with themore » firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca 2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca 2+ signaling through gap junctions seems more effective.« less

Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

PubMed Central

Fink, Doran L.; St. Geme III, Joseph W.

2003-01-01

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hapβ and a 110-kDa extracellular passenger domain called HapS. All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap. PMID:12591878

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

PubMed

Kochi, Shinsuke; Yamashiro, Keisuke; Hongo, Shoichi; Yamamoto, Tadashi; Ugawa, Yuki; Shimoe, Masayuki; Kawamura, Mari; Hirata-Yoshihara, Chiaki; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

2017-12-01

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Molecular response of Escherichia coli adhering onto nanoscale topography

NASA Astrophysics Data System (ADS)

Rizzello, Loris; Galeone, Antonio; Vecchio, Giuseppe; Brunetti, Virgilio; Sabella, Stefania; Pompa, Pier Paolo

2012-10-01

Bacterial adhesion onto abiotic surfaces is an important issue in biology and medicine since understanding the bases of such interaction represents a crucial aspect in the design of safe implant devices with intrinsic antibacterial characteristics. In this framework, we investigated the effects of nanostructured metal substrates on Escherichia coli adhesion and adaptation in order to understand the bio-molecular dynamics ruling the interactions at the interface. In particular, we show how highly controlled nanostructured gold substrates impact the bacterial behavior in terms of morphological changes and lead to modifications in the expression profile of several genes, which are crucially involved in the stress response and fimbrial synthesis. These results mainly demonstrate that E. coli cells are able to sense even slight changes in surface nanotopography and to actively respond by activating stress-related pathways. At the same time, our findings highlight the possibility of designing nanoengineered substrates able to trigger specific bio-molecular effects, thus opening the perspective of smartly tuning bacterial behavior by biomaterial design.

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood.

PubMed

Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

2017-01-01

Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. Copyright © 2016 Askarian et al.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

PubMed Central

Castiblanco-Valencia, Mónica M.; Fraga, Tatiana R.; Breda, Leandro C.D.; Vasconcellos, Sílvio A.; Figueira, Cláudio P.; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I.; Barbosa, Angela S.; Isaac, Lourdes

2017-01-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

Genetic characteristics and pathogenic mechanisms of periodontal pathogens.

PubMed

Amano, A; Chen, C; Honma, K; Li, C; Settem, R P; Sharma, A

2014-05-01

Periodontal disease is caused by a group of bacteria that utilize a variety of strategies and molecular mechanisms to evade or overcome host defenses. Recent research has uncovered new evidence illuminating interesting aspects of the virulence of these bacteria and their genomic variability. This paper summarizes some of the strategies utilized by the major species - Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis - implicated in the pathogenesis of periodontal disease. Whole-genome sequencing of 14 diverse A. actinomycetemcomitans strains has revealed variations in their genetic content (ranging between 0.4% and 19.5%) and organization. Strikingly, isolates from human periodontal sites showed no genomic changes during persistent colonization. T. forsythia manipulates the cytokine responses of macrophages and monocytes through its surface glycosylation. Studies have revealed that bacterial surface-expressed O-linked glycans modulate T-cell responses during periodontal inflammation. Periodontal pathogens belonging to the "red complex" consortium express neuraminidases, which enables them to scavenge sialic acid from host glycoconjugates. Analysis of recent data has demonstrated that the cleaved sialic acid acts as an important nutrient for bacterial growth and a molecule for the decoration of bacteria surfaces to help evade the host immune attack. In addition, bacterial entry into host cells is also an important prerequisite for the lifestyle of periodontal pathogens such as P. gingivalis. Studies have shown that, after its entry into the cell, this bacterium uses multiple sorting pathways destined for autophagy, lysosomes, or recycling pathways. In addition, P. gingivalis releases outer membrane vesicles which enter cells via endocytosis and cause cellular functional impairment.

Role of curli expression by Escherichia coli O157:H7 on the cell’s ability to attach to spinach

USDA-ARS?s Scientific Manuscript database

Introduction: Shiga-toxigenic Escherichia coli O157:H7 (STEC) outbreaks have been linked to consumption of fresh produce. Mechanisms of bacterial interaction with plant surfaces should be investigated to develop mitigation strategies. Cellular appendages, such as curli fibers have been suggested t...

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis.

PubMed

Tarazanova, Mariya; Huppertz, Thom; Kok, Jan; Bachmann, Herwig

2018-05-09

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in Myxococcus xanthus

PubMed Central

Wartel, Morgane; Czerwinski, Fabian; Le Gall, Anne-Valérie; Mauriello, Emilia M. F.; Bergam, Ptissam; Brun, Yves V.; Shaevitz, Joshua; Mignot, Tâm

2013-01-01

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories. PMID:24339744

Surface roughness and bacterial adhesion on root dentin treated with diode laser and conventional desensitizing agents.

PubMed

Cury, Maiza S; Silva, Camilla B; Nogueira, Ruchele D; Campos, Michelle G D; Palma-Dibb, Regina G; Geraldo-Martins, Vinicius R

2018-02-01

The treatments for dentin hypersensitivity (DH) may change the surface roughness of the root dentin, which can lead to biofilm accumulation, increasing the risk of root caries. The aim was to compare the surface roughness of root dentin after different treatments of DH and the biofilm formation on those surfaces. After initial surface roughness (Sa) assessment, 50 bovine root fragments received the following treatments (n = 10): G 1-no treatment; G2-5% sodium fluoride varnish; G3-professional application of a desensitizing dentifrice; G4-toothbrushing with a desensitizing dentifrice; and G5-diode laser application (908 nm; 1.5 W, 20 s). The Sa was reevaluated after treatments. Afterward, all samples were incubated in a suspension of Streptococcus mutans at 37 °C for 24 h. The colony-forming units (CFU) were counted using a stereoscope, and the results were expressed in CFU/mL. The one-way ANOVA and the Tukey's tests compared the roughness data and the results obtained on the bacterial adhesion test (α = 5%). G2 (2.3 ± 1.67%) showed similar Sa variation than G1 (0.25 ± 0.41%) and G5 (5.69 ± 0.99%), but different from group G3 (9.05 ± 2.39%). Group 4 showed the highest Sa variation (30.02 ± 3.83%; p < 0.05). Bacterial adhesion was higher in G4 (2208 ± 211.9), suggesting that bacterial growth is greater on rougher surfaces. The diode laser and the conventional treatments for DH may change the surface roughness of the root dentin, but only brushing with desensitizing dentifrice induced a higher bacteria accumulation on root dentin surface.

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Bacterial Pili exploit integrin machinery to promote immune activation and efficient blood-brain barrier penetration

PubMed Central

Banerjee, Anirban; Kim, Brandon J.; Carmona, Ellese M.; Cutting, Andrew S.; Gurney, Michael A.; Carlos, Chris; Feuer, Ralph; Prasadarao, Nemani V.; Doran, Kelly S.

2011-01-01

Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2β1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS–BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS. PMID:21897373

Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

NASA Astrophysics Data System (ADS)

Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P.; Ren, Ling; Yang, Ke

2016-07-01

Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility.

Pili of Mycobacterium tuberculosis: current knowledge and future prospects.

PubMed

Ramsugit, Saiyur; Pillay, Manormoney

2015-08-01

Many pathogenic bacteria express filamentous appendages, termed pili, on their surface. These organelles function in several important bacterial processes, including mediating bacterial interaction with, and colonization of the host, signalling events, locomotion, DNA uptake, electric conductance, and biofilm formation. In the last decade, it has been established that the tuberculosis-causing bacterium, Mycobacterium tuberculosis, produces two pili types: curli and type IV pili. In this paper, we review studies on M. tuberculosis pili, highlighting their structure and biological significance to M. tuberculosis pathogenesis, and discuss their potential as targets for therapeutic intervention and diagnostic test development.

Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

DOEpatents

Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

2007-02-27

The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis.

PubMed

Alvarez Hayes, Jimena; Oviedo, Juan Marcos; Valdez, Hugo; Laborde, Juan Martín; Maschi, Fabricio; Ayala, Miguel; Shah, Rohan; Fernandez Lahore, Marcelo; Rodriguez, Maria Eugenia

2017-10-01

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuA Bpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuA Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Mapping the Laminin Receptor Binding Domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2

PubMed Central

Mahdavi, Jafar; Oldfield, Neil J.; Wheldon, Lee M.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171–240 and 91–99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains. PMID:23049988

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

PubMed

Abouseada, Noha M; Assafi, Mahde Saleh A; Mahdavi, Jafar; Oldfield, Neil J; Wheldon, Lee M; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst.

PubMed

Tavassoli, Setareh; Hinc, Krzysztof; Iwanicki, Adam; Obuchowski, Michal; Ahmadian, Gholamreza

2013-03-01

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture.

Spatiotemporal evolution of bacterial biofilm colonies

NASA Astrophysics Data System (ADS)

Wilking, James; Koehler, Stephan; Sinha, Naveen; Seminara, Agnese; Brenner, Michael; Weitz, David

2014-03-01

Many bacteria on earth live in surface-attached communities known as biofilms. Gene expression in a biofilm is typically varied, resulting in a variety of phenotypes within a single film. These phenotypes play a critical role in biofilm physiology and development. We use time-resolved, wide-field fluorescence microscopy to image triple-labeled fluorescent Bacillus Subtilis colonies grown on agar to determine in a non-invasive fashion the evolving phenotypes. We infer their transition rates from the resulting spatiotemporal maps of gene expression. Moreover, we correlate these transition rates with local measurements of nutrient concentration to determine the influence of extracellular signals on gene expression.

A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

PubMed Central

Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H.; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

2015-01-01

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. PMID:25673765

A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections (CAUTIs).

PubMed

Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

2015-04-01

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of HBGA-expressing bacteria does not protect Tulane virus from acute heat stress

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the major cause of gastroenteritis outbreaks worldwide. Human noroviruses can interact with histo-blood group antigens (HBGAs) on the surface of mammalian cells as well as bacterial cells. HBGAs have been considered as putative receptors or co-receptors for HuNoVs in m...

Genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited IL-17RA deficiency

PubMed Central

Lévy, Romain; Okada, Satoshi; Béziat, Vivien; Moriya, Kunihiko; Liu, Caini; Chai, Louis Yi Ann; Migaud, Mélanie; Hauck, Fabian; Al Ali, Amein; Cyrus, Cyril; Vatte, Chittibabu; Patiroglu, Turkan; Unal, Ekrem; Ferneiny, Marie; Hyakuna, Nobuyuki; Nepesov, Serdar; Oleastro, Matias; Ikinciogullari, Aydan; Dogu, Figen; Asano, Takaki; Ohara, Osamu; Yun, Ling; Della Mina, Erika; Bronnimann, Didier; Itan, Yuval; Gothe, Florian; Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Tahuil, Natalia; Aytekin, Caner; Salhi, Aicha; Al Muhsen, Saleh; Kobayashi, Masao; Toubiana, Julie; Abel, Laurent; Li, Xiaoxia; Camcioglu, Yildiz; Celmeli, Fatih; Klein, Christoph; AlKhater, Suzan A.; Casanova, Jean-Laurent; Puel, Anne

2016-01-01

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC—autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency—was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface. PMID:27930337

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila▿

PubMed Central

Sha, Jian; Erova, Tatiana E.; Alyea, Rebecca A.; Wang, Shaofei; Olano, Juan P.; Pancholi, Vijay; Chopra, Ashok K.

2009-01-01

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general. PMID:19270100

MPLA inhibits release of cytotoxic mediators from human neutrophils while preserving efficient bacterial killing.

PubMed

Ruchaud-Sparagano, Marie-Hélène; Mills, Ross; Scott, Jonathan; Simpson, A John

2014-10-01

Monophosphoryl lipid A (MPLA) is a lipopolysaccharides (LPS) derivative associated with neutrophil-dependent anti-inflammatory outcomes in animal models of sepsis. Little is known about the effect of MPLA on neutrophil function. This study sought to test the hypothesis that MPLA would reduce release of cytotoxic mediators from neutrophils without impairing bacterial clearance. Neutrophils were isolated from whole blood of healthy volunteers. The effects of MPLA and LPS on autologous serum-opsonised Pseudomonas aeruginosa killing by neutrophils and phagocytosis of autologous serum-opsonised zymosan were examined. Neutrophil oxidative burst, chemotaxis, enzyme and cytokine release as well as Toll-like receptor 4 (TLR4) expression were assessed following exposure to LPS or MPLA. LPS, but not MPLA, induced significant release of superoxide and myeloperoxidase from neutrophils. However, MPLA did not impair neutrophil capacity to ingest microbial particles and kill P. aeruginosa efficiently. MPLA was directly chemotactic for neutrophils, involving TLR4, p38 mitogen-activated protein kinase and tyrosine and alkaline phosphatases. LPS, but not MPLA, impaired N-formyl-methionyl-leucyl phenylalanine-directed migration of neutrophils, increased surface expression of TLR4, increased interleukin-8 release and strongly activated the myeloid differentiation primary response 88 pathway. Phosphoinositide 3-kinase inhibition significantly augmented IL-8 release from MPLA-treated neutrophils. The addition of MPLA to LPS-preincubated neutrophils led to a significant reduction in LPS-mediated superoxide release and TLR4 surface expression. Collectively, these findings suggest that MPLA directs efficient chemotaxis and bacterial killing in human neutrophils without inducing extracellular release of cytotoxic mediators and suggest that MPLA warrants further attention as a potential therapeutic in human sepsis.

An Ribonuclease T2 Family Protein Modulates Acinetobacter baumannii Abiotic Surface Colonization

PubMed Central

Jacobs, Anna C.; Blanchard, Catlyn E.; Catherman, Seana C.; Dunman, Paul M.; Murata, Yoshihiko

2014-01-01

Acinetobacter baumannii is an emerging bacterial pathogen of considerable medical concern. The organism's transmission and ability to cause disease has been associated with its propensity to colonize and form biofilms on abiotic surfaces in health care settings. To better understand the genetic determinants that affect biomaterial attachment, we performed a transposon mutagenesis analysis of abiotic surface-colonization using A. baumannii strain 98-37-09. Disruption of an RNase T2 family gene was found to limit the organism's ability to colonize polystyrene, polypropylene, glass, and stainless steel surfaces. DNA microarray analyses revealed that in comparison to wild type and complemented cells, the RNase T2 family mutant exhibited reduced expression of 29 genes, 15 of which are predicted to be associated with bacterial attachment and surface-associated motility. Motility assays confirmed that RNase T2 mutant displays a severe motility defect. Taken together, our results indicate that the RNase T2 family protein identified in this study is a positive regulator of A. baumannii's ability to colonize inanimate surfaces and motility. Moreover, the enzyme may be an effective target for the intervention of biomaterial colonization, and consequently limit the organism's transmission within the hospital setting. PMID:24489668

Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

PubMed

Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

2014-06-15

Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Construction of recombinant Lactobacillus casei efficiently surface displayed and secreted porcine parvovirus VP2 protein and comparison of the immune responses induced by oral immunization.

PubMed

Yigang, X U; Yijing, L I

2008-05-01

Lactobacillus casei ATCC 393 was selected as a bacterial carrier for the development of mucosal vaccine against porcine parvovirus (PPV) infection. The PPV major structural polypeptide VP2 was used as the model parvovirus antigen. Two inducible expression systems, namely pPG611.1 of the cell-surface expression system and pPG612.1 of the secretion expression system based on the xylose operon promoter were used to express the VP2 protein. The immunogenicity of recombinant strains producing VP2 protein in two cellular locations, cell-surface exposed and secreted, was compared to each other by immunizing mice through the intragastric administration. The two types of constructs were able to induce strong specific immune responses against VP2 via intragastric administration and maximum titres of IgA and IgG were attained on days 46 post oral immunization, while the highest antibody levels were obtained with the strain producing the VP2 protein in extracellular milieu. The induced antibodies demonstrated neutralizing effects on PPV infection.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

PubMed

Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Dynamics of Immune System Gene Expression upon Bacterial Challenge and Wounding in a Social Insect (Bombus terrestris)

PubMed Central

Erler, Silvio; Popp, Mario; Lattorff, H. Michael G.

2011-01-01

The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge. Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment. Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the transcription factor relish, which is necessary for effector gene expression. PMID:21479237

Why Does the Healthy Cornea Resist Pseudomonas aeruginosa Infection?

PubMed Central

Evans, David J.; Fleiszig, Suzanne M. J.

2013-01-01

Purpose To provide our perspective on why the cornea is resistant to infection based on our research results with Pseudomonas aeruginosa. Perspective We focus on our current understanding of the interplay between bacteria, tear fluid and the corneal epithelium that determine health as the usual outcome, and propose a theoretical model for how contact lens wear might change those interactions to enable susceptibility to P. aeruginosa infection. Methods Use of “null-infection” in vivo models, cultured human corneal epithelial cells, contact lens-wearing animal models, and bacterial genetics help to elucidate mechanisms by which P. aeruginosa survive at the ocular surface, adheres, and traverses multilayered corneal epithelia. These models also help elucidate the molecular mechanisms of corneal epithelial innate defense. Results and Discussion Tear fluid and the corneal epithelium combine to make a formidable defense against P. aeruginosa infection of the cornea. Part of that defense involves the expression of antimicrobials such as β-defensins, the cathelicidin LL-37, cytokeratin-derived antimicrobial peptides, and RNase7. Immunomodulators such as SP-D and ST2 also contribute. Innate defenses of the cornea depend in part on MyD88, a key adaptor protein of TLR and IL-1R signaling, but the basal lamina represents the final barrier to bacterial penetration. Overcoming these defenses involves P. aeruginosa adaptation, expression of the type three secretion system, proteases, and P. aeruginosa biofilm formation on contact lenses. Conclusion After more than two decades of research focused on understanding how contact lens wear predisposes to P. aeruginosa infection, our working hypothesis places blame for microbial keratitis on bacterial adaptation to ocular surface defenses, combined with changes to the biochemistry of the corneal surface caused by trapping bacteria and tear fluid against the cornea under the lens. PMID:23601656

Common themes in microbial pathogenicity revisited.

PubMed Central

Finlay, B B; Falkow, S

1997-01-01

Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics. PMID:9184008

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

PubMed Central

Morales-Soto, Nydia; Anyan, Morgen E.; Mattingly, Anne E.; Madukoma, Chinedu S.; Harvey, Cameron W.; Alber, Mark; Déziel, Eric; Kearns, Daniel B.; Shrout, Joshua D.

2015-01-01

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment. PMID:25938934

The anti-adherence effect of Piper betle and Psidium guajava extracts on the adhesion of early settlers in dental plaque to saliva-coated glass surfaces.

PubMed

Razak, Fathilah Abdul; Rahim, Zubaidah Haji Abd

2003-12-01

The aqueous extracts of Piper betle and Psidium guajava were prepared and tested for their anti-adherence effect on the adhesion of early plaque settlers (Strep. mitis, Strep. sanguinis and Actinomyces sp.). The saliva-coated glass surfaces were used to simulate the pellicle-coated enamel surface in the oral cavity. Our results showed that the anti-adherence activities of Piper betle and Psidium guajava extracts towards the bacteria were different between the bacterial species. Psidium guajava was shown to have a slightly greater anti-adherence effect on Strep. sanguinis by 5.5% and Actinomyces sp. by 10% and a significantly higher effect on Strep. mitis (70%) compared to Piper betle. The three bacterial species are known to be highly hydrophobic, and that hydrophobic bonding seemed to be an important factor in their adherence activities. It is therefore suggested that the plant extracts, in expressing their anti-adherence activities, could have altered the hydrophobic nature of the bonding between the bacteria and the saliva-coated glass surfaces.

Can dead bacterial cells be defined and are genes expressed after cell death?

PubMed

Trevors, J T

2012-07-01

There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

PubMed Central

Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.

2012-01-01

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment. PMID:22247133

The Bacterial Cytoskeleton Modulates Motility, Type 3 Secretion, and Colonization in Salmonella

PubMed Central

Bulmer, David M.; Kharraz, Lubna; Grant, Andrew J.; Dean, Paul; Morgan, Fiona J. E.; Karavolos, Michail H.; Doble, Anne C.; McGhie, Emma J.; Koronakis, Vassilis; Daniel, Richard A.; Mastroeni, Pietro; Anjam Khan, C. M.

2012-01-01

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC. PMID:22291596

Effect of cell-surface hydrophobicity on bacterial conversion of water-immiscible chemicals in two-liquid-phase culture systems.

PubMed

Hamada, Takahiro; Maeda, Yusuke; Matsuda, Hiroyuki; Sameshima, Yuka; Honda, Kohsuke; Omasa, Takeshi; Kato, Junichi; Ohtake, Hisao

2009-08-01

The effect of bacterial cell-surface hydrophobicity on the bioconversion of water-immiscible chemicals in an aqueous-organic (A/O) two-liquid-phase culture system was investigated. Escherichia coli JM109 and Rhodococcus opacus B-4 were used as hydrophilic and hydrophobic whole-cell catalysts, respectively. Hydroxylation reactions of monoaromatics, including toluene (log P(ow)=2.9), ethylbenzene (3.1), n-propylbenzene (3.4), and sec-butylbenzene (3.7), were employed as model conversions. When the todC1C2BA genes encoding Pseudomonas putida toluene dioxygenase were expressed in E. coli JM109, the yield of hydroxylated monoaromatics decreased with increasing substrate hydrophobicity. By contrast, R. opacus transformants, which expressed the todC1C2BA genes, showed high performance in the hydroxylation of monoaromatics, irrespective of substrate hydrophobicity. When the R. opacus transformants were examined for their ability to hydroxylate monoaromatics in an aqueous single-liquid-phase culture, the reaction velocity was markedly lower than that observed in the A/O two-liquid-phase culture. These results suggested that R. opacus B-4 accessed the hydrophobic substrates in the oil phase, thus making it more effective for the bioconversion reactions.

Method and Apparatus for Detecting and Quantifying Bacterial Spores on a Surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2017-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified.

Method and apparatus for detecting and quantifying bacterial spores on a surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified.

Bacterial resistance of self-assembled surfaces using PPOm-b-PSBMAn zwitterionic copolymer - concomitant effects of surface topography and surface chemistry on attachment of live bacteria.

PubMed

Hsiao, Sheng-Wen; Venault, Antoine; Yang, Hui-Shan; Chang, Yung

2014-06-01

Three well-defined diblock copolymers made of poly(sulfobetaine methacrylate) (poly(SBMA)) and poly(propylene oxide) (PPO) groups were synthesized by atom transfer radical polymerization (ATRP) method. They were physically adsorbed onto three types of surfaces having different topography, including smooth flat surface, convex surface, and indented surface. Chemical state of surfaces was characterized by XPS while the various topographies were examined by SEM and AFM. Hydrophilicity of surfaces was dependent on both the surface chemistry and the surface topography, suggesting that orientation of copolymer brushes can be tuned in the design of surfaces aimed at resisting bacterial attachment. Escherichia coli, Staphylococcus epidermidis, Streptococcus mutans and Escherichia coli with green fluorescent protein (E. coli GFP) were used in bacterial tests to assess the resistance to bacterial attachment of poly(SBMA)-covered surfaces. Results highlighted a drastic improvement of resistance to bacterial adhesion with the increasing of poly(SBMA) to PPO ratio, as well as an important effect of surface topography. The chemical effect was directly related to the length of the hydrophilic moieties. When longer, more water could be entrapped, leading to improved anti-bacterial properties. The physical effect impacted on the orientation of the copolymer brushes, as well as on the surface contact area available. Convex surfaces as well as indented surfaces wafer presented the best resistance to bacterial adhesion. Indeed, bacterial attachment was more importantly reduced on these surfaces compared with smooth surfaces. It was explained by the non-orthogonal orientation of copolymer brushes, resulting in a more efficient surface coverage of zwitterionic molecules. This work suggests that not only the control of surface chemistry is essential in the preparation of surfaces resisting bacterial attachment, but also the control of surface topography and orientation of antifouling moieties. Copyright © 2014 Elsevier B.V. All rights reserved.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

PubMed Central

Viadas, Cristina; Ruiz de los Mozos, Igor; Valle, Jaione; Bengoechea, José Antonio; Garmendia, Junkal

2015-01-01

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence. PMID:25894755

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

PubMed

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed.

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry

PubMed Central

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed. PMID:27375566

Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

PubMed Central

Aigle, Marina; Fleurot, Renaud; Darrigo, Claire; Hennekinne, Jacques-Antoine; Gruss, Alexandra; Borezée-Durant, Elise; Delacroix-Buchet, Agnès

2014-01-01

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices. PMID:24928871

Control of bacterial adhesion and growth on honeycomb-like patterned surfaces.

PubMed

Yang, Meng; Ding, Yonghui; Ge, Xiang; Leng, Yang

2015-11-01

It is a great challenge to construct a persistent bacteria-resistant surface even though it has been demonstrated that several surface features might be used to control bacterial behavior, including surface topography. In this study, we develop micro-scale honeycomb-like patterns of different sizes (0.5-10 μm) as well as a flat area as the control on a single platform to evaluate the bacterial adhesion and growth. Bacteria strains, Escherichia coli and Staphylococcus aureus with two distinct shapes (rod and sphere) are cultured on the platforms, with the patterned surface-up and surface-down in the culture medium. The results demonstrate that the 1 μm patterns remarkably reduce bacterial adhesion and growth while suppressing bacterial colonization when compared to the flat surface. The selective adhesion of the bacterial cells on the patterns reveals that the bacterial adhesion is cooperatively mediated by maximizing the cell-substrate contact area and minimizing the cell deformation, from a thermodynamic point of view. Moreover, study of bacterial behaviors on the surface-up vs. surface-down samples shows that gravity does not apparently affect the spatial distribution of the adherent cells although it indeed facilitates bacterial adhesion. Furthermore, the experimental results suggest that two major factors, i.e. the availability of energetically favorable adhesion sites and the physical confinements, contribute to the anti-bacterial nature of the honeycomb-like patterns. Copyright © 2015 Elsevier B.V. All rights reserved.

Fabrication of a platform to isolate the influences of surface nanotopography from chemistry on bacterial attachment and growth.

PubMed

Pegalajar-Jurado, Adoracion; Easton, Christopher D; Crawford, Russell J; McArthur, Sally L

2015-03-26

Billions of dollars are spent annually worldwide to combat the adverse effects of bacterial attachment and biofilm formation in industries as varied as maritime, food, and health. While advances in the fabrication of antifouling surfaces have been reported recently, a number of the essential aspects responsible for the formation of biofilms remain unresolved, including the important initial stages of bacterial attachment to a substrate surface. The reduction of bacterial attachment to surfaces is a key concept in the prevention or minimization of biofilm formation. The chemical and physical characteristics of both the substrate and bacteria are important in understanding the attachment process, but substrate modification is likely the most practical route to enable the extent of bacterial attachment taking place to be effectively controlled. The microtopography and chemistry of the surface are known to influence bacterial attachment. The role of surface chemistry versus nanotopography and their interplay, however, remain unclear. Most methods used for imparting nanotopographical patterns onto a surface also induce changes in the surface chemistry and vice versa. In this study, the authors combine colloidal lithography and plasma polymerization to fabricate homogeneous, reproducible, and periodic nanotopographies with a controllable surface chemistry. The attachment of Escherichia coli bacteria onto carboxyl (plasma polymerized acrylic acid, ppAAc) and hydrocarbon (plasma polymerized octadiene, ppOct) rich plasma polymer films on either flat or colloidal array surfaces revealed that the surface chemistry plays a critical role in bacterial attachment, whereas the effect of surface nanotopography on the bacterial attachment appears to be more difficult to define. This platform represents a promising approach to allow a greater understanding of the role that surface chemistry and nanotopography play on bacterial attachment and the subsequent biofouling of the surface.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

PubMed

Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Refrigerated storage of platelets initiates changes in platelet surface marker expression and localization of intracellular proteins.

PubMed

Wood, Ben; Padula, Matthew P; Marks, Denese C; Johnson, Lacey

2016-10-01

Platelets (PLTs) are currently stored at room temperature (22°C), which limits their shelf life, primarily due to the risk of bacterial growth. Alternatives to room temperature storage include PLT refrigeration (2-6°C), which inhibits bacterial growth, thus potentially allowing an extension of shelf life. Additionally, refrigerated PLTs appear more hemostatically active than conventional PLTs, which may be beneficial in certain clinical situations. However, the mechanisms responsible for this hemostatic function are not well characterized. The aim of this study was to assess the protein profile of refrigerated PLTs in an effort to understand these functional consequences. Buffy coat PLTs were pooled, split, and stored either at room temperature (20-24°C) or under refrigerated (2-6°C) conditions (n = 8 in each group). PLTs were assessed for changes in external receptor expression and actin filamentation using flow cytometry. Intracellular proteomic changes were assessed using two-dimensional gel electrophoresis and Western blotting. PLT refrigeration significantly reduced the abundance of glycoproteins (GPIb, GPIX, GPIIb, and GPIV) on the external membrane. However, refrigeration resulted in the increased expression of high-affinity integrins (αIIbβ3 and β1) and activation and apoptosis markers (CD62P, CD63, and phosphatidylserine). PLT refrigeration substantially altered the abundance and localization of several cytoskeletal proteins and resulted in an increase in actin filamentation, as measured by phalloidin staining. Refrigerated storage of PLTs induces significant changes in the expression and localization of both surface-expressed and intracellular proteins. Understanding these proteomic changes may help to identify the mechanisms resulting in the refrigeration-associated alterations in PLT function and clearance. © 2016 AABB.

Flagellar motility is a key determinant of the magnitude of the inflammasome response to Pseudomonas aeruginosa.

PubMed

Patankar, Yash R; Lovewell, Rustin R; Poynter, Matthew E; Jyot, Jeevan; Kazmierczak, Barbara I; Berwin, Brent

2013-06-01

We previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronic Pseudomonas aeruginosa infections, enables bacteria to evade association and ingestion of P. aeruginosa by phagocytes both in vitro and in vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation by P. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotile P. aeruginosa. Nonmotile P. aeruginosa elicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cells in vitro. Importantly, nonmotile P. aeruginosa also elicits reduced IL-1β levels in vivo in comparison to those elicited by wild-type P. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

PubMed Central

Patankar, Yash R.; Lovewell, Rustin R.; Poynter, Matthew E.; Jyot, Jeevan; Kazmierczak, Barbara I.

2013-01-01

We previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronic Pseudomonas aeruginosa infections, enables bacteria to evade association and ingestion of P. aeruginosa by phagocytes both in vitro and in vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation by P. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotile P. aeruginosa. Nonmotile P. aeruginosa elicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cells in vitro. Importantly, nonmotile P. aeruginosa also elicits reduced IL-1β levels in vivo in comparison to those elicited by wild-type P. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system. PMID:23529619

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

PubMed

Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

2016-07-21

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Method and apparatus for detecting and quantifying bacterial spores on a surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

Method and Apparatus for Detecting and Quantifying Bacterial Spores on a Surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2016-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

PubMed Central

Sharifi, Rouhallah; Ryu, Choong-Min

2016-01-01

Biological control (biocontrol) agents act on plants via numerous mechanisms, and can be used to protect plants from pathogens. Biocontrol agents can act directly as pathogen antagonists or competitors or indirectly to promote plant induced systemic resistance (ISR). Whether a biocontrol agent acts directly or indirectly depends on the specific strain and the pathosystem type. We reported previously that bacterial volatile organic compounds (VOCs) are determinants for eliciting plant ISR. Emerging data suggest that bacterial VOCs also can directly inhibit fungal and plant growth. The aim of the current study was to differentiate direct and indirect mechanisms of bacterial VOC effects against Botrytis cinerea infection of Arabidopsis. Volatile emissions from Bacillus subtilis GB03 successfully protected Arabidopsis seedlings against B. cinerea. First, we investigated the direct effects of bacterial VOCs on symptom development and different phenological stages of B. cinerea including spore germination, mycelial attachment to the leaf surface, mycelial growth, and sporulation in vitro and in planta. Volatile emissions inhibited hyphal growth in a dose-dependent manner in vitro, and interfered with fungal attachment on the hydrophobic leaf surface. Second, the optimized bacterial concentration that did not directly inhibit fungal growth successfully protected Arabidopsis from fungal infection, which indicates that bacterial VOC-elicited plant ISR has a more important role in biocontrol than direct inhibition of fungal growth on Arabidopsis. We performed qRT-PCR to investigate the priming of the defense-related genes PR1, PDF1.2, and ChiB at 0, 12, 24, and 36 h post-infection and 14 days after the start of plant exposure to bacterial VOCs. The results indicate that bacterial VOCs potentiate expression of PR1 and PDF1.2 but not ChiB, which stimulates SA- and JA-dependent signaling pathways in plant ISR and protects plants against pathogen colonization. This study provides new evidence for bacterial VOC-elicited plant ISR that protects Arabidopsis plants from infection by the necrotrophic fungus B. cinerea. Our work reveals that bacterial VOCs primarily act via an indirect mechanism to elicit plant ISR, and have a major role in biocontrol against fungal pathogens. PMID:26941721

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene.

PubMed

Shimizu, Masami; Kimura, Tetsuya; Koyama, Takayoshi; Suzuki, Katsuhisa; Ogawa, Naoto; Miyashita, Kiyotaka; Sakka, Kazuo; Ohmiya, Kunio

2002-08-01

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.

Glycobiology of the ocular surface: Mucins and lectins

PubMed Central

Argüeso, Pablo

2013-01-01

Glycosylation is an important and common form of posttranscriptional modification of proteins in cells. A vast array of biological functions has been ascribed to glycans during the last decade thanks to a rapid evolution in glycomic technologies. Glycogenes highly expressed at the human ocular surface include families of glycosyltransferases, proteoglycans, glycan degradation proteins, as well as mucins and carbohydrate-binding proteins such as the galectins. On the apical glycocalyx, mucin O-glycans promote boundary lubrication, prevent bacterial adhesion and endocytic activity, and maintain epithelial barrier function through interactions with galectins. The emerging roles attributed to glycans are contributing to the appreciation of their biological capabilities at the ocular surface. PMID:23325272

An upgraded bacterial surface display system to express cleavable capsid protein of human noroviruses: A novel system to discover candidate receptors for HuNoV

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the dominant cause of food-borne outbreaks of gastroenteritis. However, the fundamental of the research on the viruses were restricted by the current immature system to culture HuNoVs and lacking of efficient small animal models. Previously, we demonstrated that the re...

Chemical modification of polyvinyl chloride and silicone elastomer in inhibiting adhesion of Aeromonas hydrophila.

PubMed

Kregiel, Dorota; Berlowska, Joanna; Mizerska, Urszula; Fortuniak, Witold; Chojnowski, Julian; Ambroziak, Wojciech

2013-07-01

Disease-causing bacteria of the genus Aeromonas are able to adhere to pipe materials, colonizing the surfaces and forming biofilms in water distribution systems. The aim of our research was to study how the modification of materials used commonly in the water industry can reduce bacterial cell attachment. Polyvinyl chloride and silicone elastomer surfaces were activated and modified with reactive organo-silanes by coupling or co-crosslinking silanes with the native material. Both the native and modified surfaces were tested using the bacterial strain Aeromonas hydrophila, which was isolated from the Polish water distribution system. The surface tension of both the native and modified surfaces was measured. To determine cell viability and bacterial adhesion two methods were used, namely plate count and luminometry. Results were expressed in colony-forming units (c.f.u.) and in relative light units (RLU) per cm(2). Almost all the chemically modified surfaces exhibited higher anti-adhesive and anti-microbial properties in comparison to the native surfaces. Among the modifying agents examined, poly[dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammoniopropyl chloride) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer gave the most desirable results. The surface tension of this modifier, was comparable to the non-polar native surface. However, almost half of this value was due to the result of polar forces. In this case, in an adhesion analysis, only 1 RLU cm(-2) and less than 1 c.f.u. cm(-2) were noted. For the native gumosil, the results were 9,375 RLU cm(-2) and 2.5 × 10(8) c.f.u. cm(-2), respectively. The antibacterial activity of active organo-silanes was associated only with the carrier surface because no antibacterial compounds were detected in liquid culture media, in concentrations that were able to inhibit cell growth.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis.

PubMed

Mittal, Rahul; Prasadarao, Nemani V

2011-11-22

Despite the fundamental function of neutrophils (polymorphonuclear leukocytes (PMNs)) in innate immunity, their role in Escherichia coli K1 (EC-K1) -induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1 infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by downregulating rac1, rac2 and gp91(phox) transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface-expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis

PubMed Central

Mittal, Rahul; Prasadarao, Nemani V.

2012-01-01

Despite the fundamental function of neutrophils (PMNs) in innate immunity, their role in Escherichia coli K1 (EC-K1) induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by down regulating rac1, rac2 and gp91phox transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis. PMID:22109526

Immunotoxicity of surface waters contaminated by municipal effluents to the snail Lymnaea stagnalis.

PubMed

Gust, M; Fortier, M; Garric, J; Fournier, M; Gagné, F

2013-01-15

The immunotoxic effects of surface waters contaminated by a municipal effluent dispersion plume were examined in the snail Lymnaea stagnalis. Snails were exposed to surface waters where changes in hemocyte counts, viability, levels of reactive oxygen species (ROS), reduced thiols and phagocytic activity were tracked following exposure periods of 3h and 3 and 7d. Changes in mRNA expression of some genes in the hemocytes were also assessed after 7d of exposure, as follows: genes coding for catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), selenium-dependent glutathione peroxidase (SeGPX), two isoforms of the nitric oxide synthetase (NOS1 and NOS2), molluscan defensive molecule (MDM), toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF), and heat-shock protein 70 (HSP70). At the sites closest to the discharge point, exposure led to impaired hemocyte viability and intracellular thiol levels and also an increase of hemocyte count, ROS levels and phagocytosis. Phagocytosis and ROS levels in hemocytes were correlated with heterotrophic bacterial counts in snails. We found four genes with increased mRNA expression as a response to exposure of municipal wastewaters: TLR4 (6-fold), HSP70 (2-fold), SeGPx (4-fold) and CAT (2-fold). Immunocompetence responses were analyzed by canonical analysis to seek out relationships with mRNA expression of the genes involved in stress, pattern recognition, cellular and humoral responses. The data revealed that genes involved in oxidative stress were strongly involved with immunocompetence and that the resulting immune responses were influenced both by the bacterial and pollutant loadings of the effluent. Copyright © 2012 Elsevier B.V. All rights reserved.

Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer

PubMed Central

Kristian, Sascha A.; Hwang, John H.; Hall, Bradley; Leire, Emma; Iacomini, John; Old, Robert; Galili, Uri; Roberts, Charles; Mullis, Kary B.; Westby, Mike; Nizet, Victor

2015-01-01

The ever-increasing threat of multi-drug resistant bacterial infections has spurred renewed interest in alternative approaches to classical antibiotic therapy. In contrast to other mammals, humans do not express the galactose-α-1,3-galactosyl-β-1,4-N-acetyl-glucosamine (α-Gal) epitope. As a result of exposure of humans to α-Gal in the environment, a large proportion of circulating antibodies are specific for the trisaccharide. In this study, we examine whether these anti-Gal antibodies can be recruited and redirected to exert anti-bacterial activity. We show that a specific DNA aptamer conjugated to an α-Gal epitope at its 5′ end, herein termed an alphamer, can bind to group A Streptococcus (GAS) bacteria by recognition of a conserved region of the surface-anchored M protein. The anti-GAS alphamer was shown to recruit anti-Gal antibodies to the streptococcal surface in an α-Gal-specific manner, elicit uptake and killing of the bacteria by human phagocytes, and slow growth of invasive GAS in human whole blood. These studies provide a first in vitro proof of concept that alphamers have the potential to redirect pre-existing antibodies to bacteria in a specific manner and trigger an immediate antibacterial immune response. Further validation of this novel therapeutic approach of applying α-Gal technology in in vivo models of bacterial infection is warranted. PMID:25940316

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

PubMed

Nejidat, A; Saadi, I; Ronen, Z

2008-12-01

To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Metal adsorption onto bacterial surfaces: development of a predictive approach

NASA Astrophysics Data System (ADS)

Fein, Jeremy B.; Martin, Aaron M.; Wightman, Peter G.

2001-12-01

Aqueous metal cation adsorption onto bacterial surfaces can be successfully modeled by means of a surface complexation approach. However, relatively few stability constants for metal-bacterial surface complexes have been measured. In order to determine the bacterial adsorption behavior of cations that have not been studied in the laboratory, predictive techniques are required that enable estimation of the stability constants of bacterial surface complexes. In this study, we use a linear free-energy approach to compare previously measured stability constants for Bacillus subtilis metal-carboxyl surface complexes with aqueous metal-organic acid anion stability constants. The organic acids that we consider are acetic, oxalic, citric, and tiron. We add to this limited data set by conducting metal adsorption experiments onto Bacillus subtilis, determining bacterial surface stability constants for Co, Nd, Ni, Sr, and Zn. The adsorption behavior of each of the metals studied here was described well by considering metal-carboxyl bacterial surface complexation only, except for the Zn adsorption behavior, which required carboxyl and phosphoryl complexation to obtain a suitable fit to the data. The best correlation between bacterial carboxyl surface complexes and aqueous organic acid anion stability constants was obtained by means of metal-acetate aqueous complexes, with a linear correlation coefficient of 0.97. This correlation applies only to unhydrolyzed aqueous cations and only to carboxyl binding of those cations, and it does not predict the binding behavior under conditions where metal binding to other bacterial surface site types occurs. However, the relationship derived in this study permits estimation of the carboxyl site adsorption behavior of a wide range of aqueous metal cations for which there is an absence of experimental data. This technique, coupled with the observation of similar adsorption behaviors across bacterial species (Yee and Fein, 2001), enables estimation of the effects of bacterial adsorption on metal mobilities for a large number of environmental and geologic applications.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

PubMed

Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-08-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

PubMed

Douterelo, I; Sharpe, R L; Boxall, J B

2013-02-01

Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this might have on drinking water quality. Copyright © 2012 Elsevier Ltd. All rights reserved.

A mechanism for bacterial transformation of dimethylsulfide to dimethylsulfoxide: a missing link in the marine organic sulfur cycle.

PubMed

Lidbury, Ian; Kröber, Eileen; Zhang, Zhidong; Zhu, Yijun; Murrell, J Colin; Chen, Yin; Schäfer, Hendrik

2016-09-01

The volatile organosulfur compound, dimethylsulfide (DMS), plays an important role in climate regulation and global sulfur biogeochemical cycles. Microbial oxidation of DMS to dimethylsulfoxide (DMSO) represents a major sink of DMS in surface seawater, yet the underlying molecular mechanisms and key microbial taxa involved are not known. Here, we reveal that Ruegeria pomeroyi, a model marine heterotrophic bacterium, can oxidize DMS to DMSO using trimethylamine monooxygenase (Tmm). Purified Tmm oxidizes DMS to DMSO at a 1:1 ratio. Mutagenesis of the tmm gene in R. pomeroyi completely abolished DMS oxidation and subsequent DMSO formation. Expression of Tmm and DMS oxidation in R. pomeroyi is methylamine-dependent and regulated at the post-transcriptional level. Considering that Tmm is present in approximately 20% of bacterial cells inhabiting marine surface waters, particularly the marine Roseobacter clade and the SAR11 clade, our observations contribute to a mechanistic understanding of biological DMSO production in surface seawater. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Soil Nitrogen Status Modifies Rice Root Response to Nematode-Bacteria Interactions in the Rhizosphere

PubMed Central

Wu, Yue; Valentine, Tracy A.; Li, Huixin

2016-01-01

It has been hypothesized that faunal activity in the rhizosphere influences root growth via an auxin-dependent pathway. In this study, two methods were used to adjust nematode and bacterial populations within experimental soils. One is “exclusion”, where soil mixed with pig manure was placed in two bags with different mesh sizes (1mm and 5μm diameter), and then surrounded by an outer layer of unamended soil resulting in soil with a greater populations of bacterial-feeding nematodes (1mm) and a control treatment (5μm). The second method is “inoculation”, whereby autoclaved soil was inoculated with bacteria (E. coli and Pseudomonas) and Nematodes (Cephalobus and C. elegans). In order to detect the changes in the rice’s perception of auxin under different nutrient and auxin conditions in the presence of soil bacterial-feeding nematodes, responses of soil chemistry (NH4+, NO3- and indole acetic acid (IAA)), rice root growth and the expression of an auxin responsive gene GH3-2 were measured. Results showed that, under low soil nutrient conditions (exclusion), low NO3- correlated with increased root branching and IAA correlated with increased root elongation and GH3-2 expression. However, under high soil nutrient conditions (inoculation), a high NH4+ to NO3- ratio promoted an increase in root surface area and there was an additional influence of NH4+ and NO3- on GH3-2 expression. Thus it was concluded that soil bacterial-feeding nematodes influenced soil nutritional status and soil IAA content, promoting root growth via an auxin dependent pathway that was offset by soil nitrogen status. PMID:26841062

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira.

PubMed

Hauk, P; Carvalho, E; Ho, P L

2011-04-01

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Host- and microbe determinants that may influence the success of S. aureus colonization

PubMed Central

Johannessen, Mona; Sollid, Johanna E.; Hanssen, Anne-Merethe

2012-01-01

Staphylococcus aureus may cause serious skin and soft tissue infections, deep abscesses, endocarditis, osteomyelitis, pneumonia, and sepsis. S. aureus persistently colonizes 25–30% of the adult human population, and S. aureus carriers have an increased risk for infections caused by the bacterium. The major site of colonization is the nose, i.e., the vestibulum nasi, which is covered with ordinary skin and hair follicles. Several host and microbe determinants are assumed to be associated with colonization. These include the presence and expression level of bacterial adhesins, which can adhere to various proteins in the extracellular matrix or on the cellular surface of human skin. The host expresses several antimicrobial peptides and lipids. The level of β-defensin 3, free sphingosine, and cis-6-hexadecenoic acid are found to be associated with nasal carriage of S. aureus. Other host factors are certain polymorphisms in Toll-like receptor 2, mannose-binding lectin, C-reactive protein, glucocorticoid-, and vitamin D receptor. Additional putative determinants for carriage include genetic variation and expression of microbial surface components recognizing adhesive matrix molecules and their interaction partners, as well as variation among humans in the ability of recognizing and responding appropriately to the bacteria. Moreover, the available microflora may influence the success of S. aureus colonization. In conclusion, colonization is a complex interplay between the bacteria and its host. Several bacterial and host factors are involved, and an increased molecular understanding of these are needed. PMID:22919647

Host- and microbe determinants that may influence the success of S. aureus colonization.

PubMed

Johannessen, Mona; Sollid, Johanna E; Hanssen, Anne-Merethe

2012-01-01

Staphylococcus aureus may cause serious skin and soft tissue infections, deep abscesses, endocarditis, osteomyelitis, pneumonia, and sepsis. S. aureus persistently colonizes 25-30% of the adult human population, and S. aureus carriers have an increased risk for infections caused by the bacterium. The major site of colonization is the nose, i.e., the vestibulum nasi, which is covered with ordinary skin and hair follicles. Several host and microbe determinants are assumed to be associated with colonization. These include the presence and expression level of bacterial adhesins, which can adhere to various proteins in the extracellular matrix or on the cellular surface of human skin. The host expresses several antimicrobial peptides and lipids. The level of β-defensin 3, free sphingosine, and cis-6-hexadecenoic acid are found to be associated with nasal carriage of S. aureus. Other host factors are certain polymorphisms in Toll-like receptor 2, mannose-binding lectin, C-reactive protein, glucocorticoid-, and vitamin D receptor. Additional putative determinants for carriage include genetic variation and expression of microbial surface components recognizing adhesive matrix molecules and their interaction partners, as well as variation among humans in the ability of recognizing and responding appropriately to the bacteria. Moreover, the available microflora may influence the success of S. aureus colonization. In conclusion, colonization is a complex interplay between the bacteria and its host. Several bacterial and host factors are involved, and an increased molecular understanding of these are needed.

A Single-Amino-Acid Substitution in Obg Activates a New Programmed Cell Death Pathway in Escherichia coli.

PubMed

Dewachter, Liselot; Verstraeten, Natalie; Monteyne, Daniel; Kint, Cyrielle Ines; Versées, Wim; Pérez-Morga, David; Michiels, Jan; Fauvart, Maarten

2015-12-22

Programmed cell death (PCD) is an important hallmark of multicellular organisms. Cells self-destruct through a regulated series of events for the benefit of the organism as a whole. The existence of PCD in bacteria has long been controversial due to the widely held belief that only multicellular organisms would profit from this kind of altruistic behavior at the cellular level. However, over the past decade, compelling experimental evidence has established the existence of such pathways in bacteria. Here, we report that expression of a mutant isoform of the essential GTPase ObgE causes rapid loss of viability in Escherichia coli. The physiological changes that occur upon expression of this mutant protein--including loss of membrane potential, chromosome condensation and fragmentation, exposure of phosphatidylserine on the cell surface, and membrane blebbing--point to a PCD mechanism. Importantly, key regulators and executioners of known bacterial PCD pathways were shown not to influence this cell death program. Collectively, our results suggest that the cell death pathway described in this work constitutes a new mode of bacterial PCD. Programmed cell death (PCD) is a well-known phenomenon in higher eukaryotes. In these organisms, PCD is essential for embryonic development--for example, the disappearance of the interdigital web--and also functions in tissue homeostasis and elimination of pathogen-invaded cells. The existence of PCD mechanisms in unicellular organisms like bacteria, on the other hand, has only recently begun to be recognized. We here demonstrate the existence of a bacterial PCD pathway that induces characteristics that are strikingly reminiscent of eukaryotic apoptosis, such as fragmentation of DNA, exposure of phosphatidylserine on the cell surface, and membrane blebbing. Our results can provide more insight into the mechanism and evolution of PCD pathways in higher eukaryotes. More importantly, especially in the light of the looming antibiotic crisis, they may point to a bacterial Achilles' heel and can inspire innovative ways of combating bacterial infections, directed at the targeted activation of PCD pathways. Copyright © 2015 Dewachter et al.

A study on the dynamics of the zraP gene expression profile and its application to the construction of zinc adsorption bacteria.

PubMed

Ravikumar, Sambandam; Yoo, Ik-keun; Lee, Sang Yup; Hong, Soon Ho

2011-11-01

Zinc ion plays essential roles in biological chemistry. Bacteria acquire Zn(2+) from the environment, and cellular concentration levels are controlled by zinc homeostasis systems. In comparison with other homeostatic systems, the ZraSR two-component system was found to be more efficient in responding to exogenous zinc concentrations. To understand the dynamic response of the bacterium ZraSR two-component system with respect to exogenous zinc concentrations, the genetic circuit of the ZraSR system was integrated with a reporter protein. This study was helpful in the construction of an E. coli system that can display selective metal binding peptides on the surface of the cell in response to exogenous zinc. The engineered bacterial system for monitoring exogenous zinc was successfully employed to detect levels of zinc as low as 0.001 mM, which directly activates the expression of chimeric ompC(t)--zinc binding peptide gene to remove zinc by adsorbing a maximum of 163.6 μmol of zinc per gram of dry cell weight. These results indicate that the engineered bacterial strain developed in the present study can sense the specific heavy metal and activates a cell surface display system that acts to remove the metal.

Bacteria Hold Their Breath upon Surface Contact as Shown in a Strain of Escherichia coli, Using Dispersed Surfaces and Flow Cytometry Analysis

PubMed Central

Geng, Jing; Beloin, Christophe; Ghigo, Jean-Marc; Henry, Nelly

2014-01-01

Bacteria are ubiquitously distributed throughout our planet, mainly in the form of adherent communities in which cells exhibit specific traits. The mechanisms underpinning the physiological shift in surface-attached bacteria are complex, multifactorial and still partially unclear. Here we address the question of the existence of early surface sensing through implementation of a functional response to initial surface contact. For this purpose, we developed a new experimental approach enabling simultaneous monitoring of free-floating, aggregated and adherent cells via the use of dispersed surfaces as adhesive substrates and flow cytometry analysis. With this system, we analyzed, in parallel, the constitutively expressed GFP content of the cells and production of a respiration probe—a fluorescent reduced tetrazolium ion. In an Escherichia coli strain constitutively expressing curli, a major E. coli adhesin, we found that single cell surface contact induced a decrease in the cell respiration level compared to free-floating single cells present in the same sample. Moreover, we show here that cell surface contact with an artificial surface and with another cell caused reduction in respiration. We confirm the existence of a bacterial cell “sense of touch” ensuring early signalling of surface contact formation through respiration down modulation. PMID:25054429

Impervious Surfaces Alter Soil Bacterial Communities in Urban Areas: A Case Study in Beijing, China

PubMed Central

Hu, Yinhong; Dou, Xiaolin; Li, Juanyong; Li, Feng

2018-01-01

The rapid expansion of urbanization has caused land cover change, especially the increasing area of impervious surfaces. Such alterations have significant effects on the soil ecosystem by impeding the exchange of gasses, water, and materials between soil and the atmosphere. It is unclear whether impervious surfaces have any effects on soil bacterial diversity and community composition. In the present study, we conducted an investigation of bacterial communities across five typical land cover types, including impervious surfaces (concrete), permeable pavement (bricks with round holes), shrub coverage (Buxus megistophylla Levl.), lawns (Festuca elata Keng ex E. Alexeev), and roadside trees (Sophora japonica Linn.) in Beijing, to explore the response of bacteria to impervious surfaces. The soil bacterial communities were addressed by high-throughput sequencing of the bacterial 16S rRNA gene. We found that Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, and Firmicutes were the predominant phyla in urban soils. Soil from impervious surfaces presented a lower bacterial diversity, and differed greatly from other types of land cover. Soil bacterial diversity was predominantly affected by Zn, dissolved organic carbon (DOC), and soil moisture content (SMC). The composition of the bacterial community was similar under shrub coverage, roadside trees, and lawns, but different from beneath impervious surfaces and permeable pavement. Variance partitioning analysis showed that edaphic properties contributed to 12% of the bacterial community variation, heavy metal pollution explained 3.6% of the variation, and interaction between the two explained 33% of the variance. Together, our data indicate that impervious surfaces induced changes in bacterial community composition and decrease of bacterial diversity. Interactions between edaphic properties and heavy metals were here found to change the composition of the bacterial community and diversity across areas with different types of land cover, and soil properties play a more important role than heavy metals. PMID:29545776

Band Gap Engineering of Titania Film through Cobalt Regulation for Oxidative Damage of Bacterial Respiration and Viability.

PubMed

Li, Jinhua; Wang, Jiaxing; Wang, Donghui; Guo, Geyong; Yeung, Kelvin W K; Zhang, Xianlong; Liu, Xuanyong

2017-08-23

Biomaterial-related bacterial infections cause patient suffering, mortality, and extended periods of hospitalization and impose a substantial burden on medical systems. In this context, understanding the interactions between nanomaterials and bacteria is clinically significant. Herein, TiO 2 -based heterojunctions, including Co-TiO 2 , CoO-TiO 2 , and Co 3 O 4 -TiO 2 , were first designed by optimizing magnetron sputtering to establish a platform to explore the interactions between nanomaterials and bacteria. We found that the energy band bending and band gap narrowing were effectively promoted at the contact interface of the heterojunctions, which have the ability to induce abiotic reactive oxygen species formation. Using methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, in vitro studies showed that the heterojunctions of Co-TiO 2 , CoO-TiO 2 , and especially Co 3 O 4 -TiO 2 can effectively downregulate the expression levels of bacterial respiratory genes and cause oxidative damage to bacterial membrane respiration and viability. As a result, the surfaces of the heterojunctions possess a favorable antiadherent bacterial activity. Moreover, using an osteomyelitis model, the preclinical study on rats further confirmed the favorable anti-infection effect of the elaborately designed heterojunctions (especially Co 3 O 4 -TiO 2 ). We hope this study can provide new insights into the surface antibacterial design of biomaterials using energy band engineering for both basic research and clinical needs. Meanwhile, this attempt may also contribute to expanding the biomedical applications of cobalt-based nanoparticles for the treatment of antibiotic-resistant infections.

Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen Acinetobacter baumannii

PubMed Central

Nwugo, Chika C.; Arivett, Brock A.; Zimbler, Daniel L.; Gaddy, Jennifer A.; Richards, Ashley M.; Actis, Luis A.

2012-01-01

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments. PMID:23284824

Effect of ethanol on differential protein production and expression of potential virulence functions in the opportunistic pathogen Acinetobacter baumannii.

PubMed

Nwugo, Chika C; Arivett, Brock A; Zimbler, Daniel L; Gaddy, Jennifer A; Richards, Ashley M; Actis, Luis A

2012-01-01

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.

Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

PubMed

Yang, Xiao-Yan; Sun, Bin; Zhang, Liang; Li, Nan; Han, Junlong; Zhang, Jing; Sun, Xuesong; He, Qing-Yu

2014-01-01

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

[Respiratory protection provided by N95 filtering facepiece respirators and disposable medicine masks against airborne bacteria in different working environments].

PubMed

Lu, W; Zhu, X C; Zhang, X Y; Chen, Y T; Chen, W H

2016-09-20

Objective: To determine the relative protection provided by N95 filtering facepiece respirators (FFR) and disposable medicine masks (DMM) against airborne bacteria in different working environments. Methods: The field study was performed with 12 subjects wearing an N95 filtering facepiece respirator and a disposable medicine mask for 1h, respectively. Airborne microorganisms and bacteria samples from both the external (Ce) and the inner (Ci) surface of N95 FFR and DMM are collected. The Ce: Ci ratio was used to calculate the bacterial filtering proportion. Bacterial filtering efficiency (BFE) was measured using the JWL-2A Sampler. Results: The bacterial filtration efficiency of N95 FFR and DMM were 99.93% and 91.53%, respectively. There was significant difference between the two materials ( P <0.05). In summer, airborne bacterial concentration was higher than that in winter. In the same season, airborne bacterial concentration in hospital environment is higher than that in campus. The higher the airborne bacterial concentration, the greater bacterial contaminated on the external surface of the used masks. To all masks used in different working environment, bacterial contamination on the external surface was much greater than the inner surface ( P <0.01). Compared to N95 FFR, DMM had slighter bacterial contamination on the external surface and greater bacterial contamination on the inner surface. However, this difference was not significant ( P >0.05). The bacterial filtering proportion of N95 FFR is higher than DMM. These differences were significant in samples tested in summer ( P <0.05) , but were not significant in samples tested in winter ( P >0.05). Conclusion: Bacterial filtering efficiency of N95 respirator is superior to medicine mask, and this advantage become more obvious in high airborne bacterial concentration levels.

Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants in Salmonella Vaccine Strains

PubMed Central

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B.; Lattemann, Claus T.

2003-01-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp6074-86 was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains. PMID:12654812

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

PubMed

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B; Lattemann, Claus T

2003-04-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

PARAMETERS OF TREATED STAINLESS STEEL SURFACES IMPORTANT FOR RESISTANCE TO BACTERIAL CONTAMINATION

EPA Science Inventory

Use of materials that are resistant to bacterial contamination could enhance food safety during processing. Common finishing treatments of stainless steel surfaces used for components of poultry processing equipment were tested for resistance to bacterial attachment. Surface char...

Bacterial desorption from food container and food processing surfaces.

PubMed

McEldowney, S; Fletcher, M

1988-03-01

The desorption ofStaphylococcus aureus, Acinetobacter calcoaceticus, and a coryneform from the surfaces of materials used for manufacturing food containers (glass, tin plate, and polypropylene) or postprocess canning factory conveyor belts (stainless steel and nylon) was investigated. The effect of time, pH, temperature, and adsorbed organic layers on desorption was studied.S. aureus did not detach from the substrata at any pH investigated (between pH 5 and 9).A. calcoaceticus and the coryneform in some cases detached, depending upon pH and substratum composition. The degree of bacterial detachment from the substrata was not related to bacterial respiration at experimental pH values. Bacterial desorption was not affected by temperature (4-30°C) nor by an adsorbed layer of peptone and yeast extract on the substrata. The results indicate that bacterial desorption, hence bacterial removal during cleaning or their transfer via liquids flowing over colonized surfaces, is likely to vary with the surface composition and the bacterial species colonizing the surfaces.

Claudin-3 expression in radiation-exposed rat models: A potential marker for radiation-induced intestinal barrier failure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shim, Sehwan; Lee, Jong-geol; Bae, Chang-hwan

2015-01-02

Highlights: • Irradiation increased intestinal bacterial translocation, accompanied by claudin protein expression in rats. • Neurotensin decreased the bacterial translocation and restored claudin-3 expression. • Claudin-3 can be used as a marker in evaluating radiation induced intestinal injury. - Abstract: The molecular events leading to radiation-induced intestinal barrier failure are not well known. The influence of the expression of claudin proteins in the presence and absence of neurotensin was investigated in radiation-exposed rat intestinal epithelium. Wistar rats were randomly divided into control, irradiation, and irradiation + neurotensin groups, and bacterial translocation to the mesenteric lymph node and expression of claudinsmore » were determined. Irradiation led to intestinal barrier failure as demonstrated by significant bacterial translocation. In irradiated terminal ilea, expression of claudin-3 and claudin-4 was significantly decreased, and claudin-2 expression was increased. Administration of neurotensin significantly reduced bacterial translocation and restored the structure of the villi as seen by histologic examination. Among the three subtype of claudins, only claudin-3 expression was restored. These results suggest that the therapeutic effect of neurotensin on the disruption of the intestinal barrier is associated with claudin-3 alteration and that claudin-3 could be used as a marker in evaluating radiation-induced intestinal injury.« less

Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes.

PubMed

Berglund, Björn; Khan, Ghazanfar Ali; Weisner, Stefan E B; Ehde, Per Magnus; Fick, Jerker; Lindgren, Per-Eric

2014-04-01

Recently, there have been growing concerns about pharmaceuticals including antibiotics as environmental contaminants. Antibiotics of concentrations commonly encountered in wastewater have been suggested to affect bacterial population dynamics and to promote dissemination of antibiotic resistance. Conventional wastewater treatment processes do not always adequately remove pharmaceuticals causing environmental dissemination of low levels of these compounds. Using constructed wetlands as an additional treatment step after sewage treatment plants have been proposed as a cheap alternative to increase reduction of wastewater contaminants, however this means that the natural microbial community of the wetlands becomes exposed to elevated levels of antibiotics. In this study, experimental surface-flow wetlands in Sweden were continuously exposed to antibiotics of concentrations commonly encountered in wastewater. The aim was to assess the antibiotic removal efficiency of constructed wetlands and to evaluate the impact of low levels of antibiotics on bacterial diversity, resistance development and expression in the wetland bacterial community. Antibiotic concentrations were measured using liquid chromatography-mass spectrometry and the effect on the bacterial diversity was assessed with 16S rRNA-based denaturing gradient gel electrophoresis. Real-time PCR was used to detect and quantify antibiotic resistance genes and integrons in the wetlands, during and after the exposure period. The results indicated that the antibiotic removal efficiency of constructed wetlands was comparable to conventional wastewater treatment schemes. Furthermore, short-term treatment of the constructed wetlands with environmentally relevant concentrations (i.e. 100-2000 ng×l(-1)) of antibiotics did not significantly affect resistance gene concentrations, suggesting that surface-flow constructed wetlands are well-suited for wastewater treatment purposes. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

PubMed Central

Boyd, Mary A.; Wang, Jin Y.; Tulapurkar, Mohan E.; Pasetti, Marcela F.; Levine, Myron M.; Simon, Raphael

2016-01-01

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. PMID:26998925

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

RNA-Seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid and resveratrol

PubMed Central

Qin, Nan; Tan, Xiaojuan; Jiao, Yinming; Liu, Lin; Zhao, Wangsheng; Yang, Shuang; Jia, Aiqun

2014-01-01

Bacterial biofilms are particularly problematic since they become resistant to most available antibiotics. Hence, novel potential antagonists to inhibit biofilm formation are urgent. Here the influences of two natural products, ursolic acid and resveratrol, on biofilm of the clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate were investigated using RNA-seq, and the differentially expressed genes were analyzed using Cuffdiff. The results showed that ursolic acid inhibition of biofilm formation may reduce amino acids metabolism and adhesins expression and resveratrol may disturb quorum sensing (QS) and the synthesis of surface proteins and capsular polysaccharides. In addition, the transcriptome analysis of resveratrol and the combination of resveratrol with vancomycin inhibition of established biofilm revealed that resveratrol would disturb the expression of genes related to QS, surface and secreted proteins, and capsular polysaccharides. These findings suggest that ursolic acid and resveratrol could be useful to be adjunct therapies for the treatment of MRSA biofilm-involved infections. PMID:24970710

Surface thermodynamics and adhesion forces governing bacterial transmission in contact lens related microbial keratitis.

PubMed

Qu, Wenwen; Busscher, Henk J; Hooymans, Johanna M M; van der Mei, Henny C

2011-06-15

Contact lens induced microbial keratitis results from bacterial transmission from one surface to another. We investigated the adhesion forces of Pseudomonas aeruginosa, Staphylococci and Serratia to different contact lenses, lens cases and corneal surfaces using AFM, and applied a Weibull analysis on these adhesion forces to calculate bacterial transmission probabilities from lens case to corneas with a contact lens as an intermediate. Also a new surface thermodynamic parameter was introduced, the interfacial free energy of transmission, which in essence compares the interfacial free energies of bacterial adhesion, calculated from measured contact angles with liquids on the donating and receiving surfaces in the transmission process. Bacterial adhesion forces were generally strongest among all eight strains for the lens case (-6.5 to -12.0 nN) and corneas (-3.5 to -11.5 nN), while contact lenses (-0.6 to -13.1 nN) exerted slightly smaller adhesion forces. Consequently, bacterial transmission from lens case to contact lens yielded a smaller contribution in the final transmission than from contact lens to cornea. Bacterial transmission probabilities as derived from force analyses were higher when the interfacial free energies of transmission were more negative, which is in line with surface thermodynamic principles. Therewith this parameter could provide useful in analyzing other bacterial transmission phenomena between donating and receiving surfaces as well. Copyright © 2011 Elsevier Inc. All rights reserved.

Cellulose effects on morphology and elasticity of Vibrio fischeri biofilms.

PubMed

Ziemba, Christopher; Shabtai, Yael; Piatkovsky, Maria; Herzberg, Moshe

2016-01-01

Cellulose effects on Vibrio fischeri biofilm morphology were tested for the wild-type and two of its isogenic mutants that either exhibit increased cellulose production or do not produce cellulose at all. Confocal laser scanning microscopy imaging of each biofilm revealed that total sessile volume increases with cellulose expression, but the size of colonies formed with cellulose was smaller, creating a more diffuse biofilm. These morphological differences were not attributed to variations in bacterial deposition, extracellular polymeric substances affinity to the surface or bacterial growth. A positive correlation was found between cellulose expression, Young's (elastic) modulus of the biofilm analyzed with atomic force microscope and shear modulus of the related extracellular polymeric substances layers analyzed with quartz crystal microbalance with dissipation monitoring. Cellulose production also correlated positively with concentrations of extracellular DNA. A significant negative correlation was observed between cellulose expression and rates of diffusion through the extracellular polymeric substances. The difference observed in biofilm morphology is suggested as a combined result of cellulose and likely extracellular DNA (i) increasing biofilm Young's modulus, making shear removal more difficult, and (ii) decreased diffusion rate of nutrients and wastes into and out of the biofilm, which effectively limits colony size.

Bacterial filamentation accelerates colonization of adhesive spots embedded in biopassive surfaces

NASA Astrophysics Data System (ADS)

Möller, Jens; Emge, Philippe; Avalos Vizcarra, Ima; Kollmannsberger, Philip; Vogel, Viola

2013-12-01

Sessile bacteria adhere to engineered surfaces and host tissues and pose a substantial clinical and economical risk when growing into biofilms. Most engineered and biological interfaces are of chemically heterogeneous nature and provide adhesive islands for bacterial attachment and growth. To mimic either defects in a surface coating of biomedical implants or heterogeneities within mucosal layers (Peyer's patches), we embedded micrometre-sized adhesive islands in a poly(ethylene glycol) biopassive background. We show experimentally and computationally that filamentation of Escherichia coli can significantly accelerate the bacterial surface colonization under physiological flow conditions. Filamentation can thus provide an advantage to a bacterial population to bridge non-adhesive distances exceeding 5 μm. Bacterial filamentation, caused by blocking of bacterial division, is common among bacterial species and can be triggered by environmental conditions or antibiotic treatment. While great awareness exists that the build-up of antibiotic resistance serves as intrinsic survival strategy, we show here that antibiotic treatment can actually promote surface colonization by triggering filamentation, which in turn prevents daughter cells from being washed away. Our combined microfabrication and computational approaches provide quantitative insights into mechanisms that enable biofouling of biopassive surfaces with embedded adhesive spots, even for spot distances that are multiples of the bacterial length.

A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

PubMed

Deryabin, Dmitry G; Efremova, Ludmila V; Vasilchenko, Alexey S; Saidakova, Evgeniya V; Sizova, Elena A; Troshin, Pavel A; Zhilenkov, Alexander V; Khakina, Ekaterina A; Khakina, Ekaterina E

2015-08-08

The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the activity of positively charged [60]fullerene derivatives against bacterial cells required their direct interaction. The following zeta potential inversion on the bacterial cells surface was observed as an early stage of toxicity mechanism that violates the membrane-associated energetic functions. The novel data about interrelations between physicochemical parameters and toxic properties of amphiphilic [60]fullerene derivatives make possible predicting their behavior in aquatic environment and their activity against bacterial cells.

Locomotion of bacteria in liquid flow and the boundary layer effect on bacterial attachment.

PubMed

Zhang, Chao; Liao, Qiang; Chen, Rong; Zhu, Xun

2015-06-12

The formation of biofilm greatly affects the performance of biological reactors, which highly depends on bacterial swimming and attachment that usually takes place in liquid flow. Therefore, bacterial swimming and attachment on flat and circular surfaces with the consideration of flow was studied experimentally. Besides, a mathematical model comprehensively combining bacterial swimming and motion with flow is proposed for the simulation of bacterial locomotion and attachment in flow. Both experimental and theoretical results revealed that attached bacteria density increases with decreasing boundary layer thickness on both flat and circular surfaces, the consequence of which is inherently related to the competition between bacterial swimming and the non-slip motion with flow evaluated by the Péclet number. In the boundary layer, where the Péclet number is relatively higher, bacterial locomotion mainly depends on bacterial swimming. Thinner boundary layer promotes bacterial swimming towards the surface, leading to higher attachment density. To enhance the performance of biofilm reactors, it is effective to reduce the boundary layer thickness on desired surfaces. Copyright © 2015 Elsevier Inc. All rights reserved.

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

PubMed Central

Muenzner, Petra; Kengmo Tchoupa, Arnaud; Klauser, Benedikt; Brunner, Thomas; Putze, Johannes; Dobrindt, Ulrich; Hauck, Christof R.

2016-01-01

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa. PMID:27171273

Microbiota diversity and gene expression dynamics in human oral biofilms

PubMed Central

2014-01-01

Background Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Results Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. Conclusions The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health. PMID:24767457

Microbiota diversity and gene expression dynamics in human oral biofilms.

PubMed

Benítez-Páez, Alfonso; Belda-Ferre, Pedro; Simón-Soro, Aurea; Mira, Alex

2014-04-27

Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.

Bacterial community diversity and variation in spray water sources and the tomato fruit surface.

PubMed

Telias, Adriana; White, James R; Pahl, Donna M; Ottesen, Andrea R; Walsh, Christopher S

2011-04-21

Tomato (Solanum lycopersicum) consumption has been one of the most common causes of produce-associated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454-pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production. The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant. Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices.

In vivo investigation on connective tissue healing to polished surfaces with different surface wettability.

PubMed

Kloss, Frank R; Steinmüller-Nethl, Doris; Stigler, Robert G; Ennemoser, Thomas; Rasse, Michael; Hächl, Oliver

2011-07-01

Connective tissue in contact to transgingival/-dermal implants presents itself as tight scar formation. Although rough surfaces support the attachment they increase bacterial colonisation as well. In contrast to surface roughness, little is known about the influence of surface wettability on soft-tissue healing in vivo. We therefore investigated the influence of different surface wettabilities on connective tissue healing at polished implant surfaces in vivo. Three polished experimental groups (titanium, titanium coated with hydrophobic nano-crystalline diamond (H-NCD) and titanium coated with hydrophilic nano-crystalline diamond (O-NCD) were inserted into the subcutaneous connective tissue of the abdominal wall of 24 rats. Animals were sacrificed after 1 and 4 weeks resulting in eight specimen per group per time point. Specimen were subjected to histological evaluation (van Giesson's staining) and immunohistochemistry staining for proliferating cell nuclear antigen (PCNA), fibronectin and tumour necrosis factor-alpha (TNF-α). Histological evaluation revealed dense scar formation at the titanium and H-NCD surfaces. In contrast, the connective tissue was loose at the O-NCD surface with a significantly higher number of cells after 4 weeks. O-NCD demonstrated a strong expression of PCNA and fibronectin but a weak expression of TNF-α. In contrast, the PCNA and fibronectin expression was low at the titanium and H-NCD, with a strong signal of TNF-α at the H-NCD surface. Hydrophilicity influences the connective tissue healing at polished implant surfaces in vivo positively. The attachment of connective tissue and the number of cells in contact to the surface were increased. Moreover, the inflammatory response is decreased at the hydrophilic surface. © 2010 John Wiley & Sons A/S.

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

PubMed

Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

2016-12-01

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Locomotion of bacteria in liquid flow and the boundary layer effect on bacterial attachment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chao, E-mail: zhangchao@cqu.edu.cn; Institute of Engineering Thermophysics, Chongqing University, Chongqing 400030; Liao, Qiang, E-mail: lqzx@cqu.edu.cn

The formation of biofilm greatly affects the performance of biological reactors, which highly depends on bacterial swimming and attachment that usually takes place in liquid flow. Therefore, bacterial swimming and attachment on flat and circular surfaces with the consideration of flow was studied experimentally. Besides, a mathematical model comprehensively combining bacterial swimming and motion with flow is proposed for the simulation of bacterial locomotion and attachment in flow. Both experimental and theoretical results revealed that attached bacteria density increases with decreasing boundary layer thickness on both flat and circular surfaces, the consequence of which is inherently related to the competitionmore » between bacterial swimming and the non-slip motion with flow evaluated by the Péclet number. In the boundary layer, where the Péclet number is relatively higher, bacterial locomotion mainly depends on bacterial swimming. Thinner boundary layer promotes bacterial swimming towards the surface, leading to higher attachment density. To enhance the performance of biofilm reactors, it is effective to reduce the boundary layer thickness on desired surfaces. - Highlights: • Study of bacterial locomotion in flow as an early stage in biofilm formation. • Mathematical model combining bacterial swimming and the motion with flow. • Boundary layer plays a key role in bacterial attachment under flow condition. • The competition between bacterial swimming and the motion with flow is evaluated.« less

Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

PubMed

Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Choo, Hea-Young Park; Lee, Kyung Ho

2018-05-01

Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

Binding of a C-type lectin’s coiled-coil domain to the Domeless receptor directly activates the JAK/STAT pathway in the shrimp immune response to bacterial infection

PubMed Central

Zhao, Xiao-Fan; Vasta, Gerardo R.

2017-01-01

C-type lectins (CTLs) are characterized by the presence of a C-type carbohydrate recognition domain (CTLD) that by recognizing microbial glycans, is responsible for their roles as pattern recognition receptors in the immune response to bacterial infection. In addition to the CTLD, however, some CTLs display additional domains that can carry out effector functions, such as the collagenous domain of the mannose-binding lectin. While in vertebrates, the mechanisms involved in these effector functions have been characterized in considerable detail, in invertebrates they remain poorly understood. In this study, we identified in the kuruma shrimp (Marsupenaeus japonicus) a structurally novel CTL (MjCC-CL) that in addition to the canonical CTLD, contains a coiled-coil domain (CCD) responsible for the effector functions that are key to the shrimp’s antibacterial response mediated by antimicrobial peptides (AMPs). By the use of in vitro and in vivo experimental approaches we elucidated the mechanism by which the recognition of bacterial glycans by the CTLD of MjCC-CL leads to activation of the JAK/STAT pathway via interaction of the CCD with the surface receptor Domeless, and upregulation of AMP expression. Thus, our study of the shrimp MjCC-CL revealed a striking functional difference with vertebrates, in which the JAK/STAT pathway is indirectly activated by cell death and stress signals through cytokines or growth factors. Instead, by cross-linking microbial pathogens with the cell surface receptor Domeless, a lectin directly activates the JAK/STAT pathway, which plays a central role in the shrimp antibacterial immune responses by upregulating expression of selected AMPs. PMID:28931061

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells*

PubMed Central

Agarwal, Vaibhav; Asmat, Tauseef M.; Luo, Shanshan; Jensch, Inga; Zipfel, Peter F.; Hammerschmidt, Sven

2010-01-01

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase. PMID:20504767

High density growth of T7 expression strains with auto-induction option

DOEpatents

Studier, F. William

2010-07-20

A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

Surface topography of composite restorative materials following ultrasonic scaling and its Impact on bacterial plaque accumulation. An in-vitro SEM study.

PubMed

Hossam, A Eid; Rafi, A Togoo; Ahmed, A Saleh; Sumanth, Phani Cr

2013-06-01

This is an in vitro study to investigate the effects of ultrasonic scaling on the surface roughness and quantitative bacterial count on four different types of commonly used composite restorative materials for class V cavities. Nanofilled, hybrid, silorane and flowable composites were tested. Forty extracted teeth served as specimen and were divided into 4 groups of 10 specimens, with each group receiving a different treatment and were examined by a Field emission scanning electron microscope. Bacterial suspension was then added to the pellicle-coated specimens, and then bacterial adhesion was analyzed by using image analyzing program. Flowable and silorane-based composites showed considerably smoother surfaces and lesser bacterial count in comparison to other types, proving that bacterial adhesion is directly proportional to surface roughness. The use of ultrasonic scalers affects the surfaces of composite restorative materials. Routine periodontal scaling should be carried out very carefully, and polishing of the scaled surfaces may overcome the alterations in roughness, thus preventing secondary caries, surface staining, plaque accumulation and subsequent periodontal inflammation. How to cite this article: Eid H A, Togoo R A, Saleh A A, Sumanth C R. Surface Topography of Composite Restorative Materials following Ultrasonic Scaling and its Impact on Bacterial Plaque Accumulation. An In-Vitro SEM Study. J Int Oral Health 2013; 5(3):13-19.

Surface topography of composite restorative materials following ultrasonic scaling and its Impact on bacterial plaque accumulation. An in-vitro SEM study

PubMed Central

Hossam, A. Eid; Rafi, A. Togoo; Ahmed, A Saleh; Sumanth, Phani CR

2013-01-01

Background: This is an in vitro study to investigate the effects of ultrasonic scaling on the surface roughness and quantitative bacterial count on four different types of commonly used composite restorative materials for class V cavities. Materials & Methods: Nanofilled, hybrid, silorane and flowable composites were tested. Forty extracted teeth served as specimen and were divided into 4 groups of 10 specimens, with each group receiving a different treatment and were examined by a Field emission scanning electron microscope. Bacterial suspension was then added to the pellicle-coated specimens, and then bacterial adhesion was analyzed by using image analyzing program. Results: Flowable and silorane-based composites showed considerably smoother surfaces and lesser bacterial count in comparison to other types, proving that bacterial adhesion is directly proportional to surface roughness. Conclusion: The use of ultrasonic scalers affects the surfaces of composite restorative materials. Routine periodontal scaling should be carried out very carefully, and polishing of the scaled surfaces may overcome the alterations in roughness, thus preventing secondary caries, surface staining, plaque accumulation and subsequent periodontal inflammation. How to cite this article: Eid H A, Togoo R A, Saleh A A, Sumanth C R. Surface Topography of Composite Restorative Materials following Ultrasonic Scaling and its Impact on Bacterial Plaque Accumulation. An In-Vitro SEM Study. J Int Oral Health 2013; 5(3):13-19. PMID:24155597

Reduced toll-like receptor 4 and substance P gene expression is associated with airway bacterial colonization in children.

PubMed

Grissell, Terry V; Chang, Anne B; Gibson, Peter G

2007-04-01

Neuro-immune interactions are increasingly relevant to human health and disease. The neuropeptide Substance P also has antibacterial activity and bears similarities to the innate immune antibacterial defensins. This suggests possible co-regulation of neuropeptide and innate immune mediators. In this study, non-bronchoscopic bronchoalveolar lavage (BAL) was performed on 69 children. BAL was examined for cellular profile, microbiology (bacteria, virus) and gene expression for TLRs 2, 3, 4; chemokine receptors (CCR3, CCR5, CXCR1); neurotrophins and neurokinin genes (TAC1, TAC3, CGRP, NGF). In children with bacterial colonization (n=10) there was an airway inflammatory response with increased BAL neutrophils, IL-8 protein, and CXCR1 expression. Substance P (TAC1) and TLR4 RNA expression were reduced in children with bacterial colonization. TLR3 mRNA was increased in 7.2% (n=5) children with rhinovirus, and there was a non-significant trend to increased TLR2. There is evidence for co-regulation of neurokinin (TAC1) and TLR4 gene expression in airway cells from children with airway bacterial colonization and their reduced expression may be associated with an impaired bacterial clearance. (c) 2007 Wiley-Liss, Inc.

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Genome engineering and gene expression control for bacterial strain development.

PubMed

Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

2015-01-01

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymatic Glycosylation by Transferases

NASA Astrophysics Data System (ADS)

Blixt, Ola; Razi, Nahid

Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

PubMed

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand; Apte, Shree Kumar

2016-08-15

Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

PubMed Central

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand

2016-01-01

ABSTRACT Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. PMID:27287317

Solution structure of leptospiral LigA4 Big domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mei, Song; Zhang, Jiahai; Zhang, Xuecheng

Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Bigmore » domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca{sup 2+} binding property of LigA4 Big domain. - Highlights: • Determining the solution structure of a bacterial immunoglobulin-like domain from a surface protein of Leptospira. • The solution structure shows some structural characteristics significantly different from the classic Ig-like domains. • A potential Ca{sup 2+}-binding site was identified by strains-all and NMR chemical shift perturbation.« less

Communication among Oral Bacteria

PubMed Central

Kolenbrander, Paul E.; Andersen, Roxanna N.; Blehert, David S.; Egland, Paul G.; Foster, Jamie S.; Palmer, Robert J.

2002-01-01

Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities. PMID:12209001

Microbial air quality and bacterial surface contamination in ambulances during patient services.

PubMed

Luksamijarulkul, Pipat; Pipitsangjan, Sirikun

2015-03-01

We sought to assess microbial air quality and bacterial surface contamination on medical instruments and the surrounding areas among 30 ambulance runs during service. We performed a cross-sectional study of 106 air samples collected from 30 ambulances before patient services and 212 air samples collected during patient services to assess the bacterial and fungal counts at the two time points. Additionally, 226 surface swab samples were collected from medical instrument surfaces and the surrounding areas before and after ambulance runs. Groups or genus of isolated bacteria and fungi were preliminarily identified by Gram's stain and lactophenol cotton blue. Data were analyzed using descriptive statistics, t-test, and Pearson's correlation coefficient with a p-value of less than 0.050 considered significant. The mean and standard deviation of bacterial and fungal counts at the start of ambulance runs were 318±485cfu/m(3) and 522±581cfu/m(3), respectively. Bacterial counts during patient services were 468±607cfu/m(3) and fungal counts were 656±612cfu/m(3). Mean bacterial and fungal counts during patient services were significantly higher than those at the start of ambulance runs, p=0.005 and p=0.030, respectively. For surface contamination, the overall bacterial counts before and after patient services were 0.8±0.7cfu/cm(2) and 1.3±1.1cfu/cm(2), respectively (p<0.001). The predominant isolated bacteria and fungi were Staphylococcus spp. and Aspergillus spp., respectively. Additionally, there was a significantly positive correlation between bacterial (r=0.3, p<0.010) and fungal counts (r=0.2, p=0.020) in air samples and bacterial counts on medical instruments and allocated areas. This study revealed high microbial contamination (bacterial and fungal) in ambulance air during services and higher bacterial contamination on medical instrument surfaces and allocated areas after ambulance services compared to the start of ambulance runs. Additionally, bacterial and fungal counts in ambulance air showed a significantly positive correlation with the bacterial surface contamination on medical instruments and allocated areas. Further studies should be conducted to determine the optimal intervention to reduce microbial contamination in the ambulance environment.

Bacterial attachment to RO membranes surface-modified by concentration-polarization-enhanced graft polymerization.

PubMed

Bernstein, Roy; Belfer, Sofia; Freger, Viatcheslav

2011-07-15

Concentration polarization-enhanced radical graft polymerization, a facile surface modification technique, was examined as an approach to reduce bacterial deposition onto RO membranes and thus contribute to mitigation of biofouling. For this purpose an RO membrane ESPA-1 was surface-grafted with a zwitterionic and negatively and positively charged monomers. The low monomer concentrations and low degrees of grafting employed in modifications moderately reduced flux (by 20-40%) and did not affect salt rejection, yet produced substantial changes in surface chemistry, charge and hydrophilicity. The propensity to bacterial attachment of original and modified membranes was assessed using bacterial deposition tests carried out in a parallel plate flow setup using a fluorescent strain of Pseudomonas fluorescens. Compared to unmodified ESPA-1 the deposition (mass transfer) coefficient was significantly increased for modification with the positively charged monomer. On the other hand, a substantial reduction in bacterial deposition rates was observed for membranes modified with zwitterionic monomer and, still more, with very hydrophilic negatively charged monomers. This trend is well explained by the effects of surface charge (as measured by ζ-potential) and hydrophilicity (contact angle). It also well correlated with force distance measurements by AFM using surrogate spherical probes with a negative surface charge mimicking the bacterial surface. The positively charged surface showed a strong hysteresis with a large adhesion force, which was weaker for unmodified ESPA-1 and still weaker for zwitterionic surface, while negatively charged surface showed a long-range repulsion and negligible hysteresis. These results demonstrate the potential of using the proposed surface- modification approach for varying surface characteristics, charge and hydrophilicity, and thus minimizing bacterial deposition and potentially reducing propensity biofouling.

Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.

PubMed

Tu, Zhengkun; Pierce, Robert H; Kurtis, Jonathan; Kuroki, Yoshio; Crispe, I Nicholas; Orloff, Mark S

2010-01-01

Kupffer cells (KC) are important innate immune cells of the liver, functioning as scavenging sinusoidal phagocytes and transducers of pattern recognition signals, including those of toll-like receptors (TLRs). The hepatitis C virus core protein (HCVc) engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. We examined the effects of HCVc on human primary KC functions. KC were isolated from living donor allografts and stimulated with HCVc and/or ligands for TLRs. KC were examined for production of cytokines, expression of programmed death-ligand 1 (PD-L1), secretion of type 1 interferons (IFNs), and expression of the apoptosis-inducing protein tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). HCVc acts as a ligand for TLR2 on human KC, inducing them to secrete interleukin (IL)-1beta, TNF-alpha, and IL-10 and up-regulate cell surface PD-L1. HCVc blocked TLR3-mediated secretion of IFN-alpha, IFN-beta, and cell surface expression of the cytotoxic molecule TRAIL. Inhibition of phosphoinositide 3 kinase with LY294002 blocked the up-regulation of PD-L1 by TLR ligands and the TLR3-specific induction of TRAIL and type 1 IFNs. KC are intravascular macrophages that are continuously exposed to, and tolerant of, bacterial TLR ligands, which are delivered via the portal circulation. By mimicking a bacterial TLR2 ligand and effectively blocking the TLR3-mediated, double-stranded RNA-induced antiviral response, HCVc might appear to exploit this unique aspect of immunity in the liver. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Organization and PprB-dependent control of the Pseudomonas aeruginosa tad Locus, involved in Flp pilus biology.

PubMed

Bernard, Christophe S; Bordi, Christophe; Termine, Elise; Filloux, Alain; de Bentzmann, Sophie

2009-03-01

Bacterial attachment to the substratum involves several cell surface organelles, including various types of pili. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are required for adhesion to abiotic surfaces and to eukaryotic cells. Type IVb pili consist of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase. In this study, we investigated the regulatory mechanism of the tad locus. We showed that the flp gene is expressed late in the stationary growth phase in aerobic conditions. We also showed that the tad locus was composed of five independent transcriptional units. We used transcriptional fusions to show that tad gene expression was positively controlled by the PprB response regulator. We subsequently showed that PprB bound to the promoter regions, directly controlling the expression of these genes. We then evaluated the contribution of two genes, tadF and rcpC, to type IVb pilus assembly. The deletion of these two genes had no effect on Flp production, pilus assembly, or Flp-mediated adhesion to abiotic surfaces in our conditions. However, our results suggest that the putative RcpC protein modifies the Flp pilin, thereby promoting Flp-dependent adhesion to eukaryotic cells.

Surface-structured bacterial cellulose with guided assembly-based biolithography (GAB).

PubMed

Bottan, Simone; Robotti, Francesco; Jayathissa, Prageeth; Hegglin, Alicia; Bahamonde, Nicolas; Heredia-Guerrero, José A; Bayer, Ilker S; Scarpellini, Alice; Merker, Hannes; Lindenblatt, Nicole; Poulikakos, Dimos; Ferrari, Aldo

2015-01-27

A powerful replica molding methodology to transfer on-demand functional topographies to the surface of bacterial cellulose nanofiber textures is presented. With this method, termed guided assembly-based biolithography (GAB), a surface-structured polydimethylsiloxane (PDMS) mold is introduced at the gas-liquid interface of an Acetobacter xylinum culture. Upon bacterial fermentation, the generated bacterial cellulose nanofibers are assembled in a three-dimensional network reproducing the geometric shape imposed by the mold. Additionally, GAB yields directional alignment of individual nanofibers and memory of the transferred geometrical features upon dehydration and rehydration of the substrates. Scanning electron and atomic force microscopy are used to establish the good fidelity of this facile and affordable method. Interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes illustrates the efficient control of cellular activities which are fundamental in skin wound healing and tissue regeneration. The deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant further proves the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of surface structure on skin regeneration.

Factor H-IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes.

PubMed

Blom, Anna M; Magda, Michal; Kohl, Lisa; Shaughnessy, Jutamas; Lambris, John D; Ram, Sanjay; Ermert, David

2017-12-01

Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efficiency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) that bind to S. pyogenes , linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement-dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus influenzae and Neisseria meningitidis We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes- induced sepsis in a transgenic mouse model expressing human FH ( S. pyogenes binds FH in a human-specific manner). Furthermore, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections. Copyright © 2017 by The American Association of Immunologists, Inc.

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

PubMed

Tan, Honglue; Peng, Zhaoxiang; Li, Qingtian; Xu, Xiaofen; Guo, Shengrong; Tang, Tingting

2012-01-01

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane.

PubMed

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

PubMed Central

Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

2017-01-01

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors.

PubMed

Kalograiaki, Ioanna; Campanero-Rhodes, María A; Proverbio, Davide; Euba, Begoña; Garmendia, Junkal; Aastrup, Teodor; Solís, Dolores

2018-01-01

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment. © 2018 Elsevier Inc. All rights reserved.

Bacterial biogeography influenced by shelf-basin exchange in the Arctic surface sediment at the Chukchi Borderland.

PubMed

Han, Dukki; Nam, Seung-Il; Ha, Ho Kyung; Kim, Hyoungjun; Sadowsky, Michael J; Lee, Yoo Kyung; Hur, Hor-Gil

2016-02-01

It has been known that continental shelves around the Arctic Ocean play a major role in the ventilation of the deep basins as a consequence of shelf-basin exchange. In the present study, we found that bacterial assemblage of the surface sediment was different from that of seawater while seawater harboured local bacterial assemblages in response to the Arctic hydrography. This finding suggests that the Arctic seafloor sediments may have distinctive bacterial biogeography. Moreover, the distribution of bacterial assemblages and physicochemical properties in surface sediments changed gradually from the Arctic continental shelf to deep-sea basin. Based on the results, bacterial biogeography in the Arctic seafloor sediments may be influenced by winnowing and re-deposition of surface sediments through the sediment gravity flow. The present study offers a deeper understanding of shelf convection and its role for the construction of bacterial assemblages in the Arctic Ocean. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

PubMed Central

Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

2014-01-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

PubMed

Strachan, G; Grant, S D; Learmonth, D; Longstaff, M; Porter, A J; Harris, W J

1998-09-15

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

Intestinal alkaline phosphatase: multiple biological roles in maintenance of intestinal homeostasis and modulation by diet.

PubMed

Lallès, Jean-Paul

2010-06-01

The diverse nature of intestinal alkaline phosphatase (IAP) functions has remained elusive, and it is only recently that four additional major functions of IAP have been revealed. The present review analyzes the earlier literature on the dietary factors modulating IAP activity in light of these new findings. IAP regulates lipid absorption across the apical membrane of enterocytes, participates in the regulation of bicarbonate secretion and of duodenal surface pH, limits bacterial transepithelial passage, and finally controls bacterial endotoxin-induced inflammation by dephosphorylation, thus detoxifying intestinal lipopolysaccharide. Many dietary components, including fat, protein, and carbohydrate, modulate IAP expression or activity and may be combined to sustain a high level of IAP activity. In conclusion, IAP has a pivotal role in intestinal homeostasis and its activity could be increased through the diet. This is especially true in pathological situations (e.g., inflammatory bowel diseases) in which the involvement of commensal bacteria is suspected and when intestinal AP is too low to detoxify a sufficient amount of bacterial lipopolysaccharide.

NrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence

PubMed Central

Naveen, Vankadari; Hsiao, Chwan-Deng

2016-01-01

Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion. PMID:27275780

Modulation of cell surface hydrophobicity and attachment of bacteria to abiotic surfaces and shrimp by Malaysian herb extracts.

PubMed

Hui, Yew Woh; Dykes, Gary A

2012-08-01

The use of simple crude water extracts of common herbs to reduce bacterial attachment may be a cost-effective way to control bacterial foodborne pathogens, particularly in developing countries. The ability of water extracts of three common Malaysian herbs (Andrographis paniculata, Eurycoma longifolia, and Garcinia atroviridis) to modulate hydrophobicity and attachment to surfaces of five food-related bacterial strains (Bacillus cereus ATCC 14576, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, Salmonella Enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923) were determined. The bacterial attachment to hydrocarbon assay was used to determine bacterial hydrophobicity. Staining and direct microscopic counts were used to determine attachment of bacteria to glass and stainless steel. Plating on selective media was used to determine attachment of bacteria to shrimp. All extracts were capable of either significantly ( P < 0.05) increasing or decreasing bacterial surface hydrophobicity, depending on the herb extract and bacteria combination. Bacterial attachment to all surfaces was either significantly (P < 0.05) increased or decreased, depending on the herb extract and bacteria combination. Overall, hydrophobicity did not show a significant correlation (P > 0.05) to bacterial attachment. For specific combinations of bacteria, surface material, and plant extract, significant correlations (R > 0.80) between hydrophobicity and attachment were observed. The highest of these was observed for S. aureus attachment to stainless steel and glass after treatment with the E. longifolia extract (R = 0.99, P < 0.01). The crude water herb extracts in this study were shown to have the potential to modulate specific bacterial and surface interactions and may, with further work, be useful for the simple and practical control of foodborne pathogens.

Direct observation of bacterial deposition onto clean and organic-fouled polyamide membranes.

PubMed

Subramani, Arun; Huang, Xiaofei; Hoek, Eric M V

2009-08-01

Nanofiltration (NF) and reverse osmosis (RO) membranes are commonly applied to produce highly purified water from municipal wastewater effluents. In these applications, biofouling limits overall process performance and increases the cost of operation. Initial bacteria adhesion onto a membrane surface is a critical early step in the overall process of membrane biofouling. However, adsorption of effluent organic matter onto the membrane may precede bacterial deposition and change membrane surface properties. Herein we employed direct microscopic observation to elucidate mechanisms governing bacterial cell deposition onto clean and organic-fouled NF and RO membranes. Bovine serum albumin (BSA) and alginic acid (AA) were used as models for protein and polysaccharide rich organic matter in secondary wastewater effluents. In all experiments, organic fouling increased membrane hydraulic resistance and salt rejection, in addition to interfacial hydrophilicity and roughness. Even though surface hydrophilicity increased, the rougher surfaces presented by organic-fouled membranes produced nano-scale features that promoted localized bacterial deposition. An extended DLVO analysis of bacterial cells and membrane surface properties suggested that bacterial deposition correlated most strongly with the Lewis acid-base free energy of adhesion and root mean square (RMS) roughness, whereas van der Waals and electrostatic free energies were weakly correlated. This was true for both clean and organic-fouled membranes. Bacterial deposition rates were clearly influenced by an antagonistic interplay between macroscopic surface hydrophilicity and nano-scale surface roughness.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

PubMed Central

Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments.

PubMed

Laverock, Bonnie; Smith, Cindy J; Tait, Karen; Osborn, A Mark; Widdicombe, Steve; Gilbert, Jack A

2010-12-01

Bioturbation is a key process in coastal sediments, influencing microbially driven cycling of nutrients as well as the physical characteristics of the sediment. However, little is known about the distribution, diversity and function of the microbial communities that inhabit the burrows of infaunal macroorganisms. In this study, terminal-restriction fragment length polymorphism analysis was used to investigate variation in the structure of bacterial communities in sediment bioturbated by the burrowing shrimp Upogebia deltaura or Callianassa subterranea. Analyses of 229 sediment samples revealed significant differences between bacterial communities inhabiting shrimp burrows and those inhabiting ambient surface and subsurface sediments. Bacterial communities in burrows from both shrimp species were more similar to those in surface-ambient than subsurface-ambient sediment (R=0.258, P<0.001). The presence of shrimp was also associated with changes in bacterial community structure in surrounding surface sediment, when compared with sediments uninhabited by shrimp. Bacterial community structure varied with burrow depth, and also between individual burrows, suggesting that the shrimp's burrow construction, irrigation and maintenance behaviour affect the distribution of bacteria within shrimp burrows. Subsequent sequence analysis of bacterial 16S rRNA genes from surface sediments revealed differences in the relative abundance of bacterial taxa between shrimp-inhabited and uninhabited sediments; shrimp-inhabited sediment contained a higher proportion of proteobacterial sequences, including in particular a twofold increase in Gammaproteobacteria. Chao1 and ACE diversity estimates showed that taxon richness within surface bacterial communities in shrimp-inhabited sediment was at least threefold higher than that in uninhabited sediment. This study shows that bioturbation can result in significant structural and compositional changes in sediment bacterial communities, increasing bacterial diversity in surface sediments and resulting in distinct bacterial communities even at depth within the burrow. In an area of high macrofaunal abundance, this could lead to alterations in the microbial transformations of important nutrients at the sediment-water interface.

Suppressive Potential of Paenibacillus Strains Isolated from the Tomato Phyllosphere against Fusarium Crown and Root Rot of Tomato

PubMed Central

Sato, Ikuo; Yoshida, Shigenobu; Iwamoto, Yutaka; Aino, Masataka; Hyakumachi, Mitsuro; Shimizu, Masafumi; Takahashi, Hideki; Ando, Sugihiro; Tsushima, Seiya

2014-01-01

The suppressive potentials of Bacillus and Paenibacillus strains isolated from the tomato phyllosphere were investigated to obtain new biocontrol candidates against Fusarium crown and root rot of tomato. The suppressive activities of 20 bacterial strains belonging to these genera were examined using seedlings and potted tomato plants, and two Paenibacillus strains (12HD2 and 42NP7) were selected as biocontrol candidates against the disease. These two strains suppressed the disease in the field experiment. Scanning electron microscopy revealed that the treated bacterial cells colonized the root surface, and when the roots of the seedlings were treated with strain 42NP7 cells, the cell population was maintained on the roots for at least for 4 weeks. Although the bacterial strains had no direct antifungal activity against the causal pathogen in vitro, an increase was observed in the antifungal activities of acetone extracts from tomato roots treated with the cells of both bacterial strains. Furthermore, RT-PCR analysis verified that the expression of defense-related genes was induced in both the roots and leaves of seedlings treated with the bacterial cells. Thus, the root-colonized cells of the two Paenibacillus strains were considered to induce resistance in tomato plants, which resulted in the suppression of the disease. PMID:24920171

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

PubMed

Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

2016-01-01

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Surface zwitterionization: Effective method for preventing oral bacterial biofilm formation on hydroxyapatite surfaces

NASA Astrophysics Data System (ADS)

Lee, Myoungjin; Kim, Heejin; Seo, Jiae; Kang, Minji; Kang, Sunah; Jang, Joomyung; Lee, Yan; Seo, Ji-Hun

2018-01-01

In this study, we conducted surface zwitterionization of hydroxyapatite (HA) surfaces by immersing them in the zwitterionic polymer solutions to provide anti-bacterial properties to the HA surface. Three different monomers containing various zwitterionic groups, i.e., phosphorylcholine (PC), sulfobetaine (SB), and carboxybetaine (CB), were copolymerized with the methacrylic monomer containing a Ca2+-binding moiety, using the free radical polymerization method. As a control, functionalization of the copolymer containing the Ca2+-binding moiety was synthesized using a hydroxy group. The stable immobilization of the zwitterionic functional groups was confirmed by water contact angle analysis and X-ray photoelectron spectroscopy (XPS) measurement conducted after the sonication process. The zwitterionized HA surface showed significantly decreased protein adsorption, whereas the hydroxyl group-coated HA surface showed limited efficacy. The anti-bacterial adhesion property was confirmed by conducting Streptococcus mutans (S. mutans) adhesion tests for 6 h and 24 h. When furanone C-30, a representative anti-quorum sensing molecule for S. mutans, was used, only a small amount of bacteria adhered after 6 h and the population did not increase after 24 h. In contrast, zwitterionized HA surfaces showed almost no bacterial adhesion after 6 h and the effect was retained for 24 h, resulting in the lowest level of oral bacterial adhesion. These results confirm that surface zwitterionization is a promising method to effectively prevent oral bacterial adhesion on HA-based materials.

MULTIPLE IMAGING TECHNIQUES DEMONSTRATE THE MANIPULATION OF SURFACES TO REDUCE BACTERIAL CONTAMINATION

EPA Science Inventory

Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. Stainless steel surfaces were engineered to reduce bacterial contamination, biofilm formation, and corrosion during product processing...

Effect of starch and amylase on the expression of amylase-binding protein A in Streptococcus gordonii

PubMed Central

Nikitkova, A.E.; Haase, E.M.; Scannapieco, F.A.

2012-01-01

SUMMARY Streptococcus gordonii is a common oral commensal bacterial species in tooth biofilm (dental plaque) and specifically binds to salivary amylase through the surface exposed amylase-binding protein A (AbpA). When S. gordonii cells are pretreated with amylase, amylase bound to AbpA facilitates growth with starch as a primary nutrition source. The goal of this study was to explore possible regulatory effects of starch, starch metabolites and amylase on the expression of S. gordonii AbpA. An amylase ligand-binding assay was used to assess the expression of AbpA in culture supernatants and on bacterial cells from S. gordonii grown in defined medium supplemented with 1% starch, 0.5 mg ml−1 amylase, with starch and amylase together, or with various linear malto-oligosaccharides. Transcription of abpA was determined by reverse transcription quantitative polymerase chain reaction. AbpA was not detectable in culture supernatants containing either starch alone or amylase alone. In contrast, the amount of AbpA was notably increased when starch and amylase were both present in the medium. The expression of abpA was significantly increased (P < 0.05) following 40 min of incubation in defined medium supplemented with starch and amylase. Similar results were obtained in the presence of maltose and other short-chain malto-oligosacchrides. These results suggest that the products of starch hydrolysis produced from the action of salivary α-amylase, particularly maltose and maltotriose, regulate AbpA expression in S. gordonii. PMID:22759313

The physicochemical process of bacterial attachment to abiotic surfaces: Challenges for mechanistic studies, predictability and the development of control strategies.

PubMed

Wang, Yi; Lee, Sui Mae; Dykes, Gary

2015-01-01

Bacterial attachment to abiotic surfaces can be explained as a physicochemical process. Mechanisms of the process have been widely studied but are not yet well understood due to their complexity. Physicochemical processes can be influenced by various interactions and factors in attachment systems, including, but not limited to, hydrophobic interactions, electrostatic interactions and substratum surface roughness. Mechanistic models and control strategies for bacterial attachment to abiotic surfaces have been established based on the current understanding of the attachment process and the interactions involved. Due to a lack of process control and standardization in the methodologies used to study the mechanisms of bacterial attachment, however, various challenges are apparent in the development of models and control strategies. In this review, the physicochemical mechanisms, interactions and factors affecting the process of bacterial attachment to abiotic surfaces are described. Mechanistic models established based on these parameters are discussed in terms of their limitations. Currently employed methods to study these parameters and bacterial attachment are critically compared. The roles of these parameters in the development of control strategies for bacterial attachment are reviewed, and the challenges that arise in developing mechanistic models and control strategies are assessed.

Multi-heme cytochromes provide a pathway for survival in energy-limited environments

PubMed Central

Deng, Xiao; Dohmae, Naoshi; Nealson, Kenneth H.; Hashimoto, Kazuhito; Okamoto, Akihiro

2018-01-01

Bacterial reduction of oxidized sulfur species (OSS) is critical for energy production in anaerobic marine subsurfaces. In organic-poor sediments, H2 has been considered as a major energy source for bacterial respiration. We identified outer-membrane cytochromes (OMCs) that are broadly conserved in sediment OSS-respiring bacteria and enable cells to directly use electrons from insoluble minerals via extracellular electron transport. Biochemical, transcriptomic, and microscopic analyses revealed that the identified OMCs were highly expressed on the surface of cells and nanofilaments in response to electron donor limitation. This electron uptake mechanism provides sufficient but minimum energy to drive the reduction of sulfate and other OSS. These results suggest a widespread mechanism for survival of OSS-respiring bacteria via electron uptake from solid minerals in energy-poor marine sediments. PMID:29464208

Cytotoxicity of TiO{sub 2} nanoparticles towards freshwater sediment microorganisms at low exposure concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumari, Jyoti; Kumar, Deepak; Mathur, Ankita

2014-11-15

There is a persistent need to assess the effects of TiO{sub 2} nanoparticles on the aquatic ecosystem owing to their increasing usage in consumer products and risk of environmental release. The current study is focused on TiO{sub 2} nanoparticle-induced acute toxicity at sub-ppm level (≤1 ppm) on the three different freshwater sediment bacterial isolates and their consortium under two different irradiation (visible light and dark) conditions. The consortium of the bacterial isolates was found to be less affected by the exposure to the nanoparticles compared to the individual cells. The oxidative stress contributed considerably towards the cytotoxicity under both lightmore » and dark conditions. A statistically significant increase in membrane permeability was noted under the dark conditions as compared to the light conditions. The optical and fluorescence microscopic images showed aggregation and chain formation of the bacterial cells, when exposed to the nanoparticles. The electron microscopic (SEM, TEM) observations suggested considerable damage of cells and bio-uptake of nanoparticles. The exopolysaccrides (EPS) production and biofilm formation were noted to increase in the presence of the nanoparticles, and expression of the key genes involved in biofilm formation was studied by RT-PCR. - Highlights: • Toxicity of NPs towards freshwater sediment bacteria at sub-ppm concentrations. • Decreased toxicity of the nanoparticles in the consortium of microorganisms. • Enhanced bacterial resistance through EPS and biofilm formation in the presence of NPs. • Considerable surface damage of cells and internalization of NPs. • Gene expression analyses related to biofilm formation in the presence of NPs.« less

S-Layer Protein Mediates the Stimulatory Effect of Lactobacillus helveticus MIMLh5 on Innate Immunity

PubMed Central

Taverniti, Valentina; Stuknyte, Milda; Minuzzo, Mario; Arioli, Stefania; De Noni, Ivano; Scabiosi, Christian; Cordova, Zuzet Martinez; Junttila, Ilkka; Hämäläinen, Sanna; Turpeinen, Hannu; Mora, Diego; Karp, Matti; Pesu, Marko

2013-01-01

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention. PMID:23220964

Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

PubMed

Wu, Muzo; Gibbons, John G; DeLoid, Glen M; Bedugnis, Alice S; Thimmulappa, Rajesh K; Biswal, Shyam; Kobzik, Lester

2017-07-01

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia. Copyright © 2017 the American Physiological Society.

Bacterial community diversity and variation in spray water sources and the tomato fruit surface

PubMed Central

2011-01-01

Background Tomato (Solanum lycopersicum) consumption has been one of the most common causes of produce-associated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454-pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production. Results The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant. Conclusions Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices. PMID:21510867

Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

PubMed Central

Greis, Kenneth D.; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-01-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP. PMID:16048957

Titanium Surface Chemical Composition Interferes in the Pseudomonas aeruginosa Biofilm Formation.

PubMed

Nunes Filho, Antonio; Aires, Michelle de Medeiros; Braz, Danilo Cavalcante; Hinrichs, Ruth; Macedo, Alexandre José; Alves, Clodomiro

2018-02-01

Bacterial adhesion on three different surfaces: untreated Ti, plasma nitriding, and plasma carbonitriding Ti substrates were investigated. The samples were placed in bacterial cultures of Pseudomonas aeruginosa to assess biofilm formation. The correlation between the amount of bacteria attached to the surface after a lapse of time with nanotopography and physicochemical properties was performed. TiN showed the highest capacity to avoid bacterial adhesion, while presenting intermediate roughness and wettability. Although the surface of TiCN had the highest surface roughness and low contact angle (high wettability), bacterial adhesion was intermediate on this sample. Untreated Ti, even though presenting a smooth surface and low wettability, had the highest tendency to form biofilms. © 2018 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane

PubMed Central

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

ABSTRACT Introduction: Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Methods: Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Results: Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Conclusion: Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film. PMID:28902253

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

PubMed

Úbeda, María; Lario, Margaret; Muñoz, Leticia; Borrero, María-José; Rodríguez-Serrano, Macarena; Sánchez-Díaz, Ana-María; Del Campo, Rosa; Lledó, Lourdes; Pastor, Óscar; García-Bermejo, Laura; Díaz, David; Álvarez-Mon, Melchor; Albillos, Agustín

2016-05-01

In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Changes in rhizosphere bacterial gene expression following glyphosate treatment.

PubMed

Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

2016-05-15

In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of farmhouse hotel and paper mill effluents on bacterial community structures in sediment and surface water of Nanxi River, China.

PubMed

Lu, Xiao-Ming; Lu, Peng-Zhen

2014-11-01

The pyrosequencing technique was used to evaluate bacterial community structures in sediment and surface water samples taken from Nanxi River receiving effluents from a paper mill and a farmhouse hotel, respectively. For each sample, 4,610 effective bacterial sequences were selected and used to do the analysis of diversity and abundance, respectively. Bacterial phylotype richness in the sediment sample without effluent input was higher than the other samples, and the surface water sample with addition of effluent from the paper mill contained the least richness. Effluents from both the paper mill and farmhouse hotel have a potential to reduce the bacterial diversity and abundance in the sediment and surface water, especially it is more significant in the sediment. The effect of the paper mill effluent on the sediment and surface water bacterial communities was more serious than that of the farmhouse hotel effluent. Characterization of microbial community structures in the sediment and surface water from two tributaries of the downstream river indicated that various effluents from the paper mill and farmhouse hotel have the similar potential to decrease the natural variability in riverine microbial ecosystems.

An investigation of the effect of scaling-induced surface roughness on bacterial adhesion in common fixed dental restorative materials.

PubMed

Checketts, Matthew R; Turkyilmaz, Ilser; Asar, Neset Volkan

2014-11-01

Bacterial plaque must be routinely removed from teeth, adjacent structures, and prostheses. However, the removal of this plaque can inadvertently increase the risk of future bacterial adhesion. The purpose of this investigation was to assess the change in the surface roughness of 3 different surfaces after dental prophylactic instrumentation and how this influenced bacterial adhesion. Forty specimens each of Type III gold alloy, lithium disilicate, and zirconia were fabricated in the same dimensions. The specimens were divided into 4 groups: ultrasonic scaler, stainless steel curette, prophylaxis cup, and control. Pretreatment surface roughness measurements were made with a profilometer. Surface treatments in each group were performed with a custom mechanical scaler. Posttreatment surface roughness values were measured. In turn, the specimens were inoculated with Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces viscosus. Bacterial adhesion was assessed by rinsing the specimens with sterile saline to remove unattached cells. The specimens were then placed in sterile tubes with 1 mL of sterile saline. The solution was plated and quantified. Scanning electron microscopy was performed. The statistical analysis of surface roughness was completed by using repeated-measures single-factor ANOVA with a Bonferroni correction. The surface roughness values for gold alloy specimens increased as a result of prophylaxis cup treatment (0.221 to 0.346 Ra) (P<.01) and stainless steel curette treatment (0.264 to 1.835 Ra) (P<.01). The results for bacterial adhesion to gold alloy proved inconclusive. A quantitative comparison indicated no statistically significant differences in pretreatment and posttreatment surface roughness values for lithium disilicate and zirconia specimens. In spite of these similarities, the overall bacterial adherence values for lithium disilicate were significantly greater than those recorded for gold alloy or zirconia (P<.05). Instrumentation of the lithium disilicate and zirconia with the stainless steel curette significantly increased bacterial adhesion compared with the control (P<.05). The results of this investigation indicate that Type III gold alloy exhibited increased surface roughness values after stainless steel curette and prophylaxis cup treatments. Zirconia was less susceptible to bacterial adhesion than lithium disilicate, and greater bacterial adhesion was found for the stainless steel curette than the other instrumentation methods. Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Microbial Air Quality and Bacterial Surface Contamination in Ambulances During Patient Services

PubMed Central

Luksamijarulkul, Pipat; Pipitsangjan, Sirikun

2015-01-01

Objectives We sought to assess microbial air quality and bacterial surface contamination on medical instruments and the surrounding areas among 30 ambulance runs during service. Methods We performed a cross-sectional study of 106 air samples collected from 30 ambulances before patient services and 212 air samples collected during patient services to assess the bacterial and fungal counts at the two time points. Additionally, 226 surface swab samples were collected from medical instrument surfaces and the surrounding areas before and after ambulance runs. Groups or genus of isolated bacteria and fungi were preliminarily identified by Gram’s stain and lactophenol cotton blue. Data were analyzed using descriptive statistics, t-test, and Pearson’s correlation coefficient with a p-value of less than 0.050 considered significant. Results The mean and standard deviation of bacterial and fungal counts at the start of ambulance runs were 318±485cfu/m3 and 522±581cfu/m3, respectively. Bacterial counts during patient services were 468±607cfu/m3 and fungal counts were 656±612cfu/m3. Mean bacterial and fungal counts during patient services were significantly higher than those at the start of ambulance runs, p=0.005 and p=0.030, respectively. For surface contamination, the overall bacterial counts before and after patient services were 0.8±0.7cfu/cm2 and 1.3±1.1cfu/cm2, respectively (p<0.001). The predominant isolated bacteria and fungi were Staphylococcus spp. and Aspergillus spp., respectively. Additionally, there was a significantly positive correlation between bacterial (r=0.3, p<0.010) and fungal counts (r=0.2, p=0.020) in air samples and bacterial counts on medical instruments and allocated areas. Conclusions This study revealed high microbial contamination (bacterial and fungal) in ambulance air during services and higher bacterial contamination on medical instrument surfaces and allocated areas after ambulance services compared to the start of ambulance runs. Additionally, bacterial and fungal counts in ambulance air showed a significantly positive correlation with the bacterial surface contamination on medical instruments and allocated areas. Further studies should be conducted to determine the optimal intervention to reduce microbial contamination in the ambulance environment. PMID:25960835

Diversity of Bacterial Communities on Four Frequently Used Surfaces in a Large Brazilian Teaching Hospital

PubMed Central

Pereira da Fonseca, Tairacan Augusto; Pessôa, Rodrigo; Felix, Alvina Clara; Sanabani, Sabri Saeed

2016-01-01

Frequently used hand-touch surfaces in hospital settings have been implicated as a vehicle of microbial transmission. In this study, we aimed to investigate the overall bacterial population on four frequently used surfaces using a culture-independent Illumina massively parallel sequencing approach of the 16S rRNA genes. Surface samples were collected from four sites, namely elevator buttons (EB), bank machine keyboard buttons (BMKB), restroom surfaces, and the employee biometric time clock system (EBTCS), in a large public and teaching hospital in São Paulo. Taxonomical composition revealed the abundance of Firmicutes phyla, followed by Actinobacteria and Proteobacteria, with a total of 926 bacterial families and 2832 bacterial genera. Moreover, our analysis revealed the presence of some potential pathogenic bacterial genera, including Salmonella enterica, Klebsiella pneumoniae, and Staphylococcus aureus. The presence of these pathogens in frequently used surfaces enhances the risk of exposure to any susceptible individuals. Some of the factors that may contribute to the richness of bacterial diversity on these surfaces are poor personal hygiene and ineffective routine schedules of cleaning, sanitizing, and disinfecting. Strict standards of infection control in hospitals and increased public education about hand hygiene are recommended to decrease the risk of transmission in hospitals among patients. PMID:26805866

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

PubMed

Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

2014-03-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The Pathogenesis of Staphylococcus aureus Eye Infections

PubMed Central

2018-01-01

Staphylococcus aureus is a major pathogen of the eye able to infect the tear duct, eyelid, conjunctiva, cornea, anterior and posterior chambers, and the vitreous chamber. Of these infections, those involving the cornea (keratitis) or the inner chambers of the eye (endophthalmitis) are the most threatening because of their potential to cause a loss in visual acuity or even blindness. Each of these ocular sites is protected by the constitutive expression of a variety of antimicrobial factors and these defenses are augmented by a protective host response to the organism. Such infections often involve a predisposing factor that weakens the defenses, such as the use of contact lenses prior to the development of bacterial keratitis or, for endophthalmitis, the trauma caused by cataract surgery or intravitreal injection. The structural carbohydrates of the bacterial surface induce an inflammatory response able to reduce the bacterial load, but contribute to the tissue damage. A variety of bacterial secreted proteins including alpha-toxin, beta-toxin, gamma-toxin, Panton-Valentine leukocidin and other two-component leukocidins mediate tissue damage and contribute to the induction of the inflammatory response. Quantitative animal models of keratitis and endophthalmitis have provided insights into the S. aureus virulence and host factors active in limiting such infections. PMID:29320451

Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

PubMed

Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

2013-09-03

Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

PubMed

Inouye, Satoshi; Suzuki, Takahiro

2016-12-01

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Biofilms associated with poultry processing equipment.

PubMed

Lindsay, D; Geornaras, I; von Holy, A

1996-01-01

Aerobic and Gram-negative bacteria were enumerated on non-metallic surfaces and stainless steel test pieces attached to equipment surfaces by swabbing and a mechanical dislodging procedure, respectively, in a South African grade B poultry processing plant. Changes in bacterial numbers were also monitored over time on metal test pieces. The highest bacterial counts were obtained from non-metallic surfaces such as rubber fingered pluckers and plastic defeathering curtains which exceeded the highest counts found on the metal surfaces by at least 1 log CFU cm-2. Gram-negative bacterial counts on all non-metallic surface types were at least 2 log CFU cm-2 lower than corresponding aerobic plate counts. On metal surfaces, the highest microbial numbers were obtained after 14 days exposure, with aerobic plate counts ranging from 3.57 log CFU cm-2 to 5.13 log CFU cm-2, and Gram-negative counts from 0.70 log CFU cm-2 to 3.31 log CFU cm-2. Scanning electron microscopy confirmed the presence of bacterial cells on non-metallic and metallic surfaces associated with poultry processing. Rubber 'fingers', plastic curtains, conveyor belt material and stainless steel test surfaces placed on the scald tank overflow and several chutes revealed extensive and often confluent bacterial biofilms. Extracellular polymeric substances, but few bacterial cells were visible on test pieces placed on evisceration equipment, spinchiller blades and the spinchiller outlet.

Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

PubMed

Lubin, Jean-Bernard; Lewis, Warren G; Gilbert, Nicole M; Weimer, Cory M; Almagro-Moreno, Salvador; Boyd, E Fidelma; Lewis, Amanda L

2015-08-01

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood. Vibrio vulnificus is the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle of V. vulnificus but that their emergence also resulted in a gain of function with life-threatening potential in the human host. Copyright © 2015, Lubin et al.

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

PubMed

Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L

2011-01-01

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L).

PubMed

Meng, Lingjie; Du, Yishuai; Liu, Pengfei; Li, Xian; Liu, Ying

2017-05-01

Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co-operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer-2 (AI-2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold-water teleost, little information has been known about the function of AI-2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI-2 activity and luxS defective mutant strain fail to produce AI-2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with ΔluxS strain. Overall, we hypothesis that AI-2 quorum sensing could regulate the expression of A-layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida. Copyright © 2017 Elsevier Ltd. All rights reserved.

Soft Lithography and Minimally Human Invasive Technique for Rapid Screening of Oral Biofilm Formation on New Microfabricated Dental Material Surfaces

PubMed Central

Alvarez-Escobar, Marta; Hansford, Derek; Monteiro, Fernando J.

2018-01-01

Introduction Microfabrication offers opportunities to study surface concepts focused to reduce bacterial adhesion on implants using human minimally invasive rapid screening (hMIRS). Wide information is available about cell/biomaterial interactions using eukaryotic and prokaryotic cells on surfaces of dental materials with different topographies, but studies using human being are still limited. Objective To evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials. Materials and Methods Micropatterned and flat surfaces on biomedical PDMS disks were produced by soft lithography. The hMIRS approach was used to evaluate the total oral bacterial adhesion on PDMS surfaces placed in the oral cavity of five volunteers (the study was approved by the University Ethical Committee). After 24 h, the disks were analyzed using MTT assay and light microscopy. Results In the present pilot study, microwell structures were microfabricated on the PDMS surface via soft lithography with a spacing of 5 µm. Overall, bacterial adhesion did not significantly differ between the flat and micropatterned surfaces. However, individual analysis of two subjects showed greater bacterial adhesion on the micropatterned surfaces than on the flat surfaces. Significance Microfabrication and hMIRS might be implemented to study the cell/biomaterial interactions for dental materials. PMID:29593793

Femtosecond laser induced surface modification for prevention of bacterial adhesion on 45S5 bioactive glass

NASA Astrophysics Data System (ADS)

Shaikh, Shazia; Singh, Deepti; Subramanian, Mahesh; Kedia, Sunita; Singh, Anil Kumar; Singh, Kulwant; Gupta, Nidhi; Sinha, Sucharita

2018-02-01

Bacterial attachment and biofilm formation on implant surface has been a major concern in hospital and industrial environment. Prevention of bacterial infections of implant surface through surface treatment could be a potential solution and hence this has become a key area of research. In the present study, the antibacterial and biocompatible properties of femtosecond laser surface treated 45S5 bioactive glass (BG) have been investigated. Adhesion and sustainability of both gram positive S. aureus and gram negative P.aeruginosa and E. coli nosocomial bacteria on untreated and laser treated BG samples has been explored. An imprint method has been used to visualize the growth of bacteria on the sample surface. We observed complete bacterial rejection potentially reducing risk of biofilm formation on laser treated surface. This was correlated with surface roughness, wettability and change in surface chemical composition of the samples before and after laser treatment. Biocompatibility of the laser treated BG was demonstrated by studying the anchoring and growth of human cervix cell line INT407. Our results demonstrate that, laser surface modification of BG enables enhanced bacterial rejection without affecting its biocompatibility towards growth of human cells on it. These results open a significantly potential approach towards use of laser in successfully imparting desirable characteristics to BG based bio-implants and devices.

Biophysical model of bacterial cell interactions with nanopatterned cicada wing surfaces.

PubMed

Pogodin, Sergey; Hasan, Jafar; Baulin, Vladimir A; Webb, Hayden K; Truong, Vi Khanh; Phong Nguyen, The Hong; Boshkovikj, Veselin; Fluke, Christopher J; Watson, Gregory S; Watson, Jolanta A; Crawford, Russell J; Ivanova, Elena P

2013-02-19

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on their physical surface structure. The wings provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. We propose a biophysical model of the interactions between bacterial cells and cicada wing surface structures, and show that mechanical properties, in particular cell rigidity, are key factors in determining bacterial resistance/sensitivity to the bactericidal nature of the wing surface. We confirmed this experimentally by decreasing the rigidity of surface-resistant strains through microwave irradiation of the cells, which renders them susceptible to the wing effects. Our findings demonstrate the potential benefits of incorporating cicada wing nanopatterns into the design of antibacterial nanomaterials. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus.

PubMed

Han, Jinzhi; Gao, Peng; Zhao, Shengming; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

2017-01-06

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD (+) H, NADP (+) H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries. Copyright © 2016 Elsevier B.V. All rights reserved.

Increase methylmercury accumulation in Arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE

PubMed Central

2013-01-01

The bacterial merE gene derived from the Tn21 mer operon encodes a broad-spectrum mercury transporter that governs the transport of methylmercury and mercuric ions across bacterial cytoplasmic membranes, and this gene is a potential molecular tool for improving the efficiency of methylmercury phytoremediation. A transgenic Arabidopsis engineered to express MerE was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. The subcellular localization of transiently expressed GFP-tagged MerE was examined in Arabidopsis suspension-cultured cells. The GFP-MerE was found to localize to the plasma membrane and cytosol. The transgenic Arabidopsis expressing MerE accumulated significantly more methymercury and mercuric ions into plants than the wild-type Arabidopsis did. The transgenic plants expressing MerE was significantly more resistant to mercuric ions, but only showed more resistant to methylmercury compared with the wild type Arabidopsis. These results demonstrated that expression of the bacterial mercury transporter MerE promoted the transport and accumulation of methylmercury in transgenic Arabidopsis, which may be a useful method for improving plants to facilitate the phytoremediation of methylmercury pollution. PMID:24004544

Zinc-ion implanted and deposited titanium surfaces reduce adhesion of Streptococccus mutans

NASA Astrophysics Data System (ADS)

Xu, Juan; Ding, Gang; Li, Jinlu; Yang, Shenhui; Fang, Bisong; Sun, Hongchen; Zhou, Yanmin

2010-10-01

While titanium (Ti) is a commonly used dental implant material with advantageous biocompatible and mechanical properties, native Ti surfaces do not have the ability to prevent bacterial colonization. The objective of this study was to evaluate the chemical composition and bacterial adhesive properties of zinc (Zn) ion implanted and deposited Ti surfaces (Zn-PIIID-Ti) as potential dental implant materials. Surfaces of pure Ti (cp-Ti) were modified with increasing concentrations of Zn using plasma immersion ion implantation and deposition (PIIID), and elemental surface compositions were characterized by X-ray photoelectron spectrometry (XPS). To evaluate bacterial responses, Streptococcus mutans were seeded onto the modifiedTi surfaces for 48 h and subsequently observed by scanning electron microscopy. Relative numbers of bacteria on each surface were assessed by collecting the adhered bacteria, reculturing and counting colony forming units after 48 h on bacterial grade plates. Ti, oxygen and carbon elements were detected on all surfaces by XPS. Increased Zn signals were detected on Zn-PIIID-Ti surfaces, correlating with an increase of Zn-deposition time. Substantial numbers of S. mutans adhered to cp-Ti samples, whereas bacterial adhesion on Zn-PIIID-Ti surfaces signficantly decreased as the Zn concentration increased ( p < 0.01). In conclusion, PIIID can successfully introduce Zn onto a Ti surface, forming a modified surface layer bearing Zn ions that consequently deter adhesion of S. mutans, a common bacterium in the oral environment.

Impact of pulsed xenon ultraviolet disinfection on surface contamination in a hospital facility's expressed human milk feed preparation area.

PubMed

Dippenaar, Ricky; Smith, Johan

2018-02-23

Expressed human milk (EHM) feed preparation areas represent a potential source of unintentional nosocomial infection. Daily disinfection of environmental surfaces remains an essential intervention to mitigate nosocomial infections. The inefficiency of conventional cleaning and disinfection contributes to an increased risk for the acquisition of multi-drug resistant pathogens. "Non touch" technologies such as the pulsed xenon ultraviolet (PX-UVD) light device have documented sustained reduction in surface bacterial colonization and reduced cross contamination. The impact of a PX-UVD on surface colony forming units per square centimeter (cfu/cm 2 ) in feed preparation areas was evaluated following its implementation as standard care. A quasi-experimental study was performed documenting bacterial colonization from 6 high risk feed preparation areas in a community care hospital in South Africa. Pre and post conventional cleaning neutralizing rinse swabs were collected fortnightly over a 16 week control period prior to the introduction of the PX-UVD and compared to a matching set of samples for the PX-UVD period. A 90% reduction in total surface bioburden was noted from the control period (544 cfu/cm 2 ) compared to the corresponding PX-UVD period (50 cfu/cm 2 ). Sub -analysis of both the Pre-clean Control: Pre-clean PX-UVD counts as well as the Post-clean Control: Post-clean PX-UVD counts noted significant improvements (p < 0.001). A statistically significant improvement was noted between pre-and post-cleaning total surface bioburden following exposure to the PX-UVD (p = 0.0004). The introduction of the PX-UVD was associated with a sustained reduction in the pre clean bioburden counts with a risk trend (per week) 0.19, (95% CI [0.056, 0.67], p = 0.01). The use of a PX-UVD as adjunct to standard cleaning protocols was associated with a significant decrease in surface bioburden. The study demonstrated the inefficiency of conventional cleaning. Persistence of potentially pathological species in both periods highlights current health sector challenges.

The role of type III secretion system and lens material on adhesion of Pseudomonas aeruginosa to contact lenses.

PubMed

Shen, Elizabeth P; Tsay, Ruey-Yug; Chia, Jean-San; Wu, Semon; Lee, Jing-Wen; Hu, Fung-Rong

2012-09-21

To determine the distribution of invasive and cytotoxic genotypes among ocular isolates of P. aeruginosa and investigate the influence of the type III secretion system (T3SS) on adhesion to conventional, cosmetic, and silicone hydrogel contact lenses (CL). Clinical isolates from 2001 to 2010 were analyzed by multiplex PCR for exoS, exoU, and exoT genes. Bacterial adhesion to etafilcon, nelfilcon (gray colored), balafilcon, and galyfilcon CL with or without artificial tear fluid (ATF) incubation were compared. Surface characteristics were determined with scanning electron microscopy (SEM). Among 87 total isolates, 64 strains were from microbial keratitis cases. CL-related microbial keratitis (CLMK) isolates were mostly of the cytotoxic genotype (expressing exoU) (P = 0.002). No significant differences were found in bacterial adhesion to all types of CL between the genotypes under T3SS-inducing conditions. A trend for least bacterial adhesion of galyfilcon compared to the other CL was noted for both genotypes. Needle complex pscC mutants adhered less to all materials than the wild type (P < 0.05), indicating a role of the T3SS in contact lens adhesion. ATF-incubated CL had significantly more bacterial adhesion (P < 0.05). SEM showed most of the bacteria adhering on CL surfaces. CLMK isolates were mostly of cytotoxic genotype. Different genotypes did not significantly differ in its adhesion to various CL. T3SS and other adhesins are involved in bacteria-contact lens adhesion through complex interactions. Contact lens materials may also play an important role in the adherence of both genotypes of P. aeruginosa.

Rheological effects of micropolar slime on the gliding motility of bacteria with slip boundary condition

NASA Astrophysics Data System (ADS)

Asghar, Z.; Ali, N.; Anwar Bég, O.; Javed, T.

2018-06-01

Gliding bacteria are virtually everywhere. These organisms are phylogenetically diverse with their hundreds of types, different shapes and several modes of motility. One possible mode of gliding motility in the rod shaped bacteria is that they propel themselves by producing undulating waves in their body. Few bacteria glides near the solid surface over the slime without any aid of flagella so the classical Navier-Stokes equations are incapable of explaining the slime rheology at the microscopic level. Micropolar fluid dynamics however provides a solid framework for mimicking bacterial physical phenomena at both micro and nano-scales, and therefore we use the micropolar fluid to characterize the rheology of a thin layer of slime and its dominant microrotation effects. It is also assumed that there is a certain degree of slip between slime and bacterial undulating surface and also between slime and solid substrate. The flow equations are formulated under long wavelength and low Reynolds number assumptions. Exact expressions for stream function and pressure gradient are obtained. The speed of the gliding bacteria is numerically calculated by using a modified Newton-Raphson method. Slip effects and effects of non-Newtonian slime parameters on bacterial speed and power are also quantified. In addition, when the glider is fixed, the effects of slip and rheological properties of micropolar slime parameters on the velocity, micro-rotation (angular velocity) of spherical slime particles, pressure rise per wavelength, pumping and trapping phenomena are also shown graphically and discussed in detail. The study is relevant to emerging biofuel cell technologies and also bacterial biophysics.

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis.

PubMed

Hennig, Anna; Bonfig, Katharina; Roitsch, Thomas; Warzecha, Heribert

2007-11-01

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.

Symbiotic Bacteria Direct Expression of an Intestinal Bactericidal Lectin

PubMed Central

Cash, Heather L.; Whitham, Cecilia V.; Behrendt, Cassie L.; Hooper, Lora V.

2009-01-01

The mammalian intestine harbors complex societies of beneficial bacteria that are maintained in the lumen with minimal penetration of mucosal surfaces. Microbial colonization of germ-free mice triggers epithelial expression of RegIIIγ, a secreted C-type lectin. RegIIIγ binds intestinal bacteria but lacks the complement recruitment domains present in other microbe-binding mammalian C-type lectins. We show that RegIIIγ and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind their bacterial targets via interactions with peptidoglycan carbohydrate. We propose that these proteins represent an evolutionarily primitive form of lectin-mediated innate immunity, and that they reveal intestinal strategies for maintaining symbiotic host-microbial relationships. PMID:16931762

An anti-bacterial approach to nanoscale roughening of biomimetic rice-like pattern PP by thermal annealing

NASA Astrophysics Data System (ADS)

Jafari Nodoushan, Emad; Ebrahimi, Nadereh Golshan; Ayazi, Masoumeh

2017-11-01

In this paper, we introduced thermal annealing treatment as an effective way of increasing the nanoscale roughness of a semi-crystalline polymer surface. Annealing treatment applied to a biomimetic microscale pattern of rice leaf to achieve a superhydrophobic surface with a hierarchical roughness. Resulted surfaces was characterized by XRD, AFM and FE-SEM instruments and showed an increase of roughness and cristallinity within both time and temperature of treatment. These two parameters also impact on measured static contact angle up to 158°. Bacterial attachment potency has an inverse relationship with the similarity of surface pattern dimensions and bacterial size and due to that, thermal annealing could be an effective way to create anti-bacterial surface beyond its effect on water repellency. Point in case, the anti-bacterial properties of produced water-repellence surfaces of PP were measured and counted colonies of both gram-negative (E. coli) and gram-positive (S. aureus) bacteria reduced with the nature of PP and hierarchical pattern on that. Anti-bacterial characterization of the resulted surface reveals a stunning reduction in adhesion of gram-positive bacteria to the surface. S. aureus reduction rates equaled to 95% and 66% when compared to control blank plate and smooth surface of PP. Moreover, it also could affect the other type of bacteria, gram-negative (E. coli). In the latter case, adhesion reduction rates calculated 66% and 53% when against to the same controls, respectively.

Preparation of RNA from bacteria infected with bacteriophages: a case study from the marine unicellular Synechococcus sp. WH7803 infected by phage S-PM2.

PubMed

Shan, Jinyu; Clokie, Martha

2009-01-01

Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest.

Autonomous Bacterial Localization and Gene Expression Based on Nearby Cell Receptor Density

DTIC Science & Technology

2013-01-22

synthesis of novel products (Kwok, 2010). In such cases, cell populations are aligned for optimal production . While Nature’s biosynthetic toolbox is vast...less commonly examined but equally innovative strategy envisions a repro- grammed cell itself or small collections of cells as the end products of...to surfaces. In Figure 2B, we depict the level of NF binding and AI-2 production as a function of coated avidin. We used a mathematical model for E

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2007-07-01

The T7Select 10-3b system of lytic phage display is a mid-copy vector that displays between 5-15 copies on the surface of the T7 capsid. The natural... Phage are amplified on a bacterial host that carries an ampicillin-resistant plasmid expressing additional 10A capsid protein from a T7 promoter. We... phage display library of coding fragments encompassing all open reading frames of the human genome. We designed approximately 467,000 overlapping

Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand.

PubMed

Holden, P A; LaMontagne, M G; Bruce, A K; Miller, W G; Lindow, S E

2002-05-01

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

PubMed

Nhan, Nguyen Thanh; Gonzalez de Valdivia, Ernesto; Gustavsson, Martin; Hai, Truong Nam; Larsson, Gen

2011-04-11

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Structure of a bacterial cell surface decaheme electron conduit

USDA-ARS?s Scientific Manuscript database

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

PubMed

Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

2018-04-03

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

PubMed

Lu, Yun-Yueh; Franz, Bettina; Truttmann, Matthias C; Riess, Tanja; Gay-Fraret, Jérémie; Faustmann, Marco; Kempf, Volkhard A J; Dehio, Christoph

2013-05-01

The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of Bartonella. © 2012 Blackwell Publishing Ltd.

Phyllosphere Metaproteomes of Trees from the Brazilian Atlantic Forest Show High Levels of Functional Redundancy.

PubMed

Lambais, M R; Barrera, S E; Santos, E C; Crowley, D E; Jumpponen, A

2017-01-01

The phyllosphere of the Brazilian Atlantic Forest has been estimated to contain several million bacterial species that are associated with approximately 20000 plant species. Despite the high bacterial diversity in the phyllosphere, the function of these microorganisms and the mechanisms driving their community assembly are largely unknown. In this study, we characterized the bacterial communities in the phyllospheres of four tree species of the Atlantic Forest (Mollinedia schottiana, Ocotea dispersa, Ocotea teleiandra, and Tabebuia serratifolia) and their metaproteomes to examine the basic protein functional groups expressed in the phyllosphere. Bacterial community analyses using 16S rRNA gene sequencing confirmed prior observations that plant species harbor distinct bacterial communities and that plants of the same taxon have more similar communities than more distantly related taxa. Using LC-ESI-Q-TOF, we identified 216 nonredundant proteins, based on 3503 peptide mass spectra. Most protein families were shared among the phyllosphere communities, suggesting functional redundancy despite differences in the species compositions of the bacterial communities. Proteins involved in glycolysis and anaerobic carbohydrate metabolism, solute transport, protein metabolism, cell motility, stress and antioxidant responses, nitrogen metabolism, and iron homeostasis were among the most frequently detected. In contrast to prior studies on crop plants and Arabidopsis, a low abundance of OTUs related to Methylobacterium and no proteins associated with the metabolism of one-carbon molecules were detected in the phyllospheres of the tree species studied here. Our data suggest that even though the phyllosphere bacterial communities of different tree species are phylogenetically diverse, their metaproteomes are functionally convergent with respect to traits required for survival on leaf surfaces.

Staphylococcus aureus Extracellular Adherence Protein Triggers TNFα Release, Promoting Attachment to Endothelial Cells via Protein A

PubMed Central

Edwards, Andrew M.; Bowden, Maria Gabriela; Brown, Eric L.; Laabei, Maisem; Massey, Ruth C.

2012-01-01

Staphylococcus aureus is a leading cause of bacteraemia, which frequently results in complications such as infective endocarditis, osteomyelitis and exit from the bloodstream to cause metastatic abscesses. Interaction with endothelial cells is critical to these complications and several bacterial proteins have been shown to be involved. The S. aureus extracellular adhesion protein (Eap) has many functions, it binds several host glyco-proteins and has both pro- and anti-inflammatory activity. Unfortunately its role in vivo has not been robustly tested to date, due to difficulties in complementing its activity in mutant strains. We previously found Eap to have pro-inflammatory activity, and here show that purified native Eap triggered TNFα release in whole human blood in a dose-dependent manner. This level of TNFα increased adhesion of S. aureus to endothelial cells 4-fold via a mechanism involving protein A on the bacterial surface and gC1qR/p33 on the endothelial cell surface. The contribution this and other Eap activities play in disease severity during bacteraemia was tested by constructing an isogenic set of strains in which the eap gene was inactivated and complemented by inserting an intact copy elsewhere on the bacterial chromosome. Using a murine bacteraemia model we found that Eap expressing strains cause a more severe infection, demonstrating its role in invasive disease. PMID:22905199

S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

PubMed

Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

2016-11-01

Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biofilm formation by Shiga toxin-producing Escherichia coli O157:H7 and Non-O157 strains and their tolerance to sanitizers commonly used in the food processing environment.

PubMed

Wang, Rong; Bono, James L; Kalchayanand, Norasak; Shackelford, Steven; Harhay, Dayna M

2012-08-01

Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.

Bacterial response to different surface chemistries fabricated by plasma polymerization on electrospun nanofibers.

PubMed

Abrigo, Martina; Kingshott, Peter; McArthur, Sally L

2015-12-06

Control over bacterial attachment and proliferation onto nanofibrous materials constitutes a major challenge for a variety of applications, including filtration membranes, protective clothing, wound dressings, and tissue engineering scaffolds. To develop effective devices, the interactions that occur between bacteria and nanofibers with different morphological and physicochemical properties need to be investigated. This paper explores the influence of fiber surface chemistry on bacterial behavior. Different chemical functionalities were generated on the surface of electrospun polystyrene nanofibers through plasma polymerization of four monomers (acrylic acid, allylamine, 1,7-octadiene, and 1,8-cineole). The interactions of Escherichia coli with the surface modified fibers were investigated through a combination of scanning electron microscopy and confocal laser scanning microscopy. Fiber wettability, surface charge, and chemistry were found to affect the ability of bacterial cells to attach and proliferate throughout the nanofiber meshes. The highest proportion of viable cells attachment occurred on the hydrophilic amine rich coating, followed by the hydrophobic octadiene. The acrylic acid coating rich in carboxyl groups showed a significantly lower attraction of bacterial cells. The 1,8-cineole retained the antibacterial activity of the monomer, resulting with a high proportion of dead isolated cells attached onto the fibers. Results showed that the surface chemistry properties of nanofibrous membranes can be strategically tuned to control bacterial behavior.

Biofilm formation on nanostructured titanium oxide surfaces and a micro/nanofabrication-based preventive strategy using colloidal lithography.

PubMed

Singh, Ajay Vikram; Vyas, Varun; Salve, Tushar S; Cortelli, Daniele; Dellasega, David; Podestà, Alessandro; Milani, Paolo; Gade, W N

2012-06-01

The contamination of implant devices as a result of biofilm formation through bacterial infection has instigated major research in this area, particularly to understand the mechanism of bacterial cell/implant surface interactions and their preventions. In this paper, we demonstrate a controlled method of nanostructured titanium oxide surface synthesis using supersonic cluster beam depositions. The nanoscale surface characterization using atomic force microscopy and a profilometer display a regulated evolution in nanomorphology and physical properties. X-ray photoelectron spectroscopy analyses display a stoichiometric nanostructured TiO(2) film. Measurement of the water contact angle shows a nominal increase in the hydrophilic nature of ns-TiO(2) films, whereas the surface energy increases with decreasing contact angle. Bacterial species Staphylococcus aureus and Escherichia coli interaction with nanostructured surfaces shows an increase in adhesion and biofilm formation with increasing nanoscale morphological properties. Conversely, limiting ns-TiO(2) film distribution to micro/nanopatterned designed substrates integrated with bovine serum albumin functionalization leads to a reduction in biofilm formations due to a globally decreased bacterial cell-surface interaction area. The results have potential implications in inhibiting bacterial colonization and promoting mammalian cell-implant interactions.

Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens.

PubMed

Goulter, Rebecca M; Taran, Elena; Gentle, Ian R; Gobius, Kari S; Dykes, Gary A

2014-07-01

The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry.

PubMed

Patel, Jasmine D; Ebert, Michael; Stokes, Ken; Ward, Robert; Anderson, James M

2003-01-01

Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to < or = 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of < or = 7 dyne/cm2. Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.

Diversity of Bacterial Communities of Fitness Center Surfaces in a U.S. Metropolitan Area

PubMed Central

Mukherjee, Nabanita; Dowd, Scot E.; Wise, Andy; Kedia, Sapna; Vohra, Varun; Banerjee, Pratik

2014-01-01

Public fitness centers and exercise facilities have been implicated as possible sources for transmitting community-acquired bacterial infections. However, the overall diversity of the bacterial community residing on the surfaces in these indoor environments is still unknown. In this study, we investigated the overall bacterial ecology of selected fitness centers in a metropolitan area (Memphis, TN, USA) utilizing culture-independent pyrosequencing of the 16S rRNA genes. Samples were collected from the skin-contact surfaces (e.g., exercise instruments, floor mats, handrails, etc.) within fitness centers. Taxonomical composition revealed the abundance of Firmicutes phyla, followed by Proteobacter and Actinobacteria, with a total of 17 bacterial families and 25 bacterial genera. Most of these bacterial genera are of human and environmental origin (including, air, dust, soil, and water). Additionally, we found the presence of some pathogenic or potential pathogenic bacterial genera including Salmonella, Staphylococcus, Klebsiella, and Micrococcus. Staphylococcus was found to be the most prevalent genus. Presence of viable forms of these pathogens elevates risk of exposure of any susceptible individuals. Several factors (including personal hygiene, surface cleaning and disinfection schedules of the facilities) may be the reasons for the rich bacterial diversity found in this study. The current finding underscores the need to increase public awareness on the importance of personal hygiene and sanitation for public gym users. PMID:25479039

Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection.

PubMed

Lipsky, Alexander; Joshi, Janak Raj; Carmi, Nir; Yedidia, Iris

2016-11-20

The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95-100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

PubMed

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

Variations in the Degree of d-Alanylation of Teichoic Acids in Lactococcus lactis Alter Resistance to Cationic Antimicrobials but Have No Effect on Bacterial Surface Hydrophobicity and Charge▿

PubMed Central

Giaouris, Efstathios; Briandet, Romain; Meyrand, Mickael; Courtin, Pascal; Chapot-Chartier, Marie-Pierre

2008-01-01

An increase of the degree of d-alanylation of teichoic acids in Lactococcus lactis resulted in a significant increase of bacterial resistance toward the cationic antimicrobials nisin and lysozyme, whereas the absence of d-alanylation led to a decreased resistance toward the same compounds. In contrast, the same variations of the d-alanylation degree did not modify bacterial cell surface charge and hydrophobicity. Bacterial adhesion to polystyrene and glass surfaces was not modified either. PMID:18539809

Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions

PubMed Central

Ribeiro, Marta; Monteiro, Fernando J.; Ferraz, Maria P.

2012-01-01

Staphylococcus comprises up to two-thirds of all pathogens in orthopedic implant infections and they are the principal causative agents of two major types of infection affecting bone: septic arthritis and osteomyelitis, which involve the inflammatory destruction of joint and bone. Bacterial adhesion is the first and most important step in implant infection. It is a complex process influenced by environmental factors, bacterial properties, material surface properties and by the presence of serum or tissue proteins. Properties of the substrate, such as chemical composition of the material, surface charge, hydrophobicity, surface roughness and the presence of specific proteins at the surface, are all thought to be important in the initial cell attachment process. The biofilm mode of growth of infecting bacteria on an implant surface protects the organisms from the host immune system and antibiotic therapy. The research for novel therapeutic strategies is incited by the emergence of antibiotic-resistant bacteria. This work will provide an overview of the mechanisms and factors involved in bacterial adhesion, the techniques that are currently being used studying bacterial-material interactions as well as provide insight into future directions in the field. PMID:23507884

Mechanics governs single-cell signaling and multi-cell robustness in biofilm infections

NASA Astrophysics Data System (ADS)

Gordon, Vernita

In biofilms, bacteria and other microbes are embedded in extracellular polymers (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Our work suggests that different polymers may confer distinct mechanical benefits. Our model organism is Pseudomonas aeruginosa, an opportunistic human pathogen that forms chronic biofilm infections associated with increased antibiotic resistance and evasion of the immune defense. Biofilms initiate when bacteria attach to a surface, sense the surface, and change their gene expression. Changes in gene expression are regulated by a chemical signal, cyclic-di-GMP. We find that one EPS material, called ``PEL,'' enhances surface sensing by increasing mechanical coupling of single bacteria to the surface. Measurements of bacterial motility suggest that PEL may increase frictional interactions between the surface and the bacteria. Consistent with this, we show that bacteria increase cyclic-di-GMP signaling in response to mechanical shear stress. Mechanosensing has long been known to be important to the function of cells in higher eukaryotes, but this is one of only a handful of studies showing that bacteria can sense and respond to mechanical forces. For the mature biofilm, the embedding polymer matrix can protect bacteria both chemically and mechanically. P. aeruginosa infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of another EPS material, alginate, is well-known to tend to increase over time in CF infections. Alginate chemically protects biofilms, but also makes them softer and weaker. Recently, it is being increasingly recognized that bacteria in chronic CF infections also evolve to increase PSL production. We use oscillatory bulk rheology to determine the unique contributions of EPS materials to biofilm mechanics. Unlike alginate, increased PSL stiffens biofilms. Increasing both PSL and alginate expression increases the energy cost to break the biofilm. We compare the elastic moduli of biofilms to estimated stresses exerted by phagocytotic immune cells, and infer that increased PSL could confer a mechanical fitness benefit. This work was supported by start-up funds from The University of Texas at Austin and a gift from ExxonMobile to VDG, and by Grants from the Human Frontiers Science Program (HFSP RGY0081/2012-GORDON) and the National Science Foundation (NSF 1337670).

Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

PubMed Central

Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

2013-01-01

Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces

PubMed Central

2012-01-01

Background 10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces. Methods Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically. Results 500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells. Conclusion Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely. PMID:22839724

Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces.

PubMed

Yousefi, Behnam; Ghaderi, Shahrooz; Rezapoor-Lactooyi, Alireza; Amiri, Niusha; Verdi, Javad; Shoae-Hassani, Alireza

2012-07-28

10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces. Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically. 500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells. Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it's not necessary to put down the oral flora completely.

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition

PubMed Central

Kinkead, Lauren C.; Whitmore, Laura C.; McCracken, Jenna M.; Fletcher, Joshua R.; Ketelsen, Brandi B.; Kaufman, Justin W.; Jones, Bradley D.; Weiss, David S.; Barker, Jason H.

2017-01-01

Abstract Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14C] acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function. PMID:29063667

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

PubMed

Song, Myeong-Sub; Sekhon, Simranjeet Singh; Shin, Woo-Ri; Rhee, Sung-Keun; Ko, Jung Ho; Kim, Sang Yong; Min, Jiho; Ahn, Ji-Young; Kim, Yang-Hoon

2018-05-01

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Surface-exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis.

PubMed Central

Murphy, T F; Bartos, L C

1989-01-01

The outer membrane proteins (OMPs) of Branhamella catarrhalis were studied in an effort to identify surface-exposed determinants that are conserved among strains of the bacterium. Aliquots of polyclonal antiserum were absorbed individually by strains of B. catarrhalis. The absorbed antisera were tested in comparison with unabsorbed antiserum in an immunoblot assay against OMPs of the homologous strain. The absence of a band recognized by antibodies in the absorbed antiserum compared with the unabsorbed antiserum indicated that surface-exposed determinants of the absorbing strain cross-reacted with determinants on the homologous strain. Two antisera were absorbed individually by 20 strains of B. catarrhalis, and the absorbed sera were studied in this way in immunoblot assays. OMP E (molecular weight, ca. 56,000) expresses surface-exposed determinants that are shared among 17 of the 20 strains studied. Antibodies to OMP G (molecular weight, 28,000) were absorbed from both antisera by 14 of the 20 strains. These studies demonstrate that OMP E and OMP G express determinants that are exposed on the surface of the intact bacterium. Furthermore, these determinants are antigenically conserved among a majority of strains of B. catarrhalis. On the basis of these observations, OMPs E and G should be considered when bacterial antigens are evaluated as potential vaccine candidates. Images PMID:2476393

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hesse, Cedar N.; Mueller, Rebecca C.; Vuyisich, Momchilo

Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes)more » in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.« less

Elevated numbers of SCART1+ gammadelta T cells in skin inflammation and inflammatory bowel disease.

PubMed

Fink, Dorte Rosenbek; Holm, Dorte; Schlosser, Anders; Nielsen, Ole; Latta, Markus; Lozano, Francisco; Holmskov, Uffe

2010-05-01

The members of the scavenger receptor cysteine-rich (SRCR) superfamily group B have diverse functions, including roles in the immune system. For years it has been known that the WC1 protein is expressed on the surface of bovine gammadelta T cells, and more recent studies indicate that WC1(+) gammadelta T cells respond to stimulation with bacterial antigens by producing interferon-gamma. The SRCR proteins CD5, CD6, Sp alpha, CD163, and DMBT1/gp-340 are also involved in the immune response, since they are pattern recognition receptors capable of binding directly to bacterial and/or fungal components. Here, we investigate a novel murine SRCR protein named SCART1. The ectodomain and the full-length SCART1 were expressed in mammalian cells and used to raise monoclonal antibodies against the ectodomain for immunohistochemical and FACS analysis. Immunohistochemical analysis shows that SCART1 is expressed in a range of lymphoid organs and epithelial-rich tissues by a subset of T cells identified as being gammadelta T cells by FACS analysis. SCART1 was present in 86% of the gammadelta T cells and was not found in CD4(+) or CD8(+) T cells. The numbers of SCART1(+) cells were elevated in two mouse models of human diseases: skin inflammation and inflammatory bowel disease. In the skin inflammation model, an 8.6-fold increase in SCART1(+) cells was observed. Finally, recombinant SCART1 protein was found not to bind to selected bacterial or fungal components or to whole bacteria. Our results show that SCART1 is a novel gammadelta T cell marker and it is therefore likely that SCART1 plays a role in the immune response. (c) 2010 Elsevier Ltd. All rights reserved.

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

PubMed

Araújo, Welington Luiz; Santos, Daiene Souza; Dini-Andreote, Francisco; Salgueiro-Londoño, Jennifer Katherine; Camargo-Neves, Aline Aparecida; Andreote, Fernando Dini; Dourado, Manuella Nóbrega

2015-10-01

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in bacterial colonization.

COMPARISON OF SCANNING ELECTRON AND ATOMIC FORCE MICROSCOPY OF SURFACE FINISHES ON STAINLESS STEEL THAT REDUCE BACTERIAL ATTACHMENT

EPA Science Inventory

Bacteria adhere to food products and processing surfaces that can cross-contaminate other products and work surfaces (Arnold, 1998). Using materials for food processing surfaces that are resistant to bacterial contamination could enhance food safety. Stainless steel, although sus...

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

PubMed

Arambula, Diego; Wong, Wenge; Medhekar, Bob A; Guo, Huatao; Gingery, Mari; Czornyj, Elizabeth; Liu, Minghsun; Dey, Sanghamitra; Ghosh, Partho; Miller, Jeff F

2013-05-14

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis

PubMed Central

Kuipers, Annemarie; Stapels, Daphne A. C.; Weerwind, Lleroy T.; Ko, Ya-Ping; Ruyken, Maartje; Lee, Jean C.; van Kessel, Kok P. M.

2016-01-01

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance. PMID:27112346

The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis.

PubMed

Kuipers, Annemarie; Stapels, Daphne A C; Weerwind, Lleroy T; Ko, Ya-Ping; Ruyken, Maartje; Lee, Jean C; van Kessel, Kok P M; Rooijakkers, Suzan H M

2016-07-01

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.

Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

PubMed

Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

2015-01-06

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Diagnostic test accuracy of a 2-transcript host RNA signature for discriminating bacterial vs viral infection in febrile children

PubMed Central

Shailes, Hannah; Eleftherohorinou, Hariklia; Hoggart, Clive J; Cebey-Lopez, Miriam; Carter, Michael J; Janes, Victoria A; Gormley, Stuart; Shimizu, Chisato; Tremoulet, Adriana H; Barendregt, Anouk M; Salas, Antonio; Kanegaye, John; Pollard, Andrew J; Faust, Saul N; Patel, Sanjay; Kuijpers, Taco; Martinon-Torres, Federico; Burns, Jane C; Coin, Lachlan JM; Levin, Michael

2018-01-01

Importance As clinical features do not reliably distinguish bacterial from viral infection, many children worldwide receive unnecessary antibiotic treatment whilst bacterial infection is missed in others. Objective To identify a blood RNA expression signature that distinguishes bacterial from viral infection in febrile children. Design Febrile children presenting to participating hospitals in UK, Spain, Netherlands and USA between 2009-2013 were prospectively recruited, comprising a discovery group and validation group. Each group was classified after microbiological investigation into definite bacterial, definite viral infection or indeterminate infection. RNA expression signatures distinguishing definite bacterial from viral infection were identified in the discovery group and diagnostic performance assessed in the validation group. Additional validation was undertaken in separate studies of children with meningococcal disease (n=24) inflammatory diseases (n=48), and on published gene expression datasets. Exposures A 2-transcript RNA expression signature distinguishing bacterial infection from viral infection was evaluated against clinical and microbiological diagnosis. Main Outcomes Definite Bacterial and viral infection was confirmed by culture or molecular detection of the pathogens. Performance of the RNA signature was evaluated in the definite bacterial and viral group, and the indeterminate group. Results The discovery cohort of 240 children (median age 19 months, 62% males) included 52 with definite bacterial infection of whom 36 (69%) required intensive care; and 92 with definite viral infection of whom 32 (35%) required intensive care. 96 children had indeterminate infection. Bioinformatic analysis of RNA expression data identified a 38-transcript signature distinguishing bacterial from viral infection. A smaller (2-transcript) signature (FAM89A and IFI44L) was identified by removing highly correlated transcripts. When this 2-transcript signature was implemented as a Disease Risk Score in the validation group (130 children, including 23 bacterial, 28 viral, 79 indeterminate; median age 17 months, 57% males), bacterial infection was identified in all 23 microbiologically-confirmed definite bacterial patients, with a sensitivity of 100% (95% confidence interval [CI], 100 - 100), and in 1 of 28 definite viral patients, with specificity of 96.4% (95% CI, 89.3 – 100). When applied to additional validation datasets from patients with meningococcal and inflammatory diseases, bacterial infection was identified with a sensitivity of 91.7% (79.2-100) and 90.0% (70.0-100) respectively, and with specificity of 96.0% (88.0-100) and 95.8% (89.6-100). A minority of children in the indeterminate group were classified as having bacterial infection (63 of 136, 46.3%), although most received antibiotic treatment (129 of 136, 94.9%). Conclusions and Relevance This study provides preliminary data regarding test accuracy of a 2-transcript host RNA signature discriminating bacterial from viral infection in febrile children. Further studies are needed in diverse groups of patients to assess accuracy and clinical utility of this test in different clinical settings. PMID:27552617

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

PubMed Central

Itoh, Takafumi; Hibi, Takao; Fujii, Yutaka; Sugimoto, Ikumi; Fujiwara, Akihiro; Suzuki, Fumiko; Iwasaki, Yukimoto; Kim, Jin-Kyung; Taketo, Akira

2013-01-01

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine. PMID:24077704

In vitro reduction of antibacterial activity of tigecycline against multidrug-resistant Acinetobacter baumannii with host stress hormone norepinephrine.

PubMed

Inaba, Masato; Matsuda, Naoyuki; Banno, Hirotsugu; Jin, Wanchun; Wachino, Jun-Ichi; Yamada, Keiko; Kimura, Kouji; Arakawa, Yoshichika

2016-12-01

The host stress hormone norepinephrine (NE), also called noradrenaline, is reported to augment bacterial growth and pathogenicity, but few studies have focused on the effect of NE on the activity of antimicrobials. The aim of this study was to clarify whether NE affects antimicrobial activity against multidrug-resistant Acinetobacter baumannii (MDR-AB). Time-kill studies of tigecycline (TIG) and colistin (COL) against MDR-AB as well as assays for factors contributing to antibiotic resistance were performed using MDR-AB clinical strains both in the presence and absence of 10 µM NE. In addition, expression of three efflux pump genes (adeB, adeJ and adeG) in the presence and absence of NE was analysed by quantitative reverse transcription PCR. Viable bacterial cell counts in TIG-supplemented medium containing NE were significantly increased compared with those in medium without NE. In contrast, NE had little influence on viable bacterial cell counts in the presence of COL. NE-supplemented medium resulted in an ca. 2 log increase in growth and in bacterial cell numbers adhering on polyurethane, silicone and polyvinylchloride surfaces. Amounts of biofilm in the presence of NE were ca. 3-fold higher than without NE. Expression of the adeG gene was upregulated 4-6-fold in the presence of NE. In conclusion, NE augmented factors contributing to antibiotic resistance and markedly reduced the in vitro antibacterial activity of TIG against MDR-AB. These findings suggest that NE treatment may contribute to the failure of TIG therapy in patients with MDR-AB infections. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Effect of Micro- and Nanoscale Topography on the Adhesion of Bacterial Cells to Solid Surfaces

PubMed Central

Hsu, Lillian C.; Fang, Jean; Borca-Tasciuc, Diana A.; Worobo, Randy W.

2013-01-01

Attachment and biofilm formation by bacterial pathogens on surfaces in natural, industrial, and hospital settings lead to infections and illnesses and even death. Minimizing bacterial attachment to surfaces using controlled topography could reduce the spreading of pathogens and, thus, the incidence of illnesses and subsequent human and financial losses. In this context, the attachment of key microorganisms, including Escherichia coli, Listeria innocua, and Pseudomonas fluorescens, to silica and alumina surfaces with micron and nanoscale topography was investigated. The results suggest that orientation of the attached cells occurs preferentially such as to maximize their contact area with the surface. Moreover, the bacterial cells exhibited different morphologies, including different number and size of cellular appendages, depending on the topographical details of the surface to which they attached. This suggests that bacteria may utilize different mechanisms of attachment in response to surface topography. These results are important for the design of novel microbe-repellant materials. PMID:23416997

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.

PubMed

Shea, Patrick R; Virtaneva, Kimmo; Kupko, John J; Porcella, Stephen F; Barry, William T; Wright, Fred A; Kobayashi, Scott D; Carmody, Aaron; Ireland, Robin M; Sturdevant, Daniel E; Ricklefs, Stacy M; Babar, Imran; Johnson, Claire A; Graham, Morag R; Gardner, Donald J; Bailey, John R; Parnell, Michael J; Deleo, Frank R; Musser, James M

2010-03-09

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

Restraint stress alters neutrophil and macrophage phenotypes during wound healing

PubMed Central

Tymen, Stéphanie D.; Rojas, Isolde G.; Zhou, Xiaofeng; Fang, Zong Juan; Zhao, Yan; Marucha, Phillip T.

2013-01-01

Previous studies reported that stress delays wound healing, impairs bacterial clearance, and elevates the risk for opportunistic infection. Neutrophils and macrophages are responsible for the removal of bacteria present at the wound site. The appropriate recruitment and functions of these cells are necessary for efficient bacterial clearance. In our current study we found that restraint stress induced an excessive recruitment of neutrophils extending the inflammatory phase of healing, and the gene expression of neutrophil attracting chemokines MIP-2 and KC. However, restraint stress did not affect macrophage infiltration. Stress decreased the phagocytic abilities of phagocytic cells ex vivo, yet it did not affect superoxide production. The cell surface expression of adhesion molecules CD11b and TLR4 were decreased in peripheral blood monocytes in stressed mice. The phenotype of macrophages present at the wound site was also altered. Gene expression of markers of pro-inflammatory classically activated macrophages, CXCL10 and CCL5, were down-regulated; as were markers associated with wound healing macrophages, CCL22, IGF-1, RELMα; and the regulatory macrophage marker, chemokine CCL1. Restraint stress also induced up-regulation of IL10 gene expression. In summary, our study has shown that restraint stress suppresses the phenotype shift of the macrophage population, as compared to the changes observed during normal wound healing, while the number of macrophages remains constant. We also observed a general suppression of chemokine gene expression. Modulation of the macrophage phenotype could provide a new therapeutic approach in the treatment of wounds under stress conditions in the clinical setting. PMID:22884902

Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica.

PubMed

Lee, Soo Youn; Lee, Jiho; Lee, Hye Sun; Chang, Jeong Ho

2014-03-15

We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step. © 2013 Elsevier B.V. All rights reserved.

H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

PubMed Central

Salomon, Dor; Klimko, John A.

2014-01-01

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity. PMID:24987102

Decreased bacteria activity on Si3N4 surfaces compared with PEEK or titanium

PubMed Central

Gorth, Deborah J; Puckett, Sabrina; Ercan, Batur; Webster, Thomas J; Rahaman, Mohamed; Bal, B Sonny

2012-01-01

A significant need exists for orthopedic implants that can intrinsically resist bacterial colonization. In this study, three biomaterials that are used in spinal implants – titanium (Ti), polyether-ether-ketone (PEEK), and silicon nitride (Si3N4) – were tested to understand their respective susceptibility to bacterial infection with Staphylococcus epidermidis, Staphlococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus. Specifically, the surface chemistry, wettability, and nanostructured topography of respective biomaterials, and the effects on bacterial biofilm formation, colonization, and growth were investigated. Ti and PEEK were received with as-machined surfaces; both materials are hydrophobic, with net negative surface charges. Two surface finishes of Si3N4 were examined: as-fired and polished. In contrast to Ti and PEEK, the surface of Si3N4 is hydrophilic, with a net positive charge. A decreased biofilm formation was found, as well as fewer live bacteria on both the as-fired and polished Si3N4. These differences may reflect differential surface chemistry and surface nanostructure properties between the biomaterials tested. Because protein adsorption on material surfaces affects bacterial adhesion, the adsorption of fibronectin, vitronectin, and laminin on Ti, PEEK, and Si3N4 were also examined. Significantly greater amounts of these proteins adhered to Si3N4 than to Ti or PEEK. The findings of this study suggest that surface properties of biomaterials lead to differential adsorption of physiologic proteins, and that this phenomenon could explain the observed in-vitro differences in bacterial affinity for the respective biomaterials. Intrinsic biomaterial properties as they relate to resistance to bacterial colonization may reflect a novel strategy toward designing future orthopedic implants. PMID:22973102

Bacterial Communities of Surface Mixed Layer in the Pacific Sector of the Western Arctic Ocean during Sea-Ice Melting

PubMed Central

Ha, Ho Kyung; Kim, Hyun Cheol; Kim, Ok-Sun; Lee, Bang Yong; Cho, Jang-Cheon; Hur, Hor-Gil; Lee, Yoo Kyung

2014-01-01

From July to August 2010, the IBRV ARAON journeyed to the Pacific sector of the Arctic Ocean to monitor bacterial variation in Arctic summer surface-waters, and temperature, salinity, fluorescence, and nutrient concentrations were determined during the ice-melting season. Among the measured physicochemical parameters, we observed a strong negative correlation between temperature and salinity, and consequently hypothesized that the melting ice decreased water salinity. The bacterial community compositions of 15 samples, includicng seawater, sea-ice, and melting pond water, were determined using a pyrosequencing approach and were categorized into three habitats: (1) surface seawater, (2) ice core, and (3) melting pond. Analysis of these samples indicated the presence of local bacterial communities; a deduction that was further corroborated by the discovery of seawater- and ice-specific bacterial phylotypes. In all samples, the Alphaproteobacteria, Flavobacteria, and Gammaproteobacteria taxa composed the majority of the bacterial communities. Among these, Alphaproteobacteria was the most abundant and present in all samples, and its variation differed among the habitats studied. Linear regression analysis suggested that changes in salinity could affect the relative proportion of Alphaproteobacteria in the surface water. In addition, the species-sorting model was applied to evaluate the population dynamics and environmental heterogeneity in the bacterial communities of surface mixed layer in the Arctic Ocean during sea-ice melting. PMID:24497990

Bacterial communities of surface mixed layer in the Pacific sector of the western Arctic Ocean during sea-ice melting.

PubMed

Han, Dukki; Kang, Ilnam; Ha, Ho Kyung; Kim, Hyun Cheol; Kim, Ok-Sun; Lee, Bang Yong; Cho, Jang-Cheon; Hur, Hor-Gil; Lee, Yoo Kyung

2014-01-01

From July to August 2010, the IBRV ARAON journeyed to the Pacific sector of the Arctic Ocean to monitor bacterial variation in Arctic summer surface-waters, and temperature, salinity, fluorescence, and nutrient concentrations were determined during the ice-melting season. Among the measured physicochemical parameters, we observed a strong negative correlation between temperature and salinity, and consequently hypothesized that the melting ice decreased water salinity. The bacterial community compositions of 15 samples, includicng seawater, sea-ice, and melting pond water, were determined using a pyrosequencing approach and were categorized into three habitats: (1) surface seawater, (2) ice core, and (3) melting pond. Analysis of these samples indicated the presence of local bacterial communities; a deduction that was further corroborated by the discovery of seawater- and ice-specific bacterial phylotypes. In all samples, the Alphaproteobacteria, Flavobacteria, and Gammaproteobacteria taxa composed the majority of the bacterial communities. Among these, Alphaproteobacteria was the most abundant and present in all samples, and its variation differed among the habitats studied. Linear regression analysis suggested that changes in salinity could affect the relative proportion of Alphaproteobacteria in the surface water. In addition, the species-sorting model was applied to evaluate the population dynamics and environmental heterogeneity in the bacterial communities of surface mixed layer in the Arctic Ocean during sea-ice melting.

Molecular Insights Into a Dinoflagellate Bloom Imply Bacterial Cultivation

NASA Astrophysics Data System (ADS)

Gong, W.; Hall, N.; Schruth, D.; Paerl, H. W.; Marchetti, A.

2016-02-01

In coastal waters, an increase in frequency and intensity of algal blooms worldwide has recently been observed primarily due to eutrophication, with further increases predicted as a consequence of climate change. In many marine habitats most impacted by human activities, efforts have been made to prevent conditions that promote harmful algal blooms, or HABs, although progress is limited, due in part to our current lack of understanding of the environmental and cellular processes that promote and propagate these blooms. Comparative metatranscriptomics was used to investigate the underlying molecular mechanisms associated with a dinoflagellate bloom in a highly eutrophied estuarine system. Here we show that under bloom conditions, there is increased expression of metabolic pathways indicative of rapidly growing cells, including energy production, carbon metabolism, transporters and synthesis of nucleic acids and cellular membrane components. In addition, there is a prominence of highly expressed genes involved in synthesis of membrane-associated molecules, including those for the production of glycosaminoglycans (GAGs), which may serve roles in nutrient acquisition and/or cell surface adhesion. Biotin and thiamine synthesis genes also increased expression along with several cobalamin biosynthesis-associated genes that suggests processing of B12 intermediates by dinoflagellates. The patterns in gene expression observed are consistent with bloom-forming dinoflagellates eliciting a cellular response to facilitate interactions with their surrounding bacterial consortium, possibly in an effort to cultivate for enhancement of vitamin and nutrient exchanges and/or direct consumption. Our findings provide potential molecular targets for HAB detection and remediation efforts.

Influence of Silver-hydroxyapatite Nanocomposite Coating on Biofilm Formation of Joint Prosthesis and Its Mechanism.

PubMed

Zhao, L; Ashraf, M A

2015-12-01

The main reason for biomaterial related refractory infections is biofilm formation caused by bacterial adhesion on the surface of materials. Silver-hydroxyapatite (Ag/HA) nanocomposite coating can inhibit the formation of biofilm, but its mechanism is not clear. In order to clarify the mechanism, the amounts of biofilm on the Ag/HA composite coating and HA coating were determined, the release rates of silver nanoparticles in simulated body fluid (SBF) were detected by atomic absorption spectrometry, and the expression values of atlE , fbe , sap , iapB genes of Staphylococcus aureus were studied when they grew on Ag/HA composite coating and HA coating. The amount of the biofilm on the Ag/HA composite coating was significantly less than that on the HA coating, and the bacterial adhesion was decreased. The silver nanoparticles were released continuously in SBF and the release rate decreased gradually with time. The expression values of atlE , fbe and sap were high in the initial stage of adhesion and the expression value of iapB was high in the colonies-gathering stage in the control group, but they were all significantly inhibited in the presence of Ag. These results indicated that the main antibacterial effect of Ag/HA composite coating was achieved by the release of silver nanoparticles. The addition of Ag inhibited the expression of genes related to biofilm formation, which in turn inhibited the formation of biofilms. This provided theoretical support for the clinical application of Ag/HA composite coating.

Distinct antimicrobial peptide expression determines host species-specific bacterial associations

PubMed Central

Franzenburg, Sören; Walter, Jonas; Künzel, Sven; Wang, Jun; Baines, John F.; Bosch, Thomas C. G.; Fraune, Sebastian

2013-01-01

Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations. PMID:24003149

Forest floor community metatranscriptomes identify fungal and bacterial responses to N deposition in two maple forests

DOE PAGES

Hesse, Cedar N.; Mueller, Rebecca C.; Vuyisich, Momchilo; ...

2015-04-23

Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes)more » in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.« less

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

PubMed

Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

2016-02-04

Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Biogeographical distribution and diversity of bacterial communities in surface sediments of the South China Sea.

PubMed

Li, Tao; Wang, Peng

2013-05-01

This paper aims at an investigation of the features of bacterial communities in surface sediments of the South China Sea (SCS). In particular, biogeographical distribution patterns and the phylogenetic diversity of bacteria found in sediments collected from a coral reef platform, a continental slope, and a deep-sea basin were determined. Bacterial diversity was measured by an observation of 16S rRNA genes, and 18 phylogenetic groups were identified in the bacterial clone library. Planctomycetes, Deltaproteobacteria, candidate division OP11, and Alphaproteobacteria made up the majority of the bacteria in the samples, with their mean bacterial clones being 16%, 15%, 12%, and 9%, respectively. By comparison, the bacterial communities found in the SCS surface sediments were significantly different from other previously observed deep-sea bacterial communities. This research also emphasizes the fact that geographical factors have an impact on the biogeographical distribution patterns of bacterial communities. For instance, canonical correspondence analyses illustrated that the percentage of sand weight and water depth are important factors affecting the bacterial community composition. Therefore, this study highlights the importance of adequately determining the relationship between geographical factors and the distribution of bacteria in the world's seas and oceans.

The Use of Bacterial Adherence to Hydrocarbons (BATH) Assay in Evaluation of the Hydrophobic Surface Characteristics of Potential Water Pathogens

EPA Science Inventory

Bacterial adherence to hydrocarbons, BATH, is a method for determining the hydrophobic surface characteristics of bacterial cells. The strain’s affinity for water is evaluated by thoroughly mixing a culture and hydrocarbon suspension and then evaluating the decrease in optical de...

Different nitrogen sources change the transcriptome of welan gum-producing strain Sphingomonas sp. ATCC 31555.

PubMed

Xu, Xiaopeng; Nie, Zuoming; Zheng, Zhiyong; Zhu, Li; Zhang, Hongtao; Zhan, Xiaobei

2017-09-01

To reveal effects of different nitrogen sources on the expressions and functions of genes in Sphingomonas sp. ATCC 31555, it was cultivated in medium containing inorganic nitrogen (IN), organic nitrogen (ON), or inorganic-organic combined nitrogen (CN). Welan gum production and bacterial biomass were determined, and RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEGs) between the different ATCC 31555 groups were identified, and their functions were analyzed. Welan gum production and bacterial biomass were significantly higher in the ON and CN groups compared with those in the IN group. RNA-seq produced 660 unigenes, among which 488, 731, and 844 DEGs were identified between the IN vs. ON, IN vs. CN, and ON vs. CN groups, respectively. All the DEGs were related significantly to metabolic process and signal transduction. DEGs between the IN vs. CN and ON vs. CN groups were potentially associated with bacterial chemotaxis. Real-time PCR confirmed the expressions of selected DEGs. Organic nitrogen led to higher bacterial biomass and welan gum production than inorganic nitrogen, which might reflect differences in gene expression associated with metabolic process, signal transduction, and bacterial chemotaxis induced by different nitrogen sources.

A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria*

PubMed Central

Grin, Iwan; Hartmann, Marcus D.; Sauer, Guido; Hernandez Alvarez, Birte; Schütz, Monika; Wagner, Samuel; Madlung, Johannes; Macek, Boris; Felipe-Lopez, Alfonso; Hensel, Michael; Lupas, Andrei; Linke, Dirk

2014-01-01

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins. PMID:24369174

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

PubMed Central

Pirbadian, Sahand; Barchinger, Sarah E.; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Shi, Liang; Gorby, Yuri A.; Golbeck, John H.; El-Naggar, Mohamed Y.

2014-01-01

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. PMID:25143589

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

PubMed

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.

PubMed

Mushtaq, Naseem; Redpath, Maria B; Luzio, J Paul; Taylor, Peter W

2004-05-01

Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 micro g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium.

PubMed

Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

2015-01-01

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

PubMed Central

Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

2015-01-01

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa

PubMed Central

Evans, David J.; Fleiszig, Suzanne M. J.

2017-01-01

It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen. PMID:28489917

Actin-based motility propelled by molecular motors

NASA Astrophysics Data System (ADS)

Upadyayula, Sai Pramod; Rangarajan, Murali

2012-09-01

Actin-based motility of Listeria monocytogenes propelled by filament end-tracking molecular motors has been simulated. Such systems may act as potential nanoscale actuators and shuttles useful in sorting and sensing biomolecules. Filaments are modeled as three-dimensional elastic springs distributed on one end of the capsule and persistently attached to the motile bacterial surface through an end-tracking motor complex. Filament distribution is random, and monomer concentration decreases linearly as a function of position on the bacterial surface. Filament growth rate increases with monomer concentration but decreases with the extent of compression. The growing filaments exert push-pull forces on the bacterial surface. In addition to forces, torques arise due to two factors—distribution of motors on the bacterial surface, and coupling of torsion upon growth due to the right-handed helicity of F-actin—causing the motile object to undergo simultaneous translation and rotation. The trajectory of the bacterium is simulated by performing a force and torque balance on the bacterium. All simulations use a fixed value of torsion. Simulations show strong alignment of the filaments and the long axis of the bacterium along the direction of motion. In the absence of torsion, the bacterial surface essentially moves along the direction of the long axis. When a small amount of the torsion is applied to the bacterial surface, the bacterium is seen to move in right-handed helical trajectories, consistent with experimental observations.

Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.

PubMed

Liu, Mingyu; Blinn, Christina; McLeod, Sarah M; Wiseman, John W; Newman, Joseph V; Fisher, Stewart L; Walkup, Grant K

2014-01-01

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis.

PubMed

Chen, Yanhong; Oba, Masahito; Guan, Le Luo

2012-10-12

In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Enhancing the antibacterial performance of orthopaedic implant materials by fibre laser surface engineering

NASA Astrophysics Data System (ADS)

Chan, Chi-Wai; Carson, Louise; Smith, Graham C.; Morelli, Alessio; Lee, Seunghwan

2017-05-01

Implant failure caused by bacterial infection is extremely difficult to treat and usually requires the removal of the infected components. Despite the severe consequence of bacterial infection, research into bacterial infection of orthopaedic implants is still at an early stage compared to the effort on enhancing osseointegration, wear and corrosion resistance of implant materials. In this study, the effects of laser surface treatment on enhancing the antibacterial properties of commercially pure (CP) Ti (Grade 2), Ti6Al4V (Grade 5) and CoCrMo alloy implant materials were studied and compared for the first time. Laser surface treatment was performed by a continuous wave (CW) fibre laser with a near-infrared wavelength of 1064 nm in a nitrogen-containing environment. Staphylococcus aureus, commonly implicated in infection associated with orthopaedic implants, was used to investigate the antibacterial properties of the laser-treated surfaces. The surface roughness and topography of the laser-treated materials were analysed by a 2D roughness testing and by AFM. The surface morphologies before and after 24 h of bacterial cell culture were captured by SEM, and bacterial viability was determined using live/dead staining. Surface chemistry was analysed by XPS and surface wettability was measured using the sessile drop method. The findings of this study indicated that the laser-treated CP Ti and Ti6Al4V surfaces exhibited a noticeable reduction in bacterial adhesion and possessed a bactericidal effect. Such properties were attributable to the combined effects of reduced hydrophobicity, thicker and stable oxide films and presence of laser-induced nano-features. No similar antibacterial effect was observed in the laser-treated CoCrMo.

Colloidal crystal based plasma polymer patterning to control Pseudomonas aeruginosa attachment to surfaces.

PubMed

Pingle, Hitesh; Wang, Peng-Yuan; Thissen, Helmut; McArthur, Sally; Kingshott, Peter

2015-12-02

Biofilm formation on medical implants and subsequent infections are a global problem. A great deal of effort has focused on developing chemical contrasts based on micro- and nanopatterning for studying and controlling cells and bacteria at surfaces. It has been known that micro- and nanopatterns on surfaces can influence biomolecule adsorption, and subsequent cell and bacterial adhesion. However, less focus has been on precisely controlling patterns to study the initial bacterial attachment mechanisms and subsequently how the patterning influences the role played by biomolecular adsorption on biofilm formation. In this work, the authors have used colloidal self-assembly in a confined area to pattern surfaces with colloidal crystals and used them as masks during allylamine plasma polymer (AAMpp) deposition to generate highly ordered patterns from the micro- to the nanoscale. Polyethylene glycol (PEG)-aldehyde was grafted to the plasma regions via "cloud point" grafting to prevent the attachment of bacteria on the plasma patterned surface regions, thereby controlling the adhesive sites by choice of the colloidal crystal morphology. Pseudomonas aeruginosa was chosen to study the bacterial interactions with these chemically patterned surfaces. Scanning electron microscope, x-ray photoelectron spectroscopy (XPS), atomic force microscopy, and epifluorescence microscopy were used for pattern characterization, surface chemical analysis, and imaging of attached bacteria. The AAMpp influenced bacterial attachment because of the amine groups displaying a positive charge. XPS results confirm the successful grafting of PEG on the AAMpp surfaces. The results showed that PEG patterns can be used as a surface for bacterial patterning including investigating the role of biomolecular patterning on bacterial attachment. These types of patterns are easy to fabricate and could be useful in further applications in biomedical research.

Role of surface properties in bacterial attachment

NASA Astrophysics Data System (ADS)

Conrad, Jacinta; Sharma, Sumedha

2014-03-01

Bacterial biofilms foul a wide range of engineered surfaces, from pipelines to membranes to biomedical implants, and lead to deleterious costs for industry and for human health. Designing strategies to reduce bacterial fouling requires fundamental understanding of mechanisms by which bacteria attach to surfaces. We investigate the attachment of Escherichia coli on silanized glass surfaces during flow through a linear channel at flow rates of 0.1-1 mL/min using confocal microscopy. We deposit self-assembled monolayers of organosilanes on glass and track the position and orientation of bacteria deposited on these surfaces during flow using high-throughput image processing algorithms. Here, we report differences in deposition rate and surface-tethered motion of cells as a function of surface charge and surface energy, suggesting that attachment of bacteria on these engineered surfaces is dominated by different physical mechanisms.

Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer.

PubMed

Kristian, Sascha A; Hwang, John H; Hall, Bradley; Leire, Emma; Iacomini, John; Old, Robert; Galili, Uri; Roberts, Charles; Mullis, Kary B; Westby, Mike; Nizet, Victor

2015-06-01

The ever-increasing threat of multi-drug resistant bacterial infections has spurred renewed interest in alternative approaches to classical antibiotic therapy. In contrast to other mammals, humans do not express the galactose-α-1,3-galactosyl-β-1,4-N-acetyl-glucosamine (α-Gal) epitope. As a result of exposure of humans to α-Gal in the environment, a large proportion of circulating antibodies are specific for the trisaccharide. In this study, we examine whether these anti-Gal antibodies can be recruited and redirected to exert anti-bacterial activity. We show that a specific DNA aptamer conjugated to an α-Gal epitope at its 5' end, herein termed an alphamer, can bind to group A Streptococcus (GAS) bacteria by recognition of a conserved region of the surface-anchored M protein. The anti-GAS alphamer was shown to recruit anti-Gal antibodies to the streptococcal surface in an α-Gal-specific manner, elicit uptake and killing of the bacteria by human phagocytes, and slow growth of invasive GAS in human whole blood. These studies provide a first in vitro proof of concept that alphamers have the potential to redirect pre-existing antibodies to bacteria in a specific manner and trigger an immediate antibacterial immune response. Further validation of this novel therapeutic approach of applying α-Gal technology in in vivo models of bacterial infection is warranted. . α-Gal-tagged aptamers lead to GAS opsonization with anti-Gal antibodies. . α-Gal-tagged aptamers confer phagocytosis and killing of GAS cells by human phagocytes. . α-Gal-tagged aptamers reduces replication of GAS in human blood. . α-Gal-tagged aptamers may have the potential to be used as novel passive immunization drugs.

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

PubMed Central

Williamson, Yulanda M.; Moura, Hercules; Simmons, Kaneatra; Whitmon, Jennifer; Melnick, Nikkol; Rees, Jon; Woolfitt, Adrian; Schieltz, David M.; Tondella, Maria L.; Ades, Edwin; Sampson, Jacquelyn; Carlone, George; Barr, John R.

2017-01-01

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria. PMID:22537821

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

PubMed

Römling, Ute; Galperin, Michael Y

2015-09-01

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacillus subtilis MreB Orthologs Self-Organize into Filamentous Structures underneath the Cell Membrane in a Heterologous Cell System

PubMed Central

Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L.

2011-01-01

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface. PMID:22069484

Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture.

PubMed

Yang, S; Tamai, R; Akashi, S; Takeuchi, O; Akira, S; Sugawara, S; Takada, H

2001-04-01

An analog of 1alpha,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.

Influence of nanoscale topology on bactericidal efficiency of black silicon surfaces

NASA Astrophysics Data System (ADS)

Linklater, Denver P.; Khuong Duy Nguyen, Huu; Bhadra, Chris M.; Juodkazis, Saulius; Ivanova, Elena P.

2017-06-01

The nanostructuring of materials to create bactericidal and antibiofouling surfaces presents an exciting alternative to common methods of preventing bacterial adhesion. The fabrication of synthetic bactericidal surfaces has been inspired by the anti-wetting and anti-biofouling properties of insect wings, and other topologies found in nature. Black silicon is one such synthetic surfaces which has established bactericidal properties. In this study we show that time-dependent plasma etching of silicon wafers using 15, 30, and 45 min etching intervals, is able to produce different surface geometries with linearly increasing heights of approximately 280, 430, and 610 nm, respectively. After incubation on these surfaces with Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacterial cells it was established that smaller, more densely packed pillars exhibited the greatest bactericidal activity with 85% and 89% inactivation of bacterial cells, respectively. The decrease in the pillar heights, pillar cap diameter and inter-pillar spacing corresponded to a subsequent decrease in the number of attached cells for both bacterial species.

Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

PubMed

Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

2015-10-01

Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via CR1 and CR3 in nonimmune serum

PubMed Central

Schwartz, Justin T.; Barker, Jason H.; Long, Matthew E.; Kaufman, Justin; McCracken, Jenna; Allen, Lee-Ann H.

2012-01-01

A fundamental step in the life cycle of F. tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing F. tularensis strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. PMID:22888138

Impact of bacterial biocontrol agents on aflatoxin biosynthetic genes, aflD and aflR expression, and phenotypic aflatoxin B₁ production by Aspergillus flavus under different environmental and nutritional regimes.

PubMed

Al-Saad, Labeed A; Al-Badran, Adnan I; Al-Jumayli, Sami A; Magan, Naresh; Rodríguez, Alicia

2016-01-18

The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B1 (AFB1) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (aw) on nutrient medium; 0.995, 0.98 aw on maize meal agar medium) at 35°C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway (aflD and aflR respectively) and the production of AFB1. These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 aw on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 aw and some at 0.995 aw. However, the results for AFB1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB1 production on both nutrient and maize-based media regardless of aw. However, some bacterial treatments did inhibit AFB1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB1. Copyright © 2015 Elsevier B.V. All rights reserved.

Specific Immunotherapy of Experimental Myasthenia Gravis by A Novel Mechanism

PubMed Central

Luo, Jie; Kuryatov, Alexander; Lindstrom, Jon

2009-01-01

Objective Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are antibody-mediated autoimmune diseases, in which autoantibodies bind to and cause loss of muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. To develop a specific immunotherapy of MG, we treated rats with ongoing EAMG by intraperitoneal injection of bacterially-expressed human muscle AChR constructs. Methods Rats with ongoing EAMG received intraperitoneal treatment with the constructs weekly for 5 weeks beginning after the acute phase. Autoantibody concentration, subclassification, and specificity were analyzed to address underlying therapeutic mechanism. Results EAMG was specifically suppressed by diverting autoantibody production away from pathologically relevant specificities directed at epitopes on the extracellular surface of muscle AChRs toward pathologically irrelevant epitopes on the cytoplasmic domain. A mixture of subunit cytoplasmic domains was more effective than a mixture containing both extracellular and cytoplasmic domains or than only the extracellular domain of α1 subunits. Interpretation Therapy using only cytoplasmic domains, which lack pathologically relevant epitopes, avoids the potential liability of boosting the pathological response. Use of a mixture of bacterially-expressed human muscle AChR cytoplasmic domains for antigen-specific immunosuppression of myasthenia gravis has the potential to be specific, robust, and safe. PMID:20437579

Frontline Science: HIV infection of Kupffer cells results in an amplified proinflammatory response to LPS.

PubMed

Mosoian, Arevik; Zhang, Lumin; Hong, Feng; Cunyat, Francesc; Rahman, Adeeb; Bhalla, Riti; Panchal, Ankur; Saiman, Yedidya; Fiel, M Isabel; Florman, Sander; Roayaie, Sasan; Schwartz, Myron; Branch, Andrea; Stevenson, Mario; Bansal, Meena B

2017-05-01

End-stage liver disease is a common cause of non-AIDS-related mortality in HIV + patients, despite effective anti-retroviral therapies (ARTs). HIV-1 infection causes gut CD4 depletion and is thought to contribute to increased gut permeability, bacterial translocation, and immune activation. Microbial products drain from the gut into the liver via the portal vein where Kupffer cells (KCs), the resident liver macrophage, clear translocated microbial products. As bacterial translocation is implicated in fibrogenesis in HIV patients through unclear mechanisms, we tested the hypothesis that HIV infection of KCs alters their response to LPS in a TLR4-dependent manner. We showed that HIV-1 productively infected KCs, enhanced cell-surface TLR4 and CD14 expression, and increased IL-6 and TNF-α expression, which was blocked by a small molecule TLR4 inhibitor. Our study demonstrated that HIV infection sensitizes KCs to the proinflammatory effects of LPS in a TLR4-dependent manner. These findings suggest that HIV-1-infected KCs and their dysregulated innate immune response to LPS may play a role in hepatic inflammation and fibrosis and represent a novel target for therapy. © Society for Leukocyte Biology.

Direct analysis by time-of-flight secondary ion mass spectrometry reveals action of bacterial laccase-mediator systems on both hardwood and softwood samples.

PubMed

Goacher, Robyn E; Braham, Erick J; Michienzi, Courtney L; Flick, Robert M; Yakunin, Alexander F; Master, Emma R

2017-12-29

The modification and degradation of lignin play a vital role in carbon cycling as well as production of biofuels and bioproducts. The possibility of using bacterial laccases for the oxidation of lignin offers a route to utilize existing industrial protein expression techniques. However, bacterial laccases are most frequently studied on small model compounds that do not capture the complexity of lignocellulosic materials. This work studied the action of laccases from Bacillus subtilis and Salmonella typhimurium (EC 1.10.3.2) on ground wood samples from yellow birch (Betula alleghaniensis) and red spruce (Picea rubens). The ability of bacterial laccases to modify wood can be facilitated by small molecule mediators. Herein, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), gallic acid and sinapic acid mediators were tested. Direct analysis of the wood samples was achieved by time-of-flight secondary ion mass spectrometry (ToF-SIMS), a surface sensitive mass spectrometry technique that has characteristic peaks for H, G and S lignin. The action of the bacterial laccases on both wood samples was demonstrated and revealed a strong mediator influence. The ABTS mediator led to delignification, evident in an overall increase of polysaccharide peaks in the residual solid, along with equal loss of G and S-lignin peaks. The gallic acid mediator demonstrated minimal laccase activity. Meanwhile, the sinapic acid mediator altered the S/G peak ratio consistent with mediator attaching to the wood solids. The current investigation demonstrates the action of bacterial laccase-mediator systems directly on woody materials, and the potential of using ToF-SIMS to uncover the fundamental and applied role of bacterial enzymes in lignocellulose conversion. © 2017 Scandinavian Plant Physiology Society.

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles

PubMed Central

Caswell, Clayton C.; Oglesby-Sherrouse, Amanda G.; Murphy, Erin R.

2014-01-01

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators. PMID:25389522

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

PubMed

Caswell, Clayton C; Oglesby-Sherrouse, Amanda G; Murphy, Erin R

2014-01-01

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

Regulation of bacterial virulence by Csr (Rsm) systems.

PubMed

Vakulskas, Christopher A; Potts, Anastasia H; Babitzke, Paul; Ahmer, Brian M M; Romeo, Tony

2015-06-01

Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5' untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

PubMed Central

Vakulskas, Christopher A.; Potts, Anastasia H.; Babitzke, Paul; Ahmer, Brian M. M.

2015-01-01

SUMMARY Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5′ untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. PMID:25833324

Burkholderia Hep_Hap autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

PubMed Central

Tiyawisutsri, Rachaneeporn; Holden, Matthew TG; Tumapa, Sarinna; Rengpipat, Sirirat; Clarke, Simon R; Foster, Simon J; Nierman, William C; Day, Nicholas PJ; Peacock, Sharon J

2007-01-01

Background The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Results Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Conclusion Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study. PMID:17362501

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis.

PubMed

Tiyawisutsri, Rachaneeporn; Holden, Matthew T G; Tumapa, Sarinna; Rengpipat, Sirirat; Clarke, Simon R; Foster, Simon J; Nierman, William C; Day, Nicholas P J; Peacock, Sharon J

2007-03-15

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1.

PubMed

Zelmer, Andrea; Martin, Melissa J; Gundogdu, Ozan; Birchenough, George; Lever, Rebecca; Wren, Brendan W; Luzio, J Paul; Taylor, Peter W

2010-07-01

Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C-X-C and C-C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.

Tumour necrosis factor-alpha expression by activated monocytes and altered T-cell homeostasis in ascitic alcoholic cirrhosis: amelioration with norfloxacin.

PubMed

Albillos, Agustín; Hera Ad, Antonio de la; Reyes, Eduardo; Monserrat, Jorge; Muñoz, Leticia; Nieto, Mónica; Prieto, Alfredo; Sanz, Eva; Alvarez-Mon, Melchor

2004-04-01

To investigate the distribution and activation state of circulating monocytes and T-cell subsets, their contribution to tumour necrosis factor-alpha (TNFalpha) production, and their potential relationship with bacterial products of enteric origin in alcoholic cirrhosis. Peripheral blood monocytes and T-lymphocytes from 60 cirrhotic patients and 24 controls were characterized by four-color flow-cytometry after labelling of differentiation antigens and cytokines, before and after a 4-week course of norfloxacin or placebo. Monocytes from ascitic patients showed increased number, enhanced CD80 and HLA-DR surface levels, and spontaneous intracytoplasmic TNFalpha expression, when compared to non-ascitic patients and controls. Blood TNFalpha levels directly correlated with the amount of TNFalpha expressed by monocytes. In ascitic patients, there was a collapse of virgin CD4(+) and CD8(+) T-cell subsets; and, an expansion of activated CD4(+) T-cells. The above abnormalities were mainly restricted to ascitic patients with high serum levels of lypolysaccharide-binding-protein. Norfloxacin normalized the number of monocytes, reduced their activated phenotype and ability to produce TNFalpha and improved the abnormal T-cell homeostasis. In ascitic cirrhosis with high lipolysaccharide-binding-protein, monocytes are spontaneously activated to produce TNFalpha and are major contributors to the elevated serum TNFalpha. The T-cell compartment is profoundly depleted. Enteric bacterial products play a relevant role in these immune cellular abnormalities.

Floating and Tether-Coupled Adhesion of Bacteria to Hydrophobic and Hydrophilic Surfaces

PubMed Central

2018-01-01

Models for bacterial adhesion to substratum surfaces all include uncertainty with respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterial tethers that detach from and successively reattach to a surface, eventually making bacterial adhesion irreversible. Here, we use total internal reflection microscopy to determine whether adhering bacteria also exhibit variations over time in their perpendicular distance above surfaces. Streptococci with fibrillar surface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity. Adhering, nonfibrillated streptococci vibrated with amplitudes around 20 nm above a hydrophobic surface. Amplitudes did not depend on ionic strength for either strain. Calculations of bacterial energies from their distances above the surfaces using the Boltzman equation showed that bacteria with fibrillar tethers vibrated as a harmonic oscillator. The energy of bacteria without fibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey, and Overbeek)-interaction energy. Distance variations above the surface over time of bacteria with fibrillar tethers are suggested to be governed by the harmonic oscillations, allowed by elasticity of the tethers, piercing through the potential energy barrier. Bacteria without fibrillar tethers “float” above a surface in the secondary energy minimum, with their perpendicular displacement restricted by their thermal energy and the width of the secondary minimum. The distinction between “tether-coupled” and “floating” adhesion is new, and may have implications for bacterial detachment strategies. PMID:29649869

Antibacterial Au nanostructured surfaces

NASA Astrophysics Data System (ADS)

Wu, Songmei; Zuber, Flavia; Brugger, Juergen; Maniura-Weber, Katharina; Ren, Qun

2016-01-01

We present here a technological platform for engineering Au nanotopographies by templated electrodeposition on antibacterial surfaces. Three different types of nanostructures were fabricated: nanopillars, nanorings and nanonuggets. The nanopillars are the basic structures and are 50 nm in diameter and 100 nm in height. Particular arrangement of the nanopillars in various geometries formed nanorings and nanonuggets. Flat surfaces, rough substrate surfaces, and various nanostructured surfaces were compared for their abilities to attach and kill bacterial cells. Methicillin-resistant Staphylococcus aureus, a Gram-positive bacterial strain responsible for many infections in health care system, was used as the model bacterial strain. It was found that all the Au nanostructures, regardless their shapes, exhibited similar excellent antibacterial properties. A comparison of live cells attached to nanotopographic surfaces showed that the number of live S. aureus cells was <1% of that from flat and rough reference surfaces. Our micro/nanofabrication process is a scalable approach based on cost-efficient self-organization and provides potential for further developing functional surfaces to study the behavior of microbes on nanoscale topographies.We present here a technological platform for engineering Au nanotopographies by templated electrodeposition on antibacterial surfaces. Three different types of nanostructures were fabricated: nanopillars, nanorings and nanonuggets. The nanopillars are the basic structures and are 50 nm in diameter and 100 nm in height. Particular arrangement of the nanopillars in various geometries formed nanorings and nanonuggets. Flat surfaces, rough substrate surfaces, and various nanostructured surfaces were compared for their abilities to attach and kill bacterial cells. Methicillin-resistant Staphylococcus aureus, a Gram-positive bacterial strain responsible for many infections in health care system, was used as the model bacterial strain. It was found that all the Au nanostructures, regardless their shapes, exhibited similar excellent antibacterial properties. A comparison of live cells attached to nanotopographic surfaces showed that the number of live S. aureus cells was <1% of that from flat and rough reference surfaces. Our micro/nanofabrication process is a scalable approach based on cost-efficient self-organization and provides potential for further developing functional surfaces to study the behavior of microbes on nanoscale topographies. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06157a

The role of Proteus mirabilis cell wall features in biofilm formation.

PubMed

Czerwonka, Grzegorz; Guzy, Anna; Kałuża, Klaudia; Grosicka, Michalina; Dańczuk, Magdalena; Lechowicz, Łukasz; Gmiter, Dawid; Kowalczyk, Paweł; Kaca, Wiesław

2016-11-01

Biofilms formed by Proteus mirabilis strains are a serious medical problem, especially in the case of urinary tract infections. Early stages of biofilm formation, such as reversible and irreversible adhesion, are essential for bacteria to form biofilm and avoid eradication by antibiotic therapy. Adhesion to solid surfaces is a complex process where numerous factors play a role, where hydrophobic and electrostatic interactions with solid surface seem to be substantial. Cell surface hydrophobicity and electrokinetic potential of bacterial cells depend on their surface composition and structure, where lipopolysaccharide, in Gram-negative bacteria, is prevailing. Our studies focused on clinical and laboratory P. mirabilis strains, where laboratory strains have determined LPS structures. Adherence and biofilm formation tests revealed significant differences between strains adhered in early stages of biofilm formation. Amounts of formed biofilm were expressed by the absorption of crystal violet. Higher biofilm amounts were formed by the strains with more negative values of zeta potential. In contrast, high cell surface hydrophobicity correlated with low biofilm amount.

Elaboration of antibiofilm materials by chemical grafting of an antimicrobial peptide.

PubMed

Yala, Jean-Fabrice; Thebault, Pascal; Héquet, Arnaud; Humblot, Vincent; Pradier, Claire-Marie; Berjeaud, Jean-Marc

2011-02-01

A peptide antibiotic, gramicidin A, was covalently bound to cystamine self-assembled monolayers on gold surfaces. Each step of the surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. The antimicrobial activity of the anchored gramicidin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis, and Staphylococcus aureus), the Gram-negative bacterium Escherichia coli and the yeast Candida albicans. The results revealed that the adsorbed gramicidin reduced, from 60% for E. coli to 90% for C. albicans, the number of culturable microorganisms attached to the surface. The activity was proven to be persistent overtime, up to 6 months after the first use. The bacteria attached to the functionalized surfaces were permeabilized as shown by confocal microscopy. Taken together, these results indicate a bacteriostatic mode of action of the immobilized peptide. Finally, using green fluorescent protein-expressing bacteria, it was shown that the development of a bacterial biofilm was delayed on peptide-grafted surfaces for at least 24 h.

Messing with Bacterial Quorum Sensing

PubMed Central

González, Juan E.; Keshavan, Neela D.

2006-01-01

Quorum sensing is widely recognized as an efficient mechanism to regulate expression of specific genes responsible for communal behavior in bacteria. Several bacterial phenotypes essential for the successful establishment of symbiotic, pathogenic, or commensal relationships with eukaryotic hosts, including motility, exopolysaccharide production, biofilm formation, and toxin production, are often regulated by quorum sensing. Interestingly, eukaryotes produce quorum-sensing-interfering (QSI) compounds that have a positive or negative influence on the bacterial signaling network. This eukaryotic interference could result in further fine-tuning of bacterial quorum sensing. Furthermore, recent work involving the synthesis of structural homologs to the various quorum-sensing signal molecules has resulted in the development of additional QSI compounds that could be used to control pathogenic bacteria. The creation of transgenic plants that express bacterial quorum-sensing genes is yet another strategy to interfere with bacterial behavior. Further investigation on the manipulation of quorum-sensing systems could provide us with powerful tools against harmful bacteria. PMID:17158701

Bacterial diversity of Grenache and Carignan grape surface from different vineyards at Priorat wine region (Catalonia, Spain).

PubMed

Portillo, Maria del Carmen; Franquès, Judit; Araque, Isabel; Reguant, Cristina; Bordons, Albert

2016-02-16

Epiphytic bacteria on grape berries play a critical role in grape health and quality, which decisively influence the winemaking process. Despite their importance, the bacteria related with grape berry surface remain understudied and most previous work has been based on culture-dependent methods, which offer a limited view of the actual diversity. Herein, we used high-throughput sequencing to investigate the bacterial diversity on the surface from two grape varieties, Grenache and Carignan, and compared them across five vineyards included within the Priorat region (Spain). We could detect up to 14 bacterial phyla with Firmicutes (37.6% Bacillales and 14% Lactobacillales), Proteobacteria (16.8% Pseudomonadales and 11.6% Enterobacteriales) and Actinobacteria (3.4% Actinomycetales) being the most abundant. Bacterial community was different at each vineyard being grape varietal, geographical situation and orientation related with changes in bacterial populations. The most abundant bacterial taxa and those driving differences between the vineyards and grape varietals were identified. This study indicates that bacterial community heterogeneities can be influenced by geographic factors like orientation. Copyright © 2015 Elsevier B.V. All rights reserved.

Preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid-induced colitis and bacterial translocation in mice.

PubMed

Zhao, Yuan; Zhang, Shuncai; Jiang, Li; Jiang, Jie; Liu, Hongchun

2009-11-01

To evaluate the preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid (TNBS)-induced colitis and bacterial translocation in mice. BALB/c mice were randomly divided into three groups: control group; TNBS(+)Ova(-) group; and TNBS(+)Ova(+) group. Mice of the TNBS(+)Ova(+) group were exposed to 10 000 freeze-killed S. japonicum ova by i.p. injection on day 1 and day 11. On day 15, mice were challenged with TNBS to induce colitis. The following variables were assessed: colon pathological changes; serum expression of tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and interleukin-10 (IL-10); expression of Toll-like receptor 4 (TLR4) in colon; IFN-gamma, IL-10 and TLR4 mRNA expression in colon; and the bacterial translocation rate. Compared to TNBS(+)Ova(-) group, the colonic inflammation in the TNBS(+)Ova(+) group were relieved. A highly significant elevation of IFN-gamma and TNF-alpha were observed in the TNBS-induced colitis group. After exposure to the eggs, IFN-gamma was significantly decreased, while TNF-alpha was similar to that of the TNBS(+)ova(-) group. No obvious variation was seen in IL-10 expression in TNBS-induced colitis, compared to the controls. Exposure to the eggs led to a significant upregulation of IL-10 expression. TLR4 expression was elevated after injected with TNBS and was downregulated in the eggs group. Less intestinal bacterial translocation frequency was observed when exposed to eggs. S. japonicum ova can prevent the TNBS-induced colitis and reduce the bacterial translocation frequency in mice. The mechanisms were supposed to be due to the regulation of T-helper cell 1/2 balance and TLR4 expression.

Bacterial expression of self-assembling peptide hydrogelators

NASA Astrophysics Data System (ADS)

Sonmez, Cem

For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to chemical synthesis.

The squid-Vibrio symbioses: from demes to genes.

PubMed

Kimbell, Jennifer R; McFall-Ngai, Margaret J

2003-04-01

The monospecific light organ association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri has been used as a model for the study of the most common type of coevolved animal-bacterial interaction; i.e., the association of Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. Analysis of the squid-vibrio symbiosis has ranged from characterizations of the harvesting mechanisms by which the host ensures colonization by the appropriate symbiont to identification of bacteria-induced changes in host gene expression that accompany the establishment and maintenance of the relationship. Studies of this model have been enhanced by extensive collaboration with microbiologists, who are able to manipulate the genetics of the bacterial symbiont. The results of our studies have indicated that initiation and persistence of the association requires a complex, reciprocal molecular dialogue between these two phylogenetically distant partners.

Nucleation of platelets with bloodborne pathogens on Kupffer cell precedes other innate immunity and contributes to bacterial clearance

PubMed Central

Wong, Connie H. Y.; Jenne, Craig N.; Petri, Björn; Chrobok, Navina L.; Kubes, Paul

2016-01-01

Using intravital imaging of the liver, we unveil a collaborative role for platelets with Kupffer cells (KCs) in eradicating bloodborne bacterial infections. Under basal conditions, platelets via glycoprotein Ib (GPIb) formed transient “touch-and-go” interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria, such as Bacillus cereus and Methicillin-resistant Staphylococcus aureus (MRSA), were rapidly caught by KCs and triggered platelets to switch from “touch-and-go” to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GpIbα−/− mice demonstrated increased endothelial and KC damage, leading to increased fluid leakage, significant polycythemia and rapid mortality. This study identifies a novel surveillance mechanism of intravascular macrophage by platelets that rapidly converts to a critical host response against bloodborne bacteria. PMID:23770641

Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

PubMed

Wong, Connie H Y; Jenne, Craig N; Petri, Björn; Chrobok, Navina L; Kubes, Paul

2013-08-01

Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

NASA Astrophysics Data System (ADS)

Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

2015-08-01

Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

Bacterial and cytokine mixtures predict the length of gestation and are associated with miRNA expression in the cervix.

PubMed

Sanders, Alison P; Gennings, Chris; Svensson, Katherine; Motta, Valeria; Mercado-Garcia, Adriana; Solano, Maritsa; Baccarelli, Andrea A; Tellez-Rojo, Martha M; Wright, Robert O; Burris, Heather H

2017-01-01

Bacterial vaginosis may lead to preterm birth through epigenetic programming of the inflammatory response, specifically via miRNA expression. We quantified bacterial 16S rRNA, cytokine mRNA and 800 miRNA from cervical swabs obtained from 80 women at 16-19 weeks' gestation. We generated bacterial and cytokine indices using weighted quantile sum regression and examined associations with miRNA and gestational age at delivery. Each decile of the bacterial and cytokine indices was associated with shorter gestations (p < 0.005). The bacterial index was associated with miR-494, 371a, 4286, 185, 320e, 888 and 23a (p < 0.05). miR-494 remained significant after false discovery rate correction (q < 0.1). The cytokine index was associated with 27 miRNAs (p < 0.05; q < 0.01). Future investigation into the role of bacterial vaginosis- and inflammation-associated miRNA and preterm birth is warranted.

Protein antigen in serotype k Streptococcus mutans clinical isolates.

PubMed

Nakano, K; Nomura, R; Nemoto, H; Lapirattanakul, J; Taniguchi, N; Grönroos, L; Alaluusua, S; Ooshima, T

2008-10-01

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium–host interactions

PubMed Central

Turroni, Francesca; Serafini, Fausta; Foroni, Elena; Duranti, Sabrina; O’Connell Motherway, Mary; Taverniti, Valentina; Mangifesta, Marta; Milani, Christian; Viappiani, Alice; Roversi, Tommaso; Sánchez, Borja; Santoni, Andrea; Gioiosa, Laura; Ferrarini, Alberto; Delledonne, Massimo; Margolles, Abelardo; Piazza, Laura; Palanza, Paola; Bolchi, Angelo; Guglielmetti, Simone; van Sinderen, Douwe; Ventura, Marco

2013-01-01

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity. PMID:23776216

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

PubMed Central

Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

2003-01-01

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

In Vitro Investigation of the Effect of Oral Bacteria in the Surface Oxidation of Dental Implants.

PubMed

Sridhar, Sathyanarayanan; Wilson, Thomas G; Palmer, Kelli L; Valderrama, Pilar; Mathew, Mathew T; Prasad, Shalini; Jacobs, Michael; Gindri, Izabelle M; Rodrigues, Danieli C

2015-10-01

Bacteria are major contributors to the rising number of dental implant failures. Inflammation secondary to bacterial colonization and bacterial biofilm is a major etiological factor associated with early and late implant failure (peri-implantitis). Even though there is a strong association between bacteria and bacterial biofilm and failure of dental implants, their effect on the surface of implants is yet not clear. To develop and establish an in vitro testing methodology to investigate the effect of early planktonic bacterial colonization on the surface of dental implants for a period of 60 days. Commercial dental implants were immersed in bacterial (Streptococcus mutans in brain-heart infusion broth) and control (broth only) media. Immersion testing was performed for a period of 60 days. During testing, optical density and pH of immersion media were monitored. The implant surface was surveyed with different microscopy techniques post-immersion. Metal ion release in solution was detected with an electrochemical impedance spectroscopy sensor platform called metal ion electrochemical biosensor (MIEB). Bacteria grew in the implant-containing medium and provided a sustained acidic environment. Implants immersed in bacterial culture displayed various corrosion features, including surface discoloration, deformation of rough and smooth interfaces, pitting attack, and severe surface rusting. The surface features were confirmed by microscopic techniques, and metal particle generation was detected by the MIEB. Implant surface oxidation occurred in bacteria-containing medium even at early stages of immersion (2 days). The incremental corrosion resulted in dissolution of metal ions and debris into the testing solution. Dissolution of metal ions and particles in the oral environment can trigger or contribute to the development of peri-implantitis at later stages. © 2015 Wiley Periodicals, Inc.

Molecular bacterial community analysis of clean rooms where spacecraft are assembled.

PubMed

Moissl, Christine; Osman, Shariff; La Duc, Myron T; Dekas, Anne; Brodie, Eoin; DeSantis, Todd; Desantis, Tadd; Venkateswaran, Kasthuri

2007-09-01

Molecular bacterial community composition was characterized from three geographically distinct spacecraft-associated clean rooms to determine whether such populations are influenced by the surrounding environment or the maintenance of the clean rooms. Samples were collected from facilities at the Jet Propulsion Laboratory (JPL), Kennedy Space Flight Center (KSC), and Johnson Space Center (JSC). Nine clone libraries representing different surfaces within the spacecraft facilities and three libraries from the surrounding air were created. Despite the highly desiccated, nutrient-bare conditions within these clean rooms, a broad diversity of bacteria was detected, covering all the main bacterial phyla. Furthermore, the bacterial communities were significantly different from each other, revealing only a small subset of microorganisms common to all locations (e.g. Sphingomonas, Staphylococcus). Samples from JSC assembly room surfaces showed the greatest diversity of bacteria, particularly within the Alpha- and Gammaproteobacteria and Actinobacteria. The bacterial community structure of KSC assembly surfaces revealed a high presence of proteobacterial groups, whereas the surface samples collected from the JPL assembly facility showed a predominance of Firmicutes. Our study presents the first extended molecular survey and comparison of NASA spacecraft assembly facilities, and provides new insights into the bacterial diversity of clean room environments .

Bacterial adherence to graft tissues in static and flow conditions.

PubMed

Veloso, Tiago Rafael; Claes, Jorien; Van Kerckhoven, Soetkin; Ditkowski, Bartosz; Hurtado-Aguilar, Luis G; Jockenhoevel, Stefan; Mela, Petra; Jashari, Ramadan; Gewillig, Marc; Hoylaerts, Marc F; Meyns, Bart; Heying, Ruth

2018-01-01

Various conduits and stent-mounted valves are used as pulmonary valve graft tissues for right ventricular outflow tract reconstruction with good hemodynamic results. Valve replacement carries an increased risk of infective endocarditis (IE). Recent observations have increased awareness of the risk of IE after transcatheter implantation of a stent-mounted bovine jugular vein valve. This study focused on the susceptibility of graft tissue surfaces to bacterial adherence as a potential risk factor for subsequent IE. Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguinis to bovine pericardium (BP) patch, bovine jugular vein (BJV), and cryopreserved homograft (CH) tissues was quantified under static and shear stress conditions. Microscopic analysis and histology were performed to evaluate bacterial adhesion to matrix components. In general, similar bacteria numbers were recovered from CH and BJV tissue surfaces for all strains, especially in flow conditions. Static bacterial adhesion to the CH wall was lower for S sanguinis adhesion (P < .05 vs BP patch). Adhesion to the BJV wall, CH wall, and leaflet was decreased for S epidermidis in static conditions (P < .05 vs BP patch). Bacterial adhesion under shear stress indicated similar bacterial adhesion to all tissues, except for lower adhesion to the BJV wall after S sanguinis incubation. Microscopic analysis showed the importance of matrix component exposure for bacterial adherence to CH. Our data provide evidence that the surface composition of BJV and CH tissues themselves, bacterial surface proteins, and shear forces per se are not the prime determinants of bacterial adherence. Copyright © 2017 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

A Tad pilus promotes the establishment and resistance of Vibrio vulnificus biofilms to mechanical clearance.

PubMed

Pu, Meng; Rowe-Magnus, Dean Allistair

2018-01-01

Vibrio vulnificus is autochthonous to estuaries and warm coastal waters. Infection occurs via open wounds or ingestion, where its asymptomatic colonization of seafood, most infamously oysters, provides a gateway into the human food chain. Colonization begins with initial surface contact, which is often mediated by bacterial surface appendages called pili. Type IV Tad pili are widely distributed in the Vibrionaceae, but evidence for a physiological role for these structures is scant. The V. vulnificus genome codes for three distinct tad loci. Recently, a positive correlation was demonstrated between the expression of tad-3 and the phenotypes of a V. vulnificus descendent (NT) that exhibited increased biofilm formation, auto-aggregation, and oyster colonization relative to its parent. However, the mechanism by which tad pilus expression promoted these phenotypes was not determined. Here, we show that deletion of the tad pilin gene ( flp ) altered the near-surface motility profile of NT cells from high curvature, orbital retracing patterns characteristic of cells actively probing the surface to low curvature traces indicative of wandering and diminished bacteria-surface interactions. The NT flp pilin mutant also exhibited decreased initial surface attachment, attenuated auto-aggregation and formed fragile biofilms that disintegrated under hydrodynamic flow. Thus, the tad-3 locus, designated iam , promoted i nitial surface attachment, a uto-aggregation and resistance to m echanical clearance of V. vulnificus biofilms. The prevalence of tad loci in the Vibrionaceae suggests that they may play equally important roles in other family members.

Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

PubMed

Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

2016-02-29

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

PubMed Central

Bennett, Kaila M.; Walker, Sharon L.

2014-01-01

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli (“microvillus-minus,” or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions. PMID:24778113

Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi

PubMed Central

Deng, Lingquan; Song, Jeongmin; Gao, Xiang; Wang, Jiawei; Yu, Hai; Chen, Xi; Varki, Nissi; Naito-Matsui, Yuko; Galán, Jorge E.; Varki, Ajit

2014-01-01

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here we report that typhoid toxin binds to and is toxic towards cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever. PMID:25480294

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

PubMed

Vostiar, Igor; Tkac, Jan; Mandenius, Carl-Fredrik

2004-07-15

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

PubMed

Figueira, Cláudio Pereira; Croda, Julio; Choy, Henry A; Haake, David A; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

2011-06-09

In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Immobilization of Ag nanoparticles/FGF-2 on a modified titanium implant surface and improved human gingival fibroblasts behavior.

PubMed

Ma, Qianli; Mei, Shenglin; Ji, Kun; Zhang, Yumei; Chu, Paul K

2011-08-01

The objective of this study was to form a rapid and firm soft tissue sealing around dental implants that resists bacterial invasion. We present a novel approach to modify Ti surface by immobilizing Ag nanoparticles/FGF-2 compound bioactive factors onto a titania nanotubular surface. The titanium samples were anodized to form vertically organized TiO(2) nanotube arrays and Ag nanoparticles were electrodeposited onto the nanotubular surface, on which FGF-2 was immobilized with repeated lyophilization. A uniform distribution of Ag nanoparticles/FGF-2 was observed on the TiO(2) nanotubular surface. The L929 cell line was used for cytotoxicity assessment. Human gingival fibroblasts (HGFs) were cultured on the modified surface for cytocompatibility determination. The Ag/FGF-2 immobilized samples displayed excellent cytocompatibility, negligible cytotoxicity, and enhanced HGF functions such as cell attachment, proliferation, and ECM-related gene expression. The Ag nanoparticles also exhibit some bioactivity. In conclusion, this modified TiO(2) nanotubular surface has a large potential for use in dental implant abutment. Copyright © 2011 Wiley Periodicals, Inc.

Effect of Surface Properties on Colloid Retention on Natural and Surrogate Produce Surfaces.

PubMed

Lazouskaya, Volha; Sun, Taozhu; Liu, Li; Wang, Gang; Jin, Yan

2016-12-01

Bacterial contamination of fresh produce is a growing concern in food industry. Pathogenic bacteria can attach to and colonize the surfaces of fresh produce and cause disease outbreaks among consumers. Surface properties of both bacteria and produce affect bacterial contamination; however, the effects of produce roughness, topography, and hydrophobicity on bacterial retention are still poorly understood. In this work, we used spherical polystyrene colloids as bacterial surrogates to investigate colloid retention on and removal (by rinsing) from fresh produce surfaces including tomato, orange, apple, lettuce, spinach, and cantaloupe, and from surrogate produce surface Sharklet (a micro-patterned polymer). All investigated surfaces were characterized in terms of surface roughness and hydrophobicity (including contact angle and water retention area measurements). The results showed that there was no single parameter that dominated colloid retention on fresh produce, yet strong connection was found between colloid retention and water retention and distribution on all the surfaces investigated except apple. Rinsing was generally not efficient in removing colloids from produce surfaces, which suggests the need to modify current cleaning procedures and to develop novel contamination prevention strategies. This work offers a physicochemical approach to a food safety problem and improves understanding of mechanisms leading to produce contamination. © 2016 Institute of Food Technologists®.

The Biochemistry and Physiology of Bacterial Adhesion to Surfaces

DTIC Science & Technology

1984-01-20

Organism S was isolated from surfaces incubated 33258 (Calbiochem-Behring Corp.. La Jolla, Calif.) in in an aquarium containing Instant Ocean...Abstiact /The physiologic mechanisms involved in bacterial adhesion to inert surfaces have been Investigated employing fouling isolates obtained from...of Madilyn Fletcher. Environmental Sci- A n l ms ences Department. University of Warwick. Coventry. All organisms isolated from surfaces exposed

Bacterial responses to antibiotics and their combinations.

PubMed

Mitosch, Karin; Bollenbach, Tobias

2014-12-01

Antibiotics affect bacterial cell physiology at many levels. Rather than just compensating for the direct cellular defects caused by the drug, bacteria respond to antibiotics by changing their morphology, macromolecular composition, metabolism, gene expression and possibly even their mutation rate. Inevitably, these processes affect each other, resulting in a complex response with changes in the expression of numerous genes. Genome-wide approaches can thus help in gaining a comprehensive understanding of bacterial responses to antibiotics. In addition, a combination of experimental and theoretical approaches is needed for identifying general principles that underlie these responses. Here, we review recent progress in our understanding of bacterial responses to antibiotics and their combinations, focusing on effects at the levels of growth rate and gene expression. We concentrate on studies performed in controlled laboratory conditions, which combine promising experimental techniques with quantitative data analysis and mathematical modeling. While these basic research approaches are not immediately applicable in the clinic, uncovering the principles and mechanisms underlying bacterial responses to antibiotics may, in the long term, contribute to the development of new treatment strategies to cope with and prevent the rise of resistant pathogenic bacteria.

Quorum Quenching: Enzymatic Disruption of N-Acylhomoserine Lactone-Mediated Bacterial Communication in Burkholderia thailandensis

DTIC Science & Technology

2004-10-01

lactonases failed to enhance beta-hemolytic activity. The results of this study demonstrate that heterologous expression of Bacillus sp . AiiA lactonases in...results of this study demonstrate that heterologous expression of Bacillus sp . AiiA lactonases in B. thailandensis reduced AHL accumulation, affected both...hemo- lysis, and carbon utilization by the expression of Bacillus sp . AiiA lactonases in B. thailandensis. MATERIALS AND METHODS Bacterial strains and

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

PubMed

Bachert, Beth A; Choi, Soo J; LaSala, Paul R; Harper, Tiffany I; McNitt, Dudley H; Boehm, Dylan T; Caswell, Clayton C; Ciborowski, Pawel; Keene, Douglas R; Flores, Anthony R; Musser, James M; Squeglia, Flavia; Marasco, Daniela; Berisio, Rita; Lukomski, Slawomir

2016-01-01

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

Comparative Transcriptome Analysis of Desulfovibrio Vulgaris Grown in Planktonic Culture and Mature Biofilm on a Steel Surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Weiwen; Culley, David E.; Nie, Lei

2007-08-01

The build-up of biofilms of sulphate -reducing bacteria (SRB) on metals surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of model SRB species Desulfovibrio vulgaris. Statistical analysis revealed that 472 genes were differentially expressed (1.5 fold or more with a p value less than 0.025) when comparing biofilm to planktonic cells. Among the differentially expressed genes were several that corresponded to biofilm formation genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas speciesmore » and Escherichia coli), such as down-regulation of genes encoding flagellin, flagellar motor switch protein and chemotaxis proteins involved in cell motility and induction of genes encoding sugar transferase and glycogen synthase involved in exopolysaccharide biosynthesis. In addition, D. vulgaris biofilm-bound cells exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of biofilm cells were similar to planktonic cells of stationary phases. Most notably, up-regulation of large number of outer membrane proteins was observed in D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in D. vulgaris biofilm could implicate important roles formation and maintenance of multi-cellular consortium on metal surface. The study provided insights into the metabolic networks associated with D. vulgaris biofilm formation and maintenance on an iron surface.« less

Human Tear Fluid Reduces Culturability of Contact Lens Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence Associated Type III Secretion System

PubMed Central

Wu, Yvonne T.; Tam, Connie; Zhu, Lucia S.; Evans, David J.; Fleiszig, Suzanne M. J.

2017-01-01

Purpose The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens (CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. Methods P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. Results With or without tear fluid, biofilms grew to ~108 cfu viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ~180-fold (p<.001). CL growth increased T3SS gene expression versus planktonic bacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (p=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS (p=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (p = 0.04) and 1.89 ± 0.26-fold (p<.001), respectively. Conclusions Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis. PMID:27670247

Lactobacillus rhamnosus CNCMI-4317 Modulates Fiaf/Angptl4 in Intestinal Epithelial Cells and Circulating Level in Mice

PubMed Central

Jacouton, Elsa; Mach, Núria; Cadiou, Julie; Lapaque, Nicolas; Clément, Karine; Doré, Joël; van Hylckama Vlieg, Johan E. T.; Smokvina, Tamara; Blottière, Hervé M

2015-01-01

Background and Objectives Identification of new targets for metabolic diseases treatment or prevention is required. In this context, FIAF/ANGPTL4 appears as a crucial regulator of energy homeostasis. Lactobacilli are often considered to display beneficial effect for their hosts, acting on different regulatory pathways. The aim of the present work was to study the effect of several lactobacilli strains on Fiaf gene expression in human intestinal epithelial cells (IECs) and on mice tissues to decipher the underlying mechanisms. Subjects and Methods Nineteen lactobacilli strains have been tested on HT–29 human intestinal epithelial cells for their ability to regulate Fiaf gene expression by RT-qPCR. In order to determine regulated pathways, we analysed the whole genome transcriptome of IECs. We then validated in vivo bacterial effects using C57BL/6 mono-colonized mice fed with normal chow. Results We identified one strain (Lactobacillus rhamnosus CNCMI–4317) that modulated Fiaf expression in IECs. This regulation relied potentially on bacterial surface-exposed molecules and seemed to be PPAR-γ independent but PPAR-α dependent. Transcriptome functional analysis revealed that multiple pathways including cellular function and maintenance, lymphoid tissue structure and development, as well as lipid metabolism were regulated by this strain. The regulation of immune system and lipid and carbohydrate metabolism was also confirmed by overrepresentation of Gene Ontology terms analysis. In vivo, circulating FIAF protein was increased by the strain but this phenomenon was not correlated with modulation Fiaf expression in tissues (except a trend in distal small intestine). Conclusion We showed that Lactobacillus rhamnosus CNCMI–4317 induced Fiaf expression in human IECs, and increased circulating FIAF protein level in mice. Moreover, this effect was accompanied by transcriptome modulation of several pathways including immune response and metabolism in vitro. PMID:26439630

Comparison of surface roughness and bacterial adhesion between cosmetic contact lenses and conventional contact lenses.

PubMed

Ji, Yong Woo; Cho, Young Joo; Lee, Chul Hee; Hong, Soon Ho; Chung, Dong Yong; Kim, Eung Kweon; Lee, Hyung Keun

2015-01-01

To compare physical characteristics of cosmetic contact lenses (Cos-CLs) and conventional contact lenses (Con-CLs) that might affect susceptibility to bacterial adhesion on the contact lens (CL) surface. Surface characteristics of Cos-CLs and Con-CLs made from the same material by the same manufacturer were measured by atomic force microscopy (AFM) and scanning electron microscopy. To determine the extent and rate of bacterial adhesion, Cos-CL and Con-CL were immersed in serum-free Roswell Park Memorial Institute media containing Staphylococcus aureus or Pseudomonas aeruginosa. Additionally, the rate of removal of adherent bacteria was evaluated using hand rubbing or immersion in multipurpose disinfecting solutions (MPDS). The mean surface roughness (root mean square and peak-to-valley value) measured by AFM was significantly higher for Cos-CL than for Con-CL. At each time point, significantly more S. aureus and P. aeruginosa adhered to Cos-CL than to Con-CL, which correlated with the surface roughness of CL. In Cos-CL, bacteria were mainly found on the tinted surface rather than on the noncolored or convex areas. Pseudomonas aeruginosa attached earlier than S. aureus to all types of CL. However, P. aeruginosa was more easily removed from the surface of CL than S. aureus by hand rubbing or MPDS soaking. Increased surface roughness is an important physical factor for bacterial adhesion in Cos-CL, which may explain why rates of bacterial keratitis rates are higher in Cos-CL users in CL physical characteristics.

Effect of cultivation medium on some physicochemical parameters of outer bacterial membrane.

PubMed

Horská, E; Pokorný, J; Labajová, M

1995-01-01

The changes of surface charge and hydrophobicity of the outer bacterial membrane in relation to utilization of n-hexadecane were studied. For this spectrophotometric study adsorption of methylene blue and transport of gentian violet were used. The decrease in the negative charge of the bacterial strains Pseudomonas putida CCM 3423, P. aeruginosa, and P. fluorescens CCM 2115, depended on the type of growth medium. The decrease of surface charge was in the order: meat extract peptone broth > mineral medium with glucose > mineral medium with n-hexadecane. The highest permeability of the bacterial membrane for gentian violet was determined in the case of P. fluorescens grown in meat extract peptone broth. This effect can be explained by a greater hydrophobicity of the bacterial surface for this strain. In other strains a lower permeability was observed. P. fluorescens showed a greater adherence to hexadecane.

Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages.

PubMed

Beatty, W L; Russell, D G

2000-12-01

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Sonication reduces the attachment of Salmonella Typhimurium ATCC 14028 cells to bacterial cellulose-based plant cell wall models and cut plant material.

PubMed

Tan, Michelle S F; Rahman, Sadequr; Dykes, Gary A

2017-04-01

This study investigated the removal of bacterial surface structures, particularly flagella, using sonication, and examined its effect on the attachment of Salmonella Typhimurium ATCC 14028 cells to plant cell walls. S. Typhimurium ATCC 14028 cells were subjected to sonication at 20 kHz to remove surface structures without affecting cell viability. Effective removal of flagella was determined by staining flagella of sonicated cells with Ryu's stain and enumerating the flagella remaining by direct microscopic counting. The attachment of sonicated S. Typhimurium cells to bacterial cellulose-based plant cell wall models and cut plant material (potato, apple, lettuce) was then evaluated. Varying concentrations of pectin and/or xyloglucan were used to produce a range of bacterial cellulose-based plant cell wall models. As compared to the non-sonicated controls, sonicated S. Typhimurium cells attached in significantly lower numbers (between 0.5 and 1.0 log CFU/cm 2 ) to all surfaces except to the bacterial cellulose-only composite without pectin and xyloglucan. Since attachment of S. Typhimurium to the bacterial cellulose-only composite was not affected by sonication, this suggests that bacterial surface structures, particularly flagella, could have specific interactions with pectin and xyloglucan. This study indicates that sonication may have potential applications for reducing Salmonella attachment during the processing of fresh produce. Copyright © 2016 Elsevier Ltd. All rights reserved.

Down-regulation of monocarboxylate transporter 1 (MCT1) gene expression in the colon of piglets is linked to bacterial protein fermentation and pro-inflammatory cytokine-mediated signalling.

PubMed

Villodre Tudela, Carmen; Boudry, Christelle; Stumpff, Friederike; Aschenbach, Jörg R; Vahjen, Wilfried; Zentek, Jürgen; Pieper, Robert

2015-02-28

The present study investigated the influence of bacterial metabolites on monocarboxylate transporter 1 (MCT1) expression in pigs using in vivo, ex vivo and in vitro approaches. Piglets (n 24) were fed high-protein (26 %) or low-protein (18 %) diets with or without fermentable carbohydrates. Colonic digesta samples were analysed for a broad range of bacterial metabolites. The expression of MCT1, TNF-α, interferon γ (IFN-γ) and IL-8 was determined in colonic tissue. The expression of MCT1 was lower and of TNF-α and IL-8 was higher with high-protein diets (P< 0·05). MCT1 expression was positively correlated with l-lactate, whereas negatively correlated with NH₃ and putrescine (P< 0·05). The expression of IL-8 and TNF-α was negatively correlated with l-lactate and positively correlated with NH₃ and putrescine, whereas the expression of IFN-γ was positively correlated with histamine and 4-ethylphenol (P< 0·05). Subsequently, porcine colonic tissue and Caco-2 cells were incubated with Na-butyrate, NH₄Cl or TNF-α as selected bacterial metabolites or mediators of inflammation. Colonic MCT1 expression was higher after incubation with Na-butyrate (P< 0·05) and lower after incubation with NH₄Cl or TNF-α (P< 0·05). Incubation of Caco-2 cells with increasing concentrations of these metabolites confirmed the up-regulation of MCT1 expression by Na-butyrate (linear, P< 0·05) and down-regulation by TNF-α and NH₄Cl (linear, P< 0·05). The high-protein diet decreased the expression of MCT1 in the colon of pigs, which appears to be linked to NH₃- and TNF-α-mediated signalling.

Prolonged Neutrophil Dysfunction Following Plasmodium falciparum Malaria is Related to Hemolysis and Heme Oxygenase-1 Induction1

PubMed Central

Cunnington, Aubrey J.; Njie, Madi; Correa, Simon; Takem, Ebako N.; Riley, Eleanor M.; Walther, Michael

2012-01-01

It is not known why people are more susceptible to bacterial infections such as non-Typhoid Salmonella (NTS) during and after a malaria infection but, in mice, malarial hemolysis impairs resistance to NTS by impairing the neutrophil oxidative burst. This acquired neutrophil dysfunction is a consequence of induction of the cytoprotective, heme degrading enzyme heme oxygenase-1 (HO-1) in neutrophil progenitors in bone marrow. In this study, we assessed whether neutrophil dysfunction occurs in humans with malaria and how this relates to hemolysis. We evaluated neutrophil function in 58 Gambian children with Plasmodium falciparum malaria (55 (95%) with uncomplicated disease), and examined associations with erythrocyte count, haptoglobin, hemopexin, plasma heme, expression of receptors for heme uptake, and HO-1 induction. Malaria caused the appearance of a dominant population of neutrophils with reduced oxidative burst activity, which gradually normalized over 8 weeks of follow-up. The degree of neutrophil impairment correlated significantly with markers of hemolysis and HO-1 induction. HO-1 expression was increased in blood during acute malaria, but at a cellular level HO-1 expression was modulated by changes in surface expression of the haptoglobin receptor (CD163). These findings demonstrate that neutrophil dysfunction occurs in P. falciparum malaria and support the relevance of the mechanistic studies in mice. Furthermore, they suggest the presence of a regulatory pathway to limit HO-1 induction by hemolysis in the context of infection, and indicate new targets for therapeutic intervention to abrogate the susceptibility to bacterial infection in the context of hemolysis in humans. PMID:23100518

Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

PubMed

Hill, Heather E; Pioszak, Augen A

2013-03-01

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

Influence of Silver-hydroxyapatite Nanocomposite Coating on Biofilm Formation of Joint Prosthesis and Its Mechanism

PubMed Central

Zhao, L; Ashraf, MA

2015-01-01

ABSTRACT Background: The main reason for biomaterial related refractory infections is biofilm formation caused by bacterial adhesion on the surface of materials. Silver-hydroxyapatite (Ag/HA) nanocomposite coating can inhibit the formation of biofilm, but its mechanism is not clear. Material and Method: In order to clarify the mechanism, the amounts of biofilm on the Ag/HA composite coating and HA coating were determined, the release rates of silver nanoparticles in simulated body fluid (SBF) were detected by atomic absorption spectrometry, and the expression values of atlE, fbe, sap, iapB genes of Staphylococcus aureus were studied when they grew on Ag/HA composite coating and HA coating. Results: The amount of the biofilm on the Ag/HA composite coating was significantly less than that on the HA coating, and the bacterial adhesion was decreased. The silver nanoparticles were released continuously in SBF and the release rate decreased gradually with time. The expression values of atlE, fbe and sap were high in the initial stage of adhesion and the expression value of iapB was high in the colonies-gathering stage in the control group, but they were all significantly inhibited in the presence of Ag. Conclusion: These results indicated that the main antibacterial effect of Ag/HA composite coating was achieved by the release of silver nanoparticles. The addition of Ag inhibited the expression of genes related to biofilm formation, which in turn inhibited the formation of biofilms. This provided theoretical support for the clinical application of Ag/HA composite coating. PMID:27400164

Intestinal Alkaline Phosphatase: Potential Roles in Promoting Gut Health in Weanling Piglets and Its Modulation by Feed Additives - A Review.

PubMed

Melo, A D B; Silveira, H; Luciano, F B; Andrade, C; Costa, L B; Rostagno, M H

2016-01-01

The intestinal environment plays a critical role in maintaining swine health. Many factors such as diet, microbiota, and host intestinal immune response influence the intestinal environment. Intestinal alkaline phosphatase (IAP) is an important apical brush border enzyme that is influenced by these factors. IAP dephosphorylates bacterial lipopolysaccharides (LPS), unmethylated cytosine-guanosine dinucleotides, and flagellin, reducing bacterial toxicity and consequently regulating toll-like receptors (TLRs) activation and inflammation. It also desphosphorylates extracellular nucleotides such as uridine diphosphate and adenosine triphosphate, consequently reducing inflammation, modulating, and preserving the homeostasis of the intestinal microbiota. The apical localization of IAP on the epithelial surface reveals its role on LPS (from luminal bacteria) detoxification. As the expression of IAP is reported to be downregulated in piglets at weaning, LPS from commensal and pathogenic gram-negative bacteria could increase inflammatory processes by TLR-4 activation, increasing diarrhea events during this phase. Although some studies had reported potential IAP roles to promote gut health, investigations about exogenous IAP effects or feed additives modulating IAP expression and activity yet are necessary. However, we discussed in this paper that the critical assessment reported can suggest that exogenous IAP or feed additives that could increase its expression could show beneficial effects to reduce diarrhea events during the post weaning phase. Therefore, the main goals of this review are to discuss IAP's role in intestinal inflammatory processes and present feed additives used as growth promoters that may modulate IAP expression and activity to promote gut health in piglets.

Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves.

PubMed

O'brien, R D; Lindow, S E

1988-09-01

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at -5 or -9 degrees C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.

Impact of Lactobacillus plantarum Sortase on Target Protein Sorting, Gastrointestinal Persistence, and Host Immune Response Modulation

PubMed Central

Remus, Daniela M.; Bongers, Roger S.; Meijerink, Marjolein; Fusetti, Fabrizia; Poolman, Bert; de Vos, Paul; Wells, Jerry M.; Bron, Peter A.

2013-01-01

Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L. plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiCl protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation. PMID:23175652

Dissolution of Calcite in the Twilight Zone: Bacterial Control of Dissolution of Sinking Planktonic Carbonates Is Unlikely

PubMed Central

Bissett, Andrew; Neu, Thomas R.; de Beer, Dirk

2011-01-01

We investigated the ability of bacterial communities to colonize and dissolve two biogenic carbonates (Foraminifera and oyster shells). Bacterial carbonate dissolution in the upper water column is postulated to be driven by metabolic activity of bacteria directly colonising carbonate surfaces and the subsequent development of acidic microenvironments. We employed a combination of microsensor measurements, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and image analysis and molecular documentation of colonising bacteria to monitor microbial processes and document changes in shell surface topography. Bacterial communities rapidly colonised shell surfaces, forming dense biofilms with extracellular polymeric substance (EPS) deposits. Despite this, we found no evidence of bacterially mediated carbonate dissolution. Dissolution was not indicated by Ca2+ microprofiles, nor was changes in shell surface structure related to the presence of colonizing bacteria. Given the short time (days) settling carbonate material is actually in the twilight zone (500–1000 m), it is highly unlikely that microbial metabolic activity on directly colonised shells plays a significant role in dissolving settling carbonates in the shallow ocean. PMID:22102861

Dissolution of calcite in the twilight zone: bacterial control of dissolution of sinking planktonic carbonates is unlikely.

PubMed

Bissett, Andrew; Neu, Thomas R; Beer, Dirk de

2011-01-01

We investigated the ability of bacterial communities to colonize and dissolve two biogenic carbonates (Foraminifera and oyster shells). Bacterial carbonate dissolution in the upper water column is postulated to be driven by metabolic activity of bacteria directly colonising carbonate surfaces and the subsequent development of acidic microenvironments. We employed a combination of microsensor measurements, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and image analysis and molecular documentation of colonising bacteria to monitor microbial processes and document changes in shell surface topography. Bacterial communities rapidly colonised shell surfaces, forming dense biofilms with extracellular polymeric substance (EPS) deposits. Despite this, we found no evidence of bacterially mediated carbonate dissolution. Dissolution was not indicated by Ca²⁺ microprofiles, nor was changes in shell surface structure related to the presence of colonizing bacteria. Given the short time (days) settling carbonate material is actually in the twilight zone (500-1000 m), it is highly unlikely that microbial metabolic activity on directly colonised shells plays a significant role in dissolving settling carbonates in the shallow ocean.

Role of Sulfhydryl Sites on Bacterial Cell Walls in the Biosorption, Mobility and Bioavailability of Mercury and Uranium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myneni, Satish C.; Mishra, Bhoopesh; Fein, Jeremy

2009-04-01

The goal of this exploratory study is to provide a quantitative and mechanistic understanding of the impact of bacterial sulfhydryl groups on the bacterial uptake, speciation, methylation and bioavailability of Hg and redox changes of uranium. The relative concentration and reactivity of different functional groups present on bacterial surfaces will be determined, enabling quantitative predictions of the role of biosorption of Hg under the physicochemical conditions found at contaminated DOE sites.The hypotheses we propose to test in this investigation are as follows- 1) Sulfhydryl groups on bacterial cell surfaces modify Hg speciation and solubility, and play an important role, specificallymore » in the sub-micromolar concentration ranges of metals in the natural and contaminated systems. 2) Sulfhydryl binding of Hg on bacterial surfaces significantly influences Hg transport into the cell and the methylation rates by the bacteria. 3) Sulfhydryls on cell membranes can interact with hexavalent uranium and convert to insoluble tetravalent species. 4) Bacterial sulfhydryl surface groups are inducible by the presence of metals during cell growth. Our studies focused on the first hypothesis, and we examined the nature of sulfhydryl sites on three representative bacterial species: Bacillus subtilis, a common gram-positive aerobic soil species; Shewanella oneidensis, a facultative gram-negative surface water species; and Geobacter sulfurreducens, an anaerobic iron-reducing gram-negative species that is capable of Hg methylation; and at a range of Hg concentration (and Hg:bacterial concentration ratio) in which these sites become important. A summary of our findings is as follows- Hg adsorbs more extensively to bacteria than other metals. Hg adsorption also varies strongly with pH and chloride concentration, with maximum adsorption occurring under circumneutral pH conditions for both Cl-bearing and Cl-free systems. Under these conditions, all bacterial species tested exhibit almost complete removal of Hg from the experimental solutions at relatively low bacterial concentrations. Synchrotron based X-ray spectroscopic studies of these samples indicate that the structure and the coordination environment of Hg surface complexes on bacterial cell walls change dramatically- with sulfhydryls as the dominant Hg-binding groups in the micromolar and submicromolar range, and carboxyls and phosphoryls dominating at high micromolar concentrations. Hg interactions change from a trigonal or T-shaped HgS{sub 3} complex to HgS or HgS{sub 2} type complexes as the Hg concentration increases in the submicromolar range. Although all bacterial species studied exhibited the same types of coordination environments for Hg, the relative concentrations of the complexes change as a function of Hg concentration.« less

Role of the cathelicidin-related antimicrobial peptide in inflammation and mortality in a mouse model of bacterial meningitis.

PubMed

Merres, Julika; Höss, Jonas; Albrecht, Lea-Jessica; Kress, Eugenia; Soehnlein, Oliver; Jansen, Sandra; Pufe, Thomas; Tauber, Simone C; Brandenburg, Lars-Ove

2014-01-01

Antimicrobial peptides (AP) are important components of the innate immune system, yet little is known about their expression and function in the brain. Our previous work revealed upregulated gene expression of cathelicidin-related AP (CRAMP) following bacterial meningitis in primary rat glial cells as well as bactericidal activity against frequent meningitis-causing bacteria. However, the effect of cathelicidin expression on the progression of inflammation and mortality in bacterial meningitis remains unknown. Therefore, we used CRAMP-deficient mice to investigate the effect of CRAMP on bacterial growth, inflammatory responses and mortality in meningitis. Meningitis was induced by intracerebral injection of type 3 Streptococcus pneumoniae. The degree of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time RT-PCR. CRAMP deficiency led to a higher mortality rate that was associated with increased bacterial titers in the cerebellum, blood and spleen as well as decreased meningeal neutrophil infiltration. CRAMP-deficient mice displayed a higher degree of glial cell activation that was accompanied by a more pronounced proinflammatory response. Taken together, this work provides insight into the important role of CRAMP as part of the innate immune defense against pathogens in bacterial CNS infections. Copyright © 2013 S. Karger AG, Basel

Office space bacterial abundance and diversity in three metropolitan areas.

PubMed

Hewitt, Krissi M; Gerba, Charles P; Maxwell, Sheri L; Kelley, Scott T

2012-01-01

People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded "universal" bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. "[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay." - Feazel et al. (2009).

Effects of bacterial pollution caused by a strong typhoon event and the restoration of a recreational beach: Transitions of fecal bacterial counts and bacterial flora in beach sand.

PubMed

Suzuki, Yoshihiro; Teranishi, Kotaro; Matsuwaki, Tomonori; Nukazawa, Kei; Ogura, Yoshitoshi

2018-05-28

To determine the effects of bacteria pollution associated with a strong typhoon event and to assess the restoration of the normal bacterial flora, we used conventional filtration methods and nextgeneration sequencing of 16S rRNA genes to analyze the transition of fecal and total bacterial counts in water and core sand samples collected from a recreational beach. Immediately after the typhoon event, Escherichia coli counts increased to 82 CFU/100 g in the surface beach sand. E. coli was detected through the surface to sand 85-cm deep at the land side point (10-m land side from the high-water line). However, E. coli disappeared within a month from the land side point. The composition of the bacterial flora in the beach sand at the land point was directly influenced by the typhoon event. Pseudomonas was the most prevalent genus throughout the sand layers (0-102-cm deep) during the typhoon event. After 3 months, the population of Pseudomonas significantly decreased, and the predominant genus in the surface layer was Kaistobacter, although Pseudomonas was the major genus in the 17- to 85-cm layer. When the beach conditions stabilized, the number of pollutant Pseudomonas among the 10 most abundant genera decreased to lower than the limit of detection. The bacterial population of the sand was subsequently restored to the most populous pre-event orders at the land point. A land-side beach, where users directly contact the sand, was significantly affected by bacterial pollution caused by a strong typhoon event. We show here that the normal bacterial flora of the surface sand was restored within 1 month. Copyright © 2018 Elsevier B.V. All rights reserved.

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

PubMed Central

Guinet, Françoise; Avé, Patrick; Filali, Sofia; Huon, Christèle; Savin, Cyril; Huerre, Michel; Fiette, Laurence; Carniel, Elisabeth

2015-01-01

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host. PMID:26484539

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague.

PubMed

Guinet, Françoise; Avé, Patrick; Filali, Sofia; Huon, Christèle; Savin, Cyril; Huerre, Michel; Fiette, Laurence; Carniel, Elisabeth

2015-10-01

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.

Elevated gene expression of S100A12 is correlated with the predominant clinical inflammatory factors in patients with bacterial pneumonia.

PubMed

Hou, Fei; Wang, Likui; Wang, Hong; Gu, Junchao; Li, Meiling; Zhang, Jingkai; Ling, Xiao; Gao, Xiaofang; Luo, Cheng

2015-06-01

Inflammation is the predominant characteristic of pneumonia. The present study aimed to to identify a faster and more reliable novel inflammatory marker for the diagnosis of pneumonia. The expression of the S100A12 gene was analyzed by reverse transcription quantitative polymerase chain reaction in samples obtained from 46 patients with bacterial pneumonia and other infections, compared with samples from 20 healthy individuals, using the 2‑ΔΔCt method. The expression levels of S100A12 were increased in 12 patients with bacterial pneumonia. Compared with clinical inflammatory data, a positive correlation was observed between the expression of the S100A12 gene and levels of white blood cells, C‑reactive protein (CRP), thrombocytocrit, neutrophils, erythrocyte sedimentation and soterocytes, and an inverse correlation was observed with the width of red blood cell volume distribution and platelet distribution, monocytes and hemoglobin, using Pearson's product‑moment correlation method. The P‑value of CRP and erythrocyte sedimentation were revealed to be statistically significant (P<0.05). A sporadic distribution of S100A12 was observed in a heatmap among the patients with different infections and bacterial pneumonia. Furthermore, the expression of S100A12 occurred in parallel to the number of clumps of inflamed tissue observed in chest computed tomography and X‑ray. The value of gene expression of S100A12 (>1.0) determined using the 2‑ΔΔCt method was associated with more severe respiratory diseases in the patients compromised by bacterial pneumonia, sepsis and pancreatitis. These findings suggested that S100A12 is an effective marker for inflammatory diseases.

Exploring the potential of the bacterial carotene desaturase CrtI to increase the beta-carotene content in Golden Rice.

PubMed

Al-Babili, Salim; Hoa, Tran Thi Cuc; Schaub, Patrick

2006-01-01

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.

Surface nanoporosity has a greater influence on osteogenic and bacterial cell adhesion than crystallinity and wettability

NASA Astrophysics Data System (ADS)

Rodriguez-Contreras, Alejandra; Guadarrama Bello, Dainelys; Nanci, Antonio

2018-07-01

There has been much emphasis on the influence of crystallinity and wettability for modulating cell activity, particularly for bone biomaterials. In this context, we have generated titanium oxide layers with similar mesoporous topography and surface roughness but with amorphous or crystalline oxide layers and differential wettability. We then investigated their influence on the behavior of MC3T3 osteoblastic and bacterial cells. There was no difference in cell adhesion, spreading and growth on amorphous and crystalline surfaces. The number of focal adhesions was similar, however, cells on the amorphous surface exhibited a higher frequency of mature adhesions. The crystallinity of the surface layers also had no bearing on bacterial adhesion. While it cannot be excluded that surface crystallinity, roughness and wettability contribute to some degree to determining cell behavior, our data suggest that physical characteristics of surfaces represent the major determinant.

Mechanistic study on antibacterial action of zinc oxide nanoparticles synthesized using green route.

PubMed

Happy Agarwal; Soumya Menon; Venkat Kumar, S; Rajeshkumar, S

2018-04-25

A large array of diseases caused by bacterial pathogens and origination of multidrug resistance in their gene provokes the need of developing new vectors or novel drug molecules for effective drug delivery and thus, better treatment of disease. The nanoparticle has emerged as a novel drug molecule in last decade and has been used in various industrial fields like cosmetics, healthcare, agricultural, pharmaceuticals due to their high optical, electronic, medicinal properties. Use of nanoparticles as an antibacterial agent remain in current studies with metal nanoparticles like silver, gold, copper, iron and metal oxide nanoparticles like zinc oxide, copper oxide, titanium oxide and iron oxide nanoparticles. The high anti-bacterial activity of nanoparticles is due to their large surface area to volume ratio which allows binding of a large number of ligands on nanoparticle surface and hence, its complexation with receptors present on the bacterial surface. Green synthesis of Zinc Oxide Nanoparticle (ZnO NP) and its anti-bacterial application has been particularly discussed in the review literature. The present study highlights differential nanoparticle attachment to gram + and gram - bacterial surface and different mechanism adopted by nanoparticle for bacterial control. Pharmacokinetics and applications of ZnO NP are also discussed briefly. Copyright © 2018 Elsevier B.V. All rights reserved.

Enterococcus hirae biofilm formation on hospital material surfaces and effect of new biocides.

PubMed

Di Lodovico, Silvia; Cataldi, Valentina; Di Campli, Emanuela; Ancarani, Elisabetta; Cellini, Luigina; Di Giulio, Mara

2017-08-02

Nowadays, the bacterial contamination in the hospital environment is of particular concern because the hospital-acquired infections (HAIs), also known as nosocomial infections, are responsible for significant morbidity and mortality. This work evaluated the capability of Enterococcus hirae to form biofilm on different surfaces and the action of two biocides on the produced biofilms. The biofilm formation of E. hirae ATCC 10541 was studied on polystyrene and stainless steel surfaces through the biomass quantification and the cell viability at 20 and 37 °C. The effect of LH IDROXI FAST and LH ENZYCLEAN SPRAY biocides on biomasses was expressed as percentage of biofilm reduction. E. hirae at 20 and 37 °C produced more biofilm on the stainless steel in respect to the polystyrene surface. The amount of viable cells was greater at 20 °C than with 37 °C on the two analyzed surfaces. Biocides revealed a good anti-biofilm activity with the most effect for LH ENZYCLEAN SPRAY on polystyrene and stainless steel at 37 °C with a maximum biofilm reduction of 85.72 and 86.37%, respectively. E. hirae is a moderate biofilm producer depending on surface material and temperature, and the analyzed biocides express a remarkable antibiofilm action. The capability of E. hirae to form biofilm can be associated with its increasing incidence in hospital-acquired infections, and the adoption of suitable disinfectants is strongly recommended.

Bacterial surface adaptation

NASA Astrophysics Data System (ADS)

Utada, Andrew

2014-03-01

Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

Surface-modified bacterial nanofibrillar PHB scaffolds for bladder tissue repair.

PubMed

Karahaliloğlu, Zeynep; Demirbilek, Murat; Şam, Mesut; Sağlam, Necdet; Mızrak, Alpay Koray; Denkbaş, Emir Baki

2016-01-01

The aim of the study is in vitro investigation of the feasibility of surface-modified bacterial nanofibrous poly [(R)-3-hydroxybutyrate] (PHB) graft for bladder reconstruction. In this study, the surface of electrospun bacterial PHB was modified with PEG- or EDA via radio frequency glow discharge method. After plasma modification, contact angle of EDA-modified PHB scaffolds decreased from 110 ± 1.50 to 23 ± 0.5 degree. Interestingly, less calcium oxalate stone deposition was observed on modified PHB scaffolds compared to that of non-modified group. Results of this study show that surface-modified scaffolds not only inhibited calcium oxalate growth but also enhanced the uroepithelial cell viability and proliferation.

Microbially induced separation of quartz from hematite using sulfate reducing bacteria.

PubMed

Prakasan, M R Sabari; Natarajan, K A

2010-07-01

Cells and metabolic products of Desulfovibrio desulfuricans were successfully used to separate quartz from hematite through environmentally benign microbially induced flotation. Bacterial metabolic products such as extracellular proteins and polysaccharides were isolated from both unadapted and mineral-adapted bacterial metabolite and their basic characteristics were studied in order to get insight into the changes brought about on bioreagents during adaptation. Interaction between bacterial cells and metabolites with minerals like hematite and quartz brought about significant surface-chemical changes on both the minerals. Quartz was rendered more hydrophobic, while hematite became more hydrophilic after biotreatment. The predominance of bacterial polysaccharides on interacted hematite and of proteins on quartz was responsible for the above surface-chemical changes, as attested through adsorption studies. Surface-chemical changes were also observed on bacterial cells after adaptation to the above minerals. Selective separation of quartz from hematite was achieved through interaction with quartz-adapted bacterial cells and metabolite. Mineral-specific proteins secreted by quartz-adapted cells were responsible for conferment of hydrophobicity on quartz resulting in enhanced separation from hematite through flotation. 2010 Elsevier B.V. All rights reserved.

Ellipsometric Measurement of Bacterial Films at Metal-Electrolyte Interfaces

PubMed Central

Busalmen, J. P.; de Sánchez, S. R.; Schiffrin, D. J.

1998-01-01

Ellipsometric measurements were used to monitor the formation of a bacterial cell film on polarized metal surfaces (Al-brass and Ti). Under cathodic polarization bacterial attachment was measured from changes in the ellipsometric angles. These were fitted to an effective medium model for a nonabsorbing bacterial film with an effective refractive index (nf) of 1.38 and a thickness (df) of 160 ± 10 nm. From the optical measurements a surface coverage of 17% was estimated, in agreement with direct microscopic observations. The influence of bacteria on the formation of oxide films was monitored by ellipsometry following the film growth in situ. A strong inhibition of metal oxide film formation was observed, which was assigned to the decrease in oxygen concentration due to the presence of bacteria. It is shown that the irreversible adhesion of bacteria to the surface can be monitored ellipsometrically. Electrophoretic mobility is proposed as one of the factors determining bacterial attachment. The high sensitivity of ellipsometry and its usefulness for the determination of growth of interfacial bacterial films is demonstrated. PMID:9758786

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

PubMed

Jonsson, Ing-Marie; Juuti, Jarmo T; François, Patrice; AlMajidi, Rana; Pietiäinen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J; Driessen, Arnold J M; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P

2010-12-02

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

PubMed Central

Bernasconi, Eric; Germond, Jacques-Edouard; Delley, Michèle; Fritsché, Rodolphe; Corthésy, Blaise

2002-01-01

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. PMID:12039750

Key role for scavenger receptor B-I in the integrative physiology of host defense during bacterial pneumonia.

PubMed

Gowdy, K M; Madenspacher, J H; Azzam, K M; Gabor, K A; Janardhan, K S; Aloor, J J; Fessler, M B

2015-05-01

Scavenger receptor B-I (SR-BI) is a multirecognition receptor that regulates cholesterol trafficking and cardiovascular inflammation. Although it is expressed by neutrophils (PMNs) and lung-resident cells, no role for SR-BI has been defined in pulmonary immunity. Herein, we report that, compared with SR-BI(+/+) counterparts, SR-BI(-/-) mice suffer markedly increased mortality during bacterial pneumonia associated with higher bacterial burden in the lung and blood, deficient induction of the stress glucocorticoid corticosterone, higher serum cytokines, and increased organ injury. SR-BI(-/-) mice had significantly increased PMN recruitment and cytokine production in the infected airspace. This was associated with defective hematopoietic cell-dependent clearance of lipopolysaccharide from the airspace and increased cytokine production by SR-BI(-/-) macrophages. Corticosterone replacement normalized alveolar neutrophilia but not alveolar cytokines, bacterial burden, or mortality, suggesting that adrenal insufficiency derepresses PMN trafficking to the SR-BI(-/-) airway in a cytokine-independent manner. Despite enhanced alveolar neutrophilia, SR-BI(-/-) mice displayed impaired phagocytic killing. Bone marrow chimeras revealed this defect to be independent of the dyslipidemia and adrenal insufficiency of SR-BI(-/-) mice. During infection, SR-BI(-/-) PMNs displayed deficient oxidant production and CD11b externalization, and increased surface L-selectin, suggesting defective activation. Taken together, SR-BI coordinates several steps in the integrated neutrophilic host defense response to pneumonia.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

PubMed Central

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

Reduced bacterial growth and increased osteoblast proliferation on titanium with a nanophase TiO2 surface treatment.

PubMed

Bhardwaj, Garima; Webster, Thomas J

2017-01-01

The attachment and initial growth of bacteria on an implant surface dictates the progression of infection. Treatment often requires aggressive antibiotic use, which does not always work. To overcome the difficulties faced in systemic and local antibiotic delivery, scientists have forayed into using alternative techniques, which includes implant surface modifications that prevent initial bacterial adhesion, foreign body formation, and may offer a controlled inflammatory response. The current study focused on using electrophoretic deposition to treat titanium with a nanophase titanium dioxide surface texture to reduce bacterial adhesion and growth. Two distinct nanotopographies were analyzed, Ti-160, an antimicrobial surface designed to greatly reduce bacterial colonization, and Ti-120, an antimicrobial surface with a topography that upregulates osteoblast activity while reducing bacterial colonization; the number following Ti in the nomenclature represents the atomic force microscopy root-mean-square roughness value in nanometers. There was a 95.6% reduction in Staphylococcus aureus (gram-positive bacteria) for the Ti-160-treated surfaces compared to the untreated titanium alloy controls. There was a 90.2% reduction in Pseudomonas aeruginosa (gram-negative bacteria) on Ti-160-treated surfaces compared to controls. For ampicillin-resistant Escherichia coli , there was an 81.1% reduction on the Ti-160-treated surfaces compared to controls. Similarly for surfaces treated with Ti-120, there was an 86.8% reduction in S. aureus , an 82.1% reduction in P. aeruginosa , and a 48.6% reduction in ampicillin-resistant E. coli . The Ti-120 also displayed a 120.7% increase at day 3 and a 168.7% increase at day 5 of osteoblast proliferation over standard titanium alloy control surfaces. Compared to untreated surfaces, Ti-160-treated titanium surfaces demonstrated a statistically significant 1 log reduction in S. aureus and P. aeruginosa , whereas Ti-120 provided an additional increase in osteoblast proliferation for up to 5 days, criteria, which should be further studied for a wide range of orthopedic applications.

Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies.

PubMed

Seib, Kate L; Brunelli, Brunella; Brogioni, Barbara; Palumbo, Emmanuelle; Bambini, Stefania; Muzzi, Alessandro; DiMarcello, Federica; Marchi, Sara; van der Ende, Arie; Aricó, Beatrice; Savino, Silvana; Scarselli, Maria; Comanducci, Maurizio; Rappuoli, Rino; Giuliani, Marzia M; Pizza, Mariagrazia

2011-02-01

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.

Surface Ligand Density of Antibiotic-Nanoparticle Conjugates Enhances Target Avidity and Membrane Permeabilization of Vancomycin-Resistant Bacteria.

PubMed

Hassan, Marwa M; Ranzoni, Andrea; Phetsang, Wanida; Blaskovich, Mark A T; Cooper, Matthew A

2017-02-15

Many bacterial pathogens have now acquired resistance toward commonly used antibiotics, such as the glycopeptide antibiotic vancomycin. In this study, we show that immobilization of vancomycin onto a nanometer-scale solid surface with controlled local density can potentiate antibiotic action and increase target affinity of the drug. Magnetic nanoparticles were conjugated with vancomycin and used as a model system to investigate the relationship between surface density and drug potency. We showed remarkable improvement in minimum inhibitory concentration against vancomycin-resistant strains with values of 13-28 μg/mL for conjugated vancomycin compared to 250-4000 μg/mL for unconjugated vancomycin. Higher surface densities resulted in enhanced affinity toward the bacterial target compared to that of unconjugated vancomycin, as measured by a competition experiment using a surrogate ligand for bacterial Lipid II, N-Acetyl-l-Lys-d-Ala-d-Ala. High density vancomycin nanoparticles required >64 times molar excess of ligand (relative to the vancomycin surface density) to abrogate antibacterial activity compared to only 2 molar excess for unconjugated vancomycin. Further, the drug-nanoparticle conjugates caused rapid permeabilization of the bacterial cell wall within 2 h, whereas no effect was seen with unconjugated vancomycin, suggesting additional modes of action for the nanoparticle-conjugated drug. Hence, immobilization of readily available antibiotics on nanocarriers may present a general strategy for repotentiating drugs that act on bacterial membranes or membrane-bound targets but have lost effectiveness against resistant bacterial strains.

Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

PubMed

Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

2015-12-01

We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

Identification of a two-component Class IIb bacteriocin in Streptococcus pyogenes by recombinase-based in vivo expression technology.

PubMed

Armstrong, Brent D; Herfst, Christine A; Tonial, Nicholas C; Wakabayashi, Adrienne T; Zeppa, Joseph J; McCormick, John K

2016-11-03

Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens.

Identification of a two-component Class IIb bacteriocin in Streptococcus pyogenes by recombinase-based in vivo expression technology

PubMed Central

Armstrong, Brent D.; Herfst, Christine A.; Tonial, Nicholas C.; Wakabayashi, Adrienne T.; Zeppa, Joseph J.; McCormick, John K.

2016-01-01

Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens. PMID:27808235

Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance.

PubMed

Ferguson, A L; Hughes, A D; Tufail, U; Baumann, C G; Scott, D J; Hoggett, J G

2000-09-22

The interaction between the core form of bacterial RNA polymerases and sigma factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and sigma 70 has been investigated by surface plasmon resonance. The His-tagged form of sigma 70 factor was immobilised on a Ni2+-NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9x10(-7) M, and the dissociation rate constant for release of sigma from core, in the absence of DNA or transcription, was 4x10(-3) s(-1), corresponding to a half-life of about 200 s.

Lack of Toll-like receptor 2 results in higher mortality of bacterial meningitis by impaired host resistance.

PubMed

Böhland, Martin; Kress, Eugenia; Stope, Matthias B; Pufe, Thomas; Tauber, Simone C; Brandenburg, Lars-Ove

2016-10-15

Bacterial meningitis is - despite therapeutical progress during the last decades - still characterized by high mortality and severe permanent neurogical sequelae. The brain is protected from penetrating pathogens by both the blood-brain barrier and the innate immune system. Invading pathogens are recognized by so-called pattern recognition receptors including the Toll-like receptors (TLR) which are expressed by glial immune cells in the central nervous system. Among these, TLR2 is responsible for the detection of Gram-positive bacteria such as the meningitis-causing pathogen Streptococcus pneumoniae. Here, we used TLR2-deficient mice to investigate the effects on mortality, bacterial growth and inflammation in a mouse model of pneumococcal meningitis. Our results revealed a significantly increased mortality rate and higher bacterial burden in TLR2-deficient mice with pneumococcal meningitis. Furthermore, infected TLR2-deficient mice suffered from a significantly increased pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and Chemokine (C-C motif) ligand 2 (CCL2) or CCL3 chemokine expression and decreased expression of anti-inflammatory cytokines and antimicrobial peptides. In contrast, glial cell activation assessed by glial cell marker expression was comparable to wildtype mice. Taken together, the results suggest that TLR2 is essential for an efficient immune response against Streptococcus pneumoniae meningitis since lack of the receptor led to a worse outcome by higher mortality due to increased bacterial burden, weakened innate immune response and reduced expression of antimicrobial peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries.

PubMed

Belstrøm, Daniel; Constancias, Florentin; Liu, Yang; Yang, Liang; Drautz-Moses, Daniela I; Schuster, Stephan C; Kohli, Gurjeet Singh; Jakobsen, Tim Holm; Holmstrup, Palle; Givskov, Michael

2017-01-01

The taxonomic composition of the salivary microbiota has been reported to differentiate between oral health and disease. However, information on bacterial activity and gene expression of the salivary microbiota is limited. The purpose of this study was to perform metagenomic and metatranscriptomic characterization of the salivary microbiota and test the hypothesis that salivary microbial presence and activity could be an indicator of the oral health status. Stimulated saliva samples were collected from 30 individuals (periodontitis: n  = 10, dental caries: n  = 10, oral health: n  = 10). Salivary microbiota was characterized using metagenomics and metatranscriptomics in order to compare community composition and the gene expression between the three groups. Streptococcus was the predominant bacterial genus constituting approx. 25 and 50% of all DNA and RNA reads, respectively. A significant disease-associated higher relative abundance of traditional periodontal pathogens such as Porphyromonas gingivalis and Filifactor alocis and salivary microbial activity of F . alocis was associated with periodontitis. Significantly higher relative abundance of caries-associated bacteria such as Streptococcus mutans and Lactobacillus fermentum was identified in saliva from patients with dental caries. Multiple genes involved in carbohydrate metabolism were significantly more expressed in healthy controls compared to periodontitis patients. Using metagenomics and metatranscriptomics we show that relative abundance of specific oral bacterial species and bacterial gene expression in saliva associates with periodontitis and dental caries. Further longitudinal studies are warranted to evaluate if screening of salivary microbial activity of specific oral bacterial species and metabolic gene expression can identify periodontitis and dental caries at preclinical stages.

Bacterial diversity in the oxygen minimum zone of the eastern tropical South Pacific.

PubMed

Stevens, Heike; Ulloa, Osvaldo

2008-05-01

The structure and diversity of bacterial communities associated with the oxygen minimum zone (OMZ) of the eastern tropical South Pacific was studied through phylogenetic analysis. Clone libraries of 16S rRNA gene fragments were constructed using environmental DNA collected from the OMZ (60 m and 200 m), the sea surface (10 m), and the deep oxycline (450 m). At the class level, the majority of sequences affiliated to the gamma- (53.7%) and alpha-Proteobacteria (19.7%), and to the Bacteroidetes (11.2%). A vertical partitioning of the bacterial communities was observed, with main differences between the suboxic OMZ and the more oxygenated surface and deep oxycline waters. At the surface, the microbial community was predominantly characterized by SAR86, Loktanella and unclassified Flavobacteriaceae, whereas the deeper layer was dominated by Sulfitobacter and unclassified Alteromonadaceae. In the OMZ, major constituents affiliated to the marine SAR11 clade and to thiotrophic gamma-symbionts (25% of all sequences), a group not commonly found in pelagic waters. Sequences affiliating to the phylum Chloroflexi, to the AGG47 and SAR202 clades, to the delta-Proteobacteria, to the Acidobacteria, and to the 'anammox group' of the Planctomycetes were found exclusively in the OMZ. The bacterial richness in the OMZ was higher than in the oxic surface and deeper oxycline, as revealed by rarefaction analysis and the Chao1 richness estimator (surface: 45 +/- 8, deeper oxycline: 76 +/- 26; OMZ (60 m): 97 +/- 33, OMZ (200 m): 109 +/- 31). OMZ bacterial diversity indices (Fisher's: approximately 30 +/- 5, Shannon's: approximately 3.31, inverse Simpson's: approximately 20) were similar to those found in other pelagic marine environments. Thus, our results indicate a distinct and diverse bacterial community within the OMZ, with presumably novel and yet uncultivated bacterial lineages.

Profiling bacterial diversity in a limestone cave of the western Loess Plateau of China

PubMed Central

Wu, Yucheng; Tan, Liangcheng; Liu, Wuxing; Wang, Baozhan; Wang, Jianjun; Cai, Yanjun; Lin, Xiangui

2015-01-01

Bacteria and archaea sustain subsurface cave ecosystems by dominating primary production and fueling biogeochemical cyclings, despite the permanent darkness and shortage of nutrients. However, the heterogeneity and underlying mechanism of microbial diversity in caves, in particular those well connect to surface environment are largely unexplored. In this study, we examined the bacterial abundance and composition in Jinjia Cave, a small and shallow limestone cave located on the western Loess Plateau of China, by enumerating and pyrosequencing small subunit rRNA genes. The results clearly reveal the contrasting bacterial community compositions in relation to cave habitat types, i.e., rock wall deposit, aquatic sediment, and sinkhole soil, which are differentially connected to the surface environment. The deposits on the cave walls were dominated by putative cave-specific bacterial lineages within the γ-Proteobacteria or Actinobacteria that are routinely found on cave rocks around the world. In addition, sequence identity with known functional groups suggests enrichments of chemolithotrophic bacteria potentially involved in autotrophic C fixation and inorganic N transformation on rock surfaces. By contrast, bacterial communities in aquatic sediments were more closely related to those in the overlying soils. This is consistent with the similarity in elemental composition between the cave sediment and the overlying soil, implicating the influence of mineral chemistry on cave microhabitat and bacterial composition. These findings provide compelling molecular evidence of the bacterial community heterogeneity in an East Asian cave, which might be controlled by both subsurface and surface environments. PMID:25870592

Profiling bacterial diversity in a limestone cave of the western Loess Plateau of China.

PubMed

Wu, Yucheng; Tan, Liangcheng; Liu, Wuxing; Wang, Baozhan; Wang, Jianjun; Cai, Yanjun; Lin, Xiangui

2015-01-01

Bacteria and archaea sustain subsurface cave ecosystems by dominating primary production and fueling biogeochemical cyclings, despite the permanent darkness and shortage of nutrients. However, the heterogeneity and underlying mechanism of microbial diversity in caves, in particular those well connect to surface environment are largely unexplored. In this study, we examined the bacterial abundance and composition in Jinjia Cave, a small and shallow limestone cave located on the western Loess Plateau of China, by enumerating and pyrosequencing small subunit rRNA genes. The results clearly reveal the contrasting bacterial community compositions in relation to cave habitat types, i.e., rock wall deposit, aquatic sediment, and sinkhole soil, which are differentially connected to the surface environment. The deposits on the cave walls were dominated by putative cave-specific bacterial lineages within the γ-Proteobacteria or Actinobacteria that are routinely found on cave rocks around the world. In addition, sequence identity with known functional groups suggests enrichments of chemolithotrophic bacteria potentially involved in autotrophic C fixation and inorganic N transformation on rock surfaces. By contrast, bacterial communities in aquatic sediments were more closely related to those in the overlying soils. This is consistent with the similarity in elemental composition between the cave sediment and the overlying soil, implicating the influence of mineral chemistry on cave microhabitat and bacterial composition. These findings provide compelling molecular evidence of the bacterial community heterogeneity in an East Asian cave, which might be controlled by both subsurface and surface environments.

Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data.

PubMed

Tintle, Nathan L; Sitarik, Alexandra; Boerema, Benjamin; Young, Kylie; Best, Aaron A; Dejongh, Matthew

2012-08-08

Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

Engineering the bacterial shapes for enhanced inclusion bodies accumulation.

PubMed

Jiang, Xiao-Ran; Wang, Huan; Shen, Rui; Chen, Guo-Qiang

2015-05-01

Many bacteria can accumulate inclusion bodies such as sulfur, polyphosphate, glycogen, proteins or polyhydroxyalkanoates. To exploit bacteria as factories for effective production of inclusion bodies, a larger intracellular space is needed for more inclusion body accumulation. In this study, polyhydroxybutyrate (PHB) was investigated as an inclusion bodies representative to be accumulated by Escherichia coli JM109SG. Various approaches were taken to increase the bacterial cell sizes including deletion on actin-like protein gene mreB, weak expression of mreB in mreB deletion mutant, and weak expression of mreB in mreB deletion mutant under inducible expression of SulA, the inhibitor of division ring protein FtsZ. All of the methods resulted in different levels of increases in bacterial sizes and PHB granules accumulation. Remarkably, an increase of over 100% PHB accumulation was observed in recombinant E. coli overexpressing mreB in an mreB deletion mutant under inducible expression of FtsZ inhibiting protein SulA. The molecular mechanism of enlarged bacterial size was found to be directly relate to weakened cytoskeleton which was the result of broken skeleton helix. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Detection of pathogenic organisms in food, water, and body fluids

NASA Astrophysics Data System (ADS)

Wallace, William H.; Henley, Michael V.; Sayler, Gary S.

2002-06-01

The construction of specific bioluminescent bacteriophage for detection of pathogenic organism can be developed to overcome interferences in complex matrices such as food, water and body fluids. Detection and identification of bacteria often require several days and frequently weeks by standard methods of isolation, growth and biochemical test. Immunoassay detection often requires the expression of the bacterial toxin, which can lead to non-detection of cells that may express the toxin under conditions different from testing protocols. Immunoassays require production of a specific antibody to the agent for detection and interference by contaminants frequently affects results. PCR based detection may be inhibited by substances in complex matrices. Modified methods of the PCR technique, such as magnetic capture-hybridization PCR (MCH-PCR), appear to improve the technique by removing the DNA products away from the inhibitors. However, the techniques required for PCR-based detection are slow and the procedures require skilled personnel working with labile reagents. Our approach is based on transferring bioluminescence (lux) genes into a selected bacteriophage. Bacteriophages are bacterial viruses that are widespread in nature and often are genus and species specific. This specificity eliminates or reduces false positives in a bacteriophage assay. The phage recognizes a specific receptor molecule on the surface of a susceptible bacterium, attaches and then injects the viral nucleic acid into the cell. The injected viral genome is expressed and then replicated, generating numerous exact copies of the viral genetic material including the lux genes, often resulting in an increase in bioluminescence by several hundred fold.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

PubMed

Romao, Ema; Morales-Yanez, Francisco; Hu, Yaozhong; Crauwels, Maxine; De Pauw, Pieter; Hassanzadeh, Gholamreza Ghassanzadeh; Devoogdt, Nick; Ackaert, Chloe; Vincke, Cecile; Muyldermans, Serge

2016-01-01

The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Spectral force analysis using atomic force microscopy reveals the importance of surface heterogeneity in bacterial and colloid adhesion to engineered surfaces.

PubMed

Ma, Huilian; Winslow, Charles J; Logan, Bruce E

2008-04-01

Coatings developed to reduce biofouling of engineered surfaces do not always perform as expected based on their native properties. One reason is that a relatively small number of highly adhesive sites, or the heterogeneity of the coated surface, may control the overall response of the system to initial bacterial deposition. It is shown here using an approach we call spectral force analysis (SFA), based on force volume imaging of the surface with atomic force microscopy, that the behavior of surfaces and coatings can be better understood relative to bacterial adhesion. The application of vapor deposited TiO(2) metal oxide increased bacterial and colloid adhesion, but coating the surface with silica oxide reduced adhesion in a manner consistent with SFA based on analysis of the "stickiest" sites. Application of a TiO(2)-based paint to a surface produced a relatively non-fouling surface. Addition of a hydrophilic layer coating to this surface should have decreased fouling. However, it was observed that this coating actually increased fouling. Using SFA it was shown that the reason for the increased adhesion of bacteria and particles to the hydrophilic layer was that the surface produced by this coating was highly heterogeneous, resulting in a small number of sites that created a stickier surface. These results show that while it is important to manufacture surfaces with coatings that are relatively non-adhesive to bacteria, it is also essential that these coatings have a highly uniform surface chemistry.

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

Antibacterial Au nanostructured surfaces.

PubMed

Wu, Songmei; Zuber, Flavia; Brugger, Juergen; Maniura-Weber, Katharina; Ren, Qun

2016-02-07

We present here a technological platform for engineering Au nanotopographies by templated electrodeposition on antibacterial surfaces. Three different types of nanostructures were fabricated: nanopillars, nanorings and nanonuggets. The nanopillars are the basic structures and are 50 nm in diameter and 100 nm in height. Particular arrangement of the nanopillars in various geometries formed nanorings and nanonuggets. Flat surfaces, rough substrate surfaces, and various nanostructured surfaces were compared for their abilities to attach and kill bacterial cells. Methicillin-resistant Staphylococcus aureus, a Gram-positive bacterial strain responsible for many infections in health care system, was used as the model bacterial strain. It was found that all the Au nanostructures, regardless their shapes, exhibited similar excellent antibacterial properties. A comparison of live cells attached to nanotopographic surfaces showed that the number of live S. aureus cells was <1% of that from flat and rough reference surfaces. Our micro/nanofabrication process is a scalable approach based on cost-efficient self-organization and provides potential for further developing functional surfaces to study the behavior of microbes on nanoscale topographies.

Bacterial community composition and structure in an Urban River impacted by different pollutant sources.

PubMed

Ibekwe, A Mark; Ma, Jincai; Murinda, Shelton E

2016-10-01

Microbial communities in terrestrial fresh water are diverse and dynamic in composition due to different environmental factors. The goal of this study was to undertake a comprehensive analysis of bacterial composition along different rivers and creeks and correlate these to land-use practices and pollutant sources. Here we used 454 pyrosequencing to determine the total bacterial community composition, and bacterial communities that are potentially of fecal origin, and of relevance to water quality assessment. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity, abundance, and community composition. Detrended correspondence analysis (DCA) and canonical correspondence analysis (CCA) were used to correlate bacterial composition in streams and creeks to different environmental parameters impacting bacterial communities in the sediment and surface water within the watershed. Bacteria were dominated by the phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria, with Bacteroidetes significantly (P<0.001) higher in all water samples than sediment, where as Acidobacteria and Actinobacteria where significantly higher (P<0.05) in all the sediment samples than surface water. Overall results, using the β diversity measures, coupled with PCoA and DCA showed that bacterial composition in sediment and surface water was significantly different (P<0.001). Also, there were differences in bacterial community composition between agricultural runoff and urban runoff based on parsimony tests using 454 pyrosequencing data. Fecal indicator bacteria in surface water along different creeks and channels were significantly correlated with pH (P<0.01), NO2 (P<0.03), and NH4N (P<0.005); and in the sediment with NO3 (P<0.015). Our results suggest that microbial community compositions were influenced by several environmental factors, and pH, NO2, and NH4 were the major environmental factors driving FIB in surface water based on CCA analysis, while NO3 was the only factor in sediment. Published by Elsevier B.V.

Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables.

PubMed

Jackson, Colin R; Randolph, Kevin C; Osborn, Shelly L; Tyler, Heather L

2013-12-01

Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. Total culturable bacteria on salad vegetables ranged from 8.0 × 10(3) to 5.5 × 10(8) CFU g(-1). The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 10(3) to 5.8 × 10(5) CFU g(-1). Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional culture-dependent methods. The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption.

Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables

PubMed Central

2013-01-01

Background Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. Results Total culturable bacteria on salad vegetables ranged from 8.0 × 103 to 5.5 × 108 CFU g-1. The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 103 to 5.8 × 105 CFU g-1. Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional culture-dependent methods. Conclusions The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption. PMID:24289725

Microbial specificity of metallic surfaces exposed to ambient seawater

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaidi, B.R.; Bard, R.F.; Tosteson, T.R.

1984-09-01

High-molecular-weight materials associated with the extracellular matrix and film found on titanium and aluminum surfaces after exposure to flowing coastal seawater were isolated. This material was purified by hydroxylapatite chromatography and subsequently employed to produce antibodies in the toad, Bufo marinus. The antibodies were immobilized on a solid support and employed to isolate adhesion-enhancing, high-molecular-weight materials from the laboratory culture media of bacterial strains recovered from the respective metallic surfaces during the course of their exposure to seawater. The adhesion-enhancing materials produced by the surface-associated bacterial strains were immunologically related to the extracellular biofouling matrix material found on the surfacesmore » from which these bacteria were isolated. The surface selectivity of these bacterial strains appeared to be based on the specificity of the interaction between adhesion-enhancing macromolecules produced by these bacteria and the surfaces in question. 30 references, 6 tables.« less

Dead bacteria reverse antibiotic-induced host defense impairment in burns.

PubMed

Chen, Lee-Wei; Chen, Pei-Hsuan; Fung, Chang-Phone; Hsu, Ching-Mei

2014-10-01

Burn patients can incur high rates of hospital-acquired infections. The mechanism of antibiotic exposure on inducing infection vulnerability has not been determined. This study aimed to examine the effects of antibiotic treatment on host defense mechanisms. First we treated C57/BL6 mice with combined antibiotic treatment after 30% to 35% total body surface area burn. Animals were sacrificed at 48 hours after sham or thermal injury treatment. Bacterial counts in intestinal lumen and mucosa were measured. Next, we treated animals with or without oral dead Escherichia coli or Staphylococcus aureus supplementation to stimulate Toll-like receptor in the intestinal mucosa. Toll-like receptor 4, antibacterial protein expression, nuclear factor (NF)-κB DNA-binding activity, and bacteria-killing activity in the intestinal mucosa; intestinal permeability; bacterial translocation to mesenteric lymph nodes; Klebsiella pneumoniae translocation; interleukin-6 in the blood; and phagocytic activity of alveolar macrophages, were assessed. Thermal injury increased microflora and NF-κB DNA-binding activity of the intestine. Systemic antibiotic treatment decreased gut microflora and increased bacterial translocation to mesenteric lymph nodes, intestinal permeability, and interleukin-6 levels in the blood. Antibiotic treatment also decreased bacteria-killing activity in intestinal mucosa and phagocytic activity of alveolar macrophages. Oral dead E coli and S aureus supplementation induced NF-κB DNA-binding activity, Toll-like receptor 4, and antibacterial protein expression of the intestinal mucosa. Taken together with the fact that dead bacteria reversed antibiotic-induced K pneumoniae translocation and intestinal and pulmonary defense impairment, we conclude that combined antibiotic treatment results in systemic host defense impairment in burns through the decrease in intestinal flora. We suggest that dead bacteria supplementation could induce nondefensin protein expression and reverse antibiotic-induced gut and lung defense impairment in burn patients. Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Gut-derived commensal bacterial products inhibit liver dendritic cell maturation by stimulating hepatic interleukin-6/signal transducer and activator of transcription 3 activity.

PubMed

Lunz, John G; Specht, Susan M; Murase, Noriko; Isse, Kumiko; Demetris, Anthony J

2007-12-01

Intraorgan dendritic cells (DCs) monitor the environment and help translate triggers of innate immunity into adaptive immune responses. Liver-based DCs are continually exposed, via gut-derived portal venous blood, to potential antigens and bacterial products that can trigger innate immunity. However, somehow the liver avoids a state of perpetual inflammation and protects central immune organs from overstimulation. In this study, we tested the hypothesis that hepatic interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) activity increases the activation/maturation threshold of hepatic DCs toward innate immune signals. The results show that the liver nuclear STAT3 activity is significantly higher than that of other organs and is IL-6-dependent. Hepatic DCs in normal IL-6 wild-type (IL-6(+/+)) mice are phenotypically and functionally less mature than DCs from IL-6-deficient (IL-6(-/-)) or STAT3-inhibited IL-6(+/+) mice, as determined by surface marker expression, proinflammatory cytokine secretion, and allogeneic T-cell stimulation. IL-6(+/+) liver DCs produce IL-6 in response to exposure to lipopolysaccharide (LPS) and cytidine phosphate guanosine oligonucleotides (CpG) but are resistant to maturation compared with IL-6(-/-) liver DCs. Conversely, exogenous IL-6 inhibits LPS-induced IL-6(-/-) liver DC maturation. IL-6/STAT3 signaling influences the liver DC expression of toll-like receptor 9 and IL-1 receptor associated kinase-M. The depletion of gut commensal bacteria in IL-6(+/+) mice with oral antibiotics decreased portal blood endotoxin levels, lowered the expression of IL-6 and phospho-STAT3, and significantly increased liver DC maturation. Gut-derived bacterial products, by stimulating hepatic IL-6/STAT3 signaling, inhibit hepatic DC activation/maturation and thereby elevate the threshold needed for translating triggers of innate immunity into adaptive immune responses. Manipulating gut bacteria may therefore be an effective strategy for altering intrahepatic immune responses.

Limonene inhibits streptococcal biofilm formation by targeting surface-associated virulence factors.

PubMed

Subramenium, Ganapathy Ashwinkumar; Vijayakumar, Karuppiah; Pandian, Shunmugiah Karutha

2015-08-01

The present study explores the efficacy of limonene, a cyclic terpene found in the rind of citrus fruits, for antibiofilm potential against species of the genus Streptococcus, which have been deeply studied worldwide owing to their multiple pathogenic efficacy. Limonene showed a concentration-dependent reduction in the biofilm formation of Streptococcus pyogenes (SF370), with minimal biofilm inhibitory concentration (MBIC) of 400 μg ml - 1. Limonene was found to possess about 75-95 % antibiofilm activity against all the pathogens tested, viz. Streptococcus pyogenes (SF370 and 5 clinical isolates), Streptococcus mutans (UA159) and Streptococcus mitis (ATCC 6249) at 400 μg ml - 1 concentration. Microscopic analysis of biofilm architecture revealed a quantitative breach in biofilm formation. Results of a surface-coating assay suggested that the possible mode of action of limonene could be by inhibiting bacterial adhesion to surfaces, thereby preventing the biofilm formation cascade. Susceptibility of limonene-treated Streptococcus pyogenes to healthy human blood goes in unison with gene expression studies in which the mga gene was found to be downregulated. Anti-cariogenic efficacy of limonene against Streptococcus mutans was confirmed, with inhibition of acid production and downregulation of the vicR gene. Downregulation of the covR, mga and vicR genes, which play a critical role in regulating surface-associated proteins in Streptococcus pyogenes and Streptococcus mutans, respectively, is yet further evidence to show that limonene targets surface-associated proteins. The results of physiological assays and gene expression studies clearly show that the surface-associated antagonistic mechanism of limonene also reduces surface-mediated virulence factors.

Identification of Mycobacterial Surface Proteins Released into Subcellular Compartments of Infected Macrophages

PubMed Central

Beatty, Wandy L.; Russell, David G.

2000-01-01

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome. PMID:11083824

Natural Sunlight Shapes Crude Oil-Degrading Bacterial Communities in Northern Gulf of Mexico Surface Waters

PubMed Central

Bacosa, Hernando P.; Liu, Zhanfei; Erdner, Deana L.

2015-01-01

Following the Deepwater Horizon (DWH) spill in 2010, an enormous amount of oil was observed in the deep and surface waters of the northern Gulf of Mexico. Surface waters are characterized by intense sunlight and high temperature during summer. While the oil-degrading bacterial communities in the deep-sea plume have been widely investigated, the effect of natural sunlight on those in oil polluted surface waters remains unexplored to date. In this study, we incubated surface water from the DWH site with amendments of crude oil, Corexit dispersant, or both for 36 days under natural sunlight in the northern Gulf of Mexico. The bacterial community was analyzed over time for total abundance, density of alkane and polycyclic aromatic hydrocarbon degraders, and community composition via pyrosequencing. Our results showed that, for treatments with oil and/or Corexit, sunlight significantly reduced bacterial diversity and evenness and was a key driver of shifts in bacterial community structure. In samples containing oil or dispersant, sunlight greatly reduced abundance of the Cyanobacterium Synechococcus but increased the relative abundances of Alteromonas, Marinobacter, Labrenzia, Sandarakinotalea, Bartonella, and Halomonas. Dark samples with oil were represented by members of Thalassobius, Winogradskyella, Alcanivorax, Formosa, Pseudomonas, Eubacterium, Erythrobacter, Natronocella, and Coxiella. Both oil and Corexit inhibited the Candidatus Pelagibacter with or without sunlight exposure. For the first time, we demonstrated the effects of light in structuring microbial communities in water with oil and/or Corexit. Overall, our findings improve understanding of oil pollution in surface water, and provide unequivocal evidence that sunlight is a key factor in determining bacterial community composition and dynamics in oil polluted marine waters. PMID:26648916

Natural Sunlight Shapes Crude Oil-Degrading Bacterial Communities in Northern Gulf of Mexico Surface Waters.

PubMed

Bacosa, Hernando P; Liu, Zhanfei; Erdner, Deana L

2015-01-01

Following the Deepwater Horizon (DWH) spill in 2010, an enormous amount of oil was observed in the deep and surface waters of the northern Gulf of Mexico. Surface waters are characterized by intense sunlight and high temperature during summer. While the oil-degrading bacterial communities in the deep-sea plume have been widely investigated, the effect of natural sunlight on those in oil polluted surface waters remains unexplored to date. In this study, we incubated surface water from the DWH site with amendments of crude oil, Corexit dispersant, or both for 36 days under natural sunlight in the northern Gulf of Mexico. The bacterial community was analyzed over time for total abundance, density of alkane and polycyclic aromatic hydrocarbon degraders, and community composition via pyrosequencing. Our results showed that, for treatments with oil and/or Corexit, sunlight significantly reduced bacterial diversity and evenness and was a key driver of shifts in bacterial community structure. In samples containing oil or dispersant, sunlight greatly reduced abundance of the Cyanobacterium Synechococcus but increased the relative abundances of Alteromonas, Marinobacter, Labrenzia, Sandarakinotalea, Bartonella, and Halomonas. Dark samples with oil were represented by members of Thalassobius, Winogradskyella, Alcanivorax, Formosa, Pseudomonas, Eubacterium, Erythrobacter, Natronocella, and Coxiella. Both oil and Corexit inhibited the Candidatus Pelagibacter with or without sunlight exposure. For the first time, we demonstrated the effects of light in structuring microbial communities in water with oil and/or Corexit. Overall, our findings improve understanding of oil pollution in surface water, and provide unequivocal evidence that sunlight is a key factor in determining bacterial community composition and dynamics in oil polluted marine waters.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

PubMed

Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

2008-01-01

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; Péchoux, Christine; Trieu-Cuot, Patrick; Hols, Pascal; Mistou, Michel-Yves; Dufrêne, Yves F.

2014-11-01

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the ``pellicle'') overlaid by a 15-45 nm thick layer of CPS (the ``capsule''). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

A light-regulated type I pilus contributes to Acinetobacter baumannii biofilm, motility and virulence functions.

PubMed

Wood, Cecily R; Ohneck, Emily J; Edelmann, Richard E; Actis, Luis A

2018-06-11

Transcriptional analyses of A. baumannii ATCC 17978 showed that the expression of A1S_2091 was enhanced in cells cultured in darkness at 24°C through a process that depended on the BlsA photoreceptor. Disruption of A1S_2091, a component of the A1S_2088-A1S_2091 polycistronic operon predicted to code for a type I chaperone/usher pilus assembly system, abolished surface motility and pellicle formation but significantly enhanced biofilm formation on plastic by bacteria cultured under darkness. Based on these observations, the A1S_2088-A1S_2091 operon was named the p hoto- r egulated p ilus ABCD ( prpABCD ) operon, with A1S_2091 coding for the PrpA pilin subunit. Unexpectedly, the comparative analyses of ATCC 17978 and prpA isogenic mutant cells cultured at 37°C showed the expression of light-regulated biofilm biogenesis and motility functions under a temperature condition that drastically affects BlsA production and its light sensing activity. These assays also suggest that 17978 cells produce alternative light-regulated adhesins and/or pili systems that enhance bacterial adhesion and biofilm formation both at 24°C and 37°C on plastic as well as on the surface of polarized A549 alveolar epithelial cells, where formation of bacterial filaments and cell chains was significantly enhanced. The inactivation of prpA also resulted in a significant reduction in virulence when tested using the Galleria mellonella virulence model. All these observations provide strong evidence showing the capacity of A. baumannii to sense light and interact with biotic and abiotic surfaces using undetermined alternative sensing and regulatory systems as well as alternative adherence and motility cellular functions that allow this pathogen to persist in different ecological niches. Copyright © 2018 American Society for Microbiology.

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes

PubMed Central

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex

2016-01-01

ABSTRACT DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. IMPORTANCE Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes. PMID:27402625

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes.

PubMed

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex; Becker, Anke

2016-09-15

DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Escherichia coli growth changes by the mediated effects after low-intensity electromagnetic irradiation of extremely high frequencies.

PubMed

Torgomyan, Heghine; Hovnanyan, Karlen; Trchounian, Armen

2013-04-01

Water is the major constituent of environmental medium and biological systems. The effects occurring in water as a result of low-intensity electromagnetic irradiation (EMI) in extremely high frequencies are supposed to be the primary mechanism to create conditions for biological responses. The EMI effects on Escherichia coli, after irradiation of their suspension, are most probably water-mediated. Indirect effects of EMI at 51.8, 53, 70.6, and 73 GHz frequencies on bacteria, through water, assay buffer (Tris-phosphate buffer with inorganic salts at low or moderate concentrations), or peptone growth medium were studied. The mediated effects of 70.6 and 73 GHz irradiated water, assay buffer, and growth medium on E. coli growth characteristics were insignificant. But the results were different for 51.8 and 53 GHz. EMI mediated effects on bacterial growth were clearly demonstrated. The effects were more strongly expressed with 53 GHz. Moreover, it was shown that 70.6 and 73 GHz similarly suppressed the cell growth after direct irradiation of E. coli in water or on solid medium. Interestingly, for 51.8 and 53 GHz the bacterial growth decreases after suspension irradiation was less, compared to the direct irradiation of bacteria on solid medium. Especially, it was also more expressed in case of 53 GHz. Also with electron microscopy, EMI-induced bacterial cell sizes and structure different changes were detected. In addition, the distinguished changes in surface tension, oxidation-reduction potential and pH of water, assay buffer, growth medium, and bacterial suspension were determined. They depended on EMI frequency used. The differences could be associated with the partial absorbance of EMI energy by the surrounding medium, which depends on a specific frequency. The results are crucial to understand biophysical mechanisms of EMI effects on bacteria.

Repressor-mediated tissue-specific gene expression in plants

DOEpatents

Meagher, Richard B [Athens, GA; Balish, Rebecca S [Oxford, OH; Tehryung, Kim [Athens, GA; McKinney, Elizabeth C [Athens, GA

2009-02-17

Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association.

PubMed

Chun, Carlene K; Troll, Joshua V; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E; Bonaldo, Maria de Fatima; Casavant, Thomas L; Soares, M Bento; Ruby, Edward G; McFall-Ngai, Margaret J

2008-08-12

The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association

PubMed Central

Chun, Carlene K.; Troll, Joshua V.; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E.; de Fatima Bonaldo, Maria; Casavant, Thomas L.; Soares, M. Bento; Ruby, Edward G.; McFall-Ngai, Margaret J.

2008-01-01

The light–organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution. PMID:18682555

DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

PubMed Central

Halverson, Tyler W.R.; Wilton, Mike; Poon, Karen K. H.; Petri, Björn; Lewenza, Shawn

2015-01-01

Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies. PMID:25590621

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

PubMed

Singh, Anirudh K; Woodiga, Shireen A; Grau, Margaret A; King, Samantha J

2017-03-01

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis , like Streptococcus gordonii and Streptococcus sanguinis , binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. Copyright © 2017 American Society for Microbiology.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin

PubMed Central

2011-01-01

Background In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. Results The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. Conclusions This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis. PMID:21658265

Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.

2011-02-17

Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular {alpha}-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins ({approx}99%) with known functions were expressed, whereas only approximately 80% of hypothetical proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in bothmore » bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.« less

Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

PubMed

Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

2013-01-01

Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

CRISPR Perturbation of Gene Expression Alters Bacterial Fitness under Stress and Reveals Underlying Epistatic Constraints.

PubMed

Otoupal, Peter B; Erickson, Keesha E; Escalas-Bordoy, Antoni; Chatterjee, Anushree

2017-01-20

The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance. We examine the impact of gene expression changes on bacterial adaptation by constructing a library of deactivated CRISPR-Cas9 synthetic devices to tune the expression of a set of stress-response genes in Escherichia coli. We show that artificially inducing perturbations in gene expression imparts significant synthetic control over fitness and growth during stress exposure. We present evidence that these impacts are reversible; strains with synthetically perturbed gene expression regained wild-type growth phenotypes upon stress removal, while maintaining divergent growth characteristics under stress. Furthermore, we demonstrate a prevailing trend toward negative epistatic interactions when multiple gene perturbations are combined simultaneously, thereby posing an intrinsic constraint on gene expression underlying adaptive trajectories. Together, these results emphasize how CRISPR-Cas9 can be employed to engineer gene expression changes that shape bacterial adaptation, and present a novel approach to synthetically control the evolution of antimicrobial resistance.

Factors influencing bacterial adhesion to contact lenses.

PubMed

Dutta, Debarun; Cole, Nerida; Willcox, Mark

2012-01-01

The process of any contact lens related keratitis generally starts with the adhesion of opportunistic pathogens to contact lens surface. This article focuses on identifying the factors which have been reported to affect bacterial adhesion to contact lenses. Adhesion to lenses differs between various genera/species/strains of bacteria. Pseudomonas aeruginosa, which is the predominant causative organism, adheres in the highest numbers to both hydrogel and silicone hydrogel lenses in vitro. The adhesion of this strain reaches maximum numbers within 1h in most in vitro studies and a biofilm has generally formed within 24 h of cells adhering to the lens surface. Physical and chemical properties of contact lens material affect bacterial adhesion. The water content of hydroxyethylmethacrylate (HEMA)-based lenses and their iconicity affect the ability of bacteria to adhere. The higher hydrophobicity of silicone hydrogel lenses compared to HEMA-based lenses has been implicated in the higher numbers of bacteria that can adhere to their surfaces. Lens wear has different effects on bacterial adhesion, partly due to differences between wearers, responses of bacterial strains and the ability of certain tear film proteins when bound to a lens surface to kill certain types of bacteria.

Kinetics of Pseudomonas aeruginosa adhesion to 304 and 316-L stainless steel: role of cell surface hydrophobicity.

PubMed Central

Vanhaecke, E; Remon, J P; Moors, M; Raes, F; De Rudder, D; Van Peteghem, A

1990-01-01

Fifteen different isolates of Pseudomonas aeruginosa were used to study the kinetics of adhesion to 304 and 316-L stainless steel. Stainless steel plates were incubated with approximately 1.5 X 10(7) CFU/ml in 0.01 M phosphate-buffered saline (pH 7.4). After the plates were rinsed with the buffer, the number of adhering bacteria was determined by a bioluminescence assay. Measurable adhesion, even to the electropolished surfaces, occurred within 30 s. Bacterial cell surface hydrophobicity, as determined by the bacterial adherence to hydrocarbons test and the contact angle measurement test, was the major parameter influencing the adhesion rate constant for the first 30 min of adhesion. A parabolic relationship between the CAM values and the logarithm of the adhesion rate constants (In k) was established. No correlation between either the salt aggregation or the improved salt aggregation values and the bacterial adhesion rate constants could be found. Since there was no significant correlation between the bacterial electrophoretic mobilities and the In k values, the bacterial cell surface charge seemed of minor importance in the process of adhesion of P. aeruginosa to 304 and 316-L stainless steel. PMID:2107796

Factors influencing bacterial adhesion to contact lenses

PubMed Central

Dutta, Debarun; Willcox, Mark

2012-01-01

The process of any contact lens related keratitis generally starts with the adhesion of opportunistic pathogens to contact lens surface. This article focuses on identifying the factors which have been reported to affect bacterial adhesion to contact lenses. Adhesion to lenses differs between various genera/species/strains of bacteria. Pseudomonas aeruginosa, which is the predominant causative organism, adheres in the highest numbers to both hydrogel and silicone hydrogel lenses in vitro. The adhesion of this strain reaches maximum numbers within 1h in most in vitro studies and a biofilm has generally formed within 24 h of cells adhering to the lens surface. Physical and chemical properties of contact lens material affect bacterial adhesion. The water content of hydroxyethylmethacrylate (HEMA)-based lenses and their iconicity affect the ability of bacteria to adhere. The higher hydrophobicity of silicone hydrogel lenses compared to HEMA-based lenses has been implicated in the higher numbers of bacteria that can adhere to their surfaces. Lens wear has different effects on bacterial adhesion, partly due to differences between wearers, responses of bacterial strains and the ability of certain tear film proteins when bound to a lens surface to kill certain types of bacteria. PMID:22259220

Active screen cage pulsed dc discharge for implanting copper in polytetrafluoroethylene (PTFE)

NASA Astrophysics Data System (ADS)

Zaka-ul-Islam, Mujahid; Naeem, Muhammad; Shafiq, Muhammad; Sitara; Jabbar Al-Rajab, Abdul; Zakaullah, Muhammad

2017-07-01

Polymers such as polytetrafluoroethylene (PTFE) are widely used in artificial organs where long-term anti-bacterial properties are required to avoid bacterial proliferation. Copper or silver ion implantation on the polymer surface is known as a viable method to generate long-term anti-bacterial properties. Here, we have tested pulsed DC plasma with copper cathodic cage for the PTFE surface treatment. The surface analysis of the treated specimens suggests that the surface, structural properties, crystallinity and chemical structure of the PTFE have been changed, after the plasma treatment. The copper release tests show that copper ions are released from the polymer at a slow rate and quantity of the released copper increases with the plasma treatment time.

Effects of Material Properties on Bacterial Adhesion and Biofilm Formation.

PubMed

Song, F; Koo, H; Ren, D

2015-08-01

Adhesion of microbes, such as bacteria and fungi, to surfaces and the subsequent formation of biofilms cause multidrug-tolerant infections in humans and fouling of medical devices. To address these challenges, it is important to understand how material properties affect microbe-surface interactions and engineer better nonfouling materials. Here we review the recent progresses in this field and discuss the main challenges and opportunities. In particular, we focus on bacterial biofilms and review the effects of surface energy, charge, topography, and stiffness of substratum material on bacterial adhesion. We summarize how these surface properties influence oral biofilm formation, and we discuss the important findings from nondental systems that have potential applications in dental medicine. © International & American Associations for Dental Research 2015.

Effect of UV-photofunctionalization on Oral Bacterial Attachment and Biofilm Formation to Titanium Implant Material

PubMed Central

de Avila, Erica Dorigatti; Lima, Bruno P.; Sekiya, Takeo; Torii, Yasuyoshi; Ogawa, Takahiro; Shi, Wenyuan; Lux, Renate

2015-01-01

Bacterial biofilm infections remain prevalent reasons for implant failure. Dental implant placement occurs in the oral environment, which harbors a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part of the implant structure is exposed to oral cavity and there is no effective measure to prevent bacterial attachment to implant materials. Here, we demonstrated that UV treatment of titanium immediately prior to use (photofunctionalization) affects the ability of human polymicrobial oral biofilm communities to colonize in the presence of salivary and blood components. UV-treatment of machined titanium transformed the surface from hydrophobic to superhydrophilic. UV-treated surfaces exhibited a significant reduction in bacterial attachment as well as subsequent biofilm formation compared to untreated ones, even though overall bacterial viability was not affected. The function of reducing bacterial colonization was maintained on UV-treated titanium that had been stored in a liquid environment before use. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed. Our findings confirm that UV-photofunctionalization of titanium has a strong potential to improve outcome of implant placement by creating and maintaining antimicrobial surfaces. PMID:26210175

Bacterial Abundance and Activity across Sites within Two Northern Wisconsin Sphagnum Bogs.

PubMed

Fisher; Graham; Graham

1998-11-01

Abstract Bacterial abundance, temperature, pH, and dissolved organic carbon (DOC) concentration were compared across surface sites within and between two northern Wisconsin Sphagnum peatlands over the summer seasons in 1995 and 1996. Sites of interest were the Sphagnum mat surface, the water-filled moat (lagg) at the bog margin, and the bog lake littoral zone. Significant differences in both bacterial populations and water chemistry were observed between sites. pH was highest in the lake and lowest in the mat at both bogs; the opposite was true for DOC. Large populations of bacteria were present in surface interstitial water from the mat; abundance in this site was consistently higher than in the moat or lake. Bacterial abundance also increased across sites of increasing DOC concentration and declining pH. Bacterial activities (rates of [3H]leucine incorporation) and growth in dilution cultures (with grazers removed) were also assessed in lake, moat, and mat sites. Results using these measures generally supported the trends observed in abundance, although high rates of [3H]leucine incorporation were recorded in the moat at one of the bogs. Our results indicate that bacterial populations in Sphagnum peatlands are not adversely affected by acidity, and that DOC may be more important than pH in determining bacterial abundance in these environments.

The effects of interfacial potential on antimicrobial propensity of ZnO nanoparticle

PubMed Central

Arakha, Manoranjan; Saleem, Mohammed; Mallick, Bairagi C.; Jha, Suman

2015-01-01

The work investigates the role of interfacial potential in defining antimicrobial propensity of ZnO nanoparticle (ZnONP) against different Gram positive and Gram negative bacteria. ZnONPs with positive and negative surface potential are tested against different bacteria with varying surface potentials, ranging −14.7 to −23.6 mV. Chemically synthesized ZnONPs with positive surface potential show very high antimicrobial propensity with minimum inhibitory concentration of 50 and 100 μg/mL for Gram negative and positive bacterium, respectively. On other hand, ZnONPs of the same size but with negative surface potential show insignificant antimicrobial propensity against the studied bacteria. Unlike the positively charged nanoparticles, neither Zn2+ ion nor negatively charged ZnONP shows any significant inhibition in growth or morphology of the bacterium. Potential neutralization and colony forming unit studies together proved adverse effect of the resultant nano-bacterial interfacial potential on bacterial viability. Thus, ZnONP with positive surface potential upon interaction with negative surface potential of bacterial membrane enhances production of the reactive oxygen species and exerts mechanical stress on the membrane, resulting in the membrane depolarization. Our results show that the antimicrobial propensity of metal oxide nanoparticle mainly depends upon the interfacial potential, the potential resulting upon interaction of nanoparticle surface with bacterial membrane. PMID:25873247

Athletic equipment microbiota are shaped by interactions with human skin

DOE PAGES

Wood, Mariah; Gibbons, Sean M.; Lax, Simon; ...

2015-06-19

Background: Americans spend the vast majority of their lives in built environments. Even traditionally outdoor pursuits, such as exercising, are often now performed indoors. Bacteria that colonize these indoor ecosystems are primarily derived from the human microbiome. The modes of human interaction with indoor surfaces and the physical conditions associated with each surface type determine the steady-state ecology of the microbial community. Results: Bacterial assemblages associated with different surfaces in three athletic facilities, including floors, mats, benches, free weights, and elliptical handles, were sampled every other hour (8 am to 6 pm) for 2 days. Surface and equipment type hadmore » a stronger influence on bacterial community composition than the facility in which they were housed. Surfaces that were primarily in contact with human skin exhibited highly dynamic bacterial community composition and non-random co-occurrence patterns, suggesting that different host microbiomes—shaped by selective forces—were being deposited on these surfaces through time. Bacterial assemblages found on the floors and mats changed less over time, and species co-occurrence patterns appeared random, suggesting more neutral community assembly. Conclusions: These longitudinal patterns highlight the dramatic turnover of microbial communities on surfaces in regular contact with human skin. By uncovering these longitudinal patterns, this study promotes a better understanding of microbe-human interactions within the built environment.« less

Role of gravity in the formation of bacterial colonies with a hydrophobic surface layer

NASA Astrophysics Data System (ADS)

Puzyr, A. P.; Tirranen, L. K.; Krylova, T. Y.; Borodina, E. V.

A simple technique for determining hydrophobic-hydrophilic properties of bacterial colonies surface, which involves putting a drop of liquid with known properties (e.g. water, oil) on their surface, has been described. This technique allows quick estimate of wettability of bacterial colony surface, i.e. its hydrophobic-hydrophilic properties. The behaviour of water drops on colonies of bacteria Bacillus five strains (of different types) has been studied. It was revealed that 1) orientation in the Earth gravity field during bacterial growth can define the form of colonies with hydrophobic surface; 2) the form and size of the colony are dependent on the extention ability, most probably, of the hydrophobic layer; 3) the Earth gravity field (gravity) serves as a 'pump' providing and keeping water within the colony. We suppose that at growing colonies on agar media the inflow of water-soluble nutrient materials takes place both due to diffusion processes and directed water current produced by the gravity. The revealed effect probably should be taken into consideration while constructing the models of colonies growing on dense nutrient media. The easily determined hydrophobic properties of colonies surface can become a systematic feature after collecting more extensive data on the surface hydrophobic-hydrophilic properties of microorganism colonies of other types and species.

Air, water, and surface bacterial contamination in a university-hospital autopsy room.

PubMed

Maujean, Géraldine; Malicier, Daniel; Fanton, Laurent

2012-03-01

Today, little is known about the bacteriological environment of the autopsy room and its potential interest for medico-legal practices. Seven hundred fifty microbiological samples were taken from surface (n = 660), air (n = 48), and water (n = 42) to evaluate it in a French University Forensic Department. Median bacterial counts were compared before and during autopsy for air samples, and before and after autopsy for surface samples, using Wilcoxon matched pairs signed ranks test. Bacterial identification relied on traditional phenotypic methods. Bacterial counts in the air were low before autopsy, increased significantly during procedure, and seemed more linked to the number of people in the room than to an important production of aerosol-containing bacteria. Despite cleaning, human fecal flora was omnipresent on surfaces, which revealed insufficient disinfection. Bacteriological sampling is an easy way to monitor cleaning practices in postmortem rooms, but chiefly a way to improve the reliability of medico-legal proofs of infectious deaths. © 2012 American Academy of Forensic Sciences.

MicroRNA-155 regulates host immune response to postviral bacterial pneumonia via IL-23/IL-17 pathway.

PubMed

Podsiad, Amy; Standiford, Theodore J; Ballinger, Megan N; Eakin, Richard; Park, Pauline; Kunkel, Steven L; Moore, Bethany B; Bhan, Urvashi

2016-03-01

Postinfluenza bacterial pneumonia is associated with significant mortality and morbidity. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. miR-155 has recently emerged as a crucial regulator of innate immunity and inflammatory responses and is induced in macrophages during infection. We hypothesized upregulation of miR-155 inhibits IL-17 and increases susceptibility to secondary bacterial pneumonia. Mice were challenged with 100 plaque-forming units H1N1 intranasally and were infected with 10(7) colony-forming units of MRSA intratracheally at day 5 postviral challenge. Lungs were harvested 24 h later, and expression of miR-155, IL-17, and IL-23 was measured by real-time RT-PCR. Induction of miR-155 was 3.6-fold higher in dual-infected lungs compared with single infection. miR-155(-/-) mice were protected with significantly lower (4-fold) bacterial burden and no differences in viral load, associated with robust induction of IL-23 and IL-17 (2.2- and 4.8-fold, respectively) postsequential challenge with virus and bacteria, compared with WT mice. Treatment with miR-155 antagomir improved lung bacterial clearance by 4.2-fold compared with control antagomir postsequential infection with virus and bacteria. Moreover, lung macrophages collected from patients with postviral bacterial pneumonia also had upregulation of miR-155 expression compared with healthy controls, consistent with observations in our murine model. This is the first demonstration that cellular miRNAs regulate postinfluenza immune response to subsequent bacterial challenge by suppressing the IL-17 pathway in the lung. Our findings suggest that antagonizing certain microRNA might serve as a potential therapeutic strategy against secondary bacterial infection. Copyright © 2016 the American Physiological Society.

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus

PubMed Central

Hieggelke, Lena; Schneiker, Bianca; Lysson, Mariola; Stoffels, Burkhard; Nuding, Sabine; Wehkamp, Jan; Kikhney, Judith; Moter, Annette; Kalff, Joerg C.

2018-01-01

Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery. PMID:29652914

Patterns and drivers of bacterial α- and β-diversity across vertical profiles from surface to subsurface sediments.

PubMed

Luna, Gian Marco; Corinaldesi, Cinzia; Rastelli, Eugenio; Danovaro, Roberto

2013-10-01

We investigated the patterns and drivers of bacterial α- and β-diversity, along with viral and prokaryotic abundance and the carbon production rates, in marine surface and subsurface sediments (down to 1 m depth) in two habitats: vegetated sediments (seagrass meadow) and non-vegetated sediments. Prokaryotic abundance and production decreased with depth in the sediment, but cell-specific production rates and the virus-to-prokaryote ratio increased, highlighting unexpectedly high activity in the subsurface. The highest diversity was observed in vegetated sediments. Bacterial β-diversity between sediment horizons was high, and only a minor number of taxa was shared between surface and subsurface layers. Viruses significantly contributed to explain α- and β-diversity patterns. Despite potential limitations due to the only use of fingerprinting techniques, this study indicates that the coastal subsurface host highly active and diversified bacterial assemblages, that subsurface cells are more active than expected and that viruses promote β-diversity and stimulate bacterial metabolism in subsurface layers. The limited number of taxa shared between habitats, and between surface and subsurface sediment horizons, suggests that future investigations of the shallow subsurface will provide insights into the census of bacterial diversity, and the comprehension of the patterns and drivers of prokaryotic diversity in marine ecosystems. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli

PubMed Central

Brandl, Maria T.; Kudva, Indira T.; Katani, Robab; Moreau, Matthew R.; Kapur, Vivek

2017-01-01

ABSTRACT Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH. The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments. PMID:29054868

Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children.

PubMed

Wong, Hector R; Cvijanovich, Natalie Z; Hall, Mark; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Lin, Richard; Bigham, Michael T; Sen, Anita; Nowak, Jeffrey; Quasney, Michael; Henricksen, Jared W; Chopra, Arun; Banschbach, Sharon; Beckman, Eileen; Harmon, Kelli; Lahni, Patrick; Shanley, Thomas P

2012-10-29

Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27. The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607.

Tuning bacterial hydrodynamics with magnetic fields

NASA Astrophysics Data System (ADS)

Pierce, C. J.; Mumper, E.; Brown, E. E.; Brangham, J. T.; Lower, B. H.; Lower, S. K.; Yang, F. Y.; Sooryakumar, R.

2017-06-01

Magnetotactic bacteria are a group of motile prokaryotes that synthesize chains of lipid-bound, magnetic nanoparticles called magnetosomes. This study exploits their innate magnetism to investigate previously unexplored facets of bacterial hydrodynamics at surfaces. Through use of weak, uniform, external magnetic fields and local, micromagnetic surface patterns, the relative strength of hydrodynamic, magnetic, and flagellar force components is tuned through magnetic control of the bacteria's orientation. The resulting swimming behaviors provide a means to experimentally determine hydrodynamic parameters and offer a high degree of control over large numbers of living microscopic entities. The implications of this controlled motion for studies of bacterial motility near surfaces and for micro- and nanotechnology are discussed.

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

PubMed Central

Wang, Ying; Hougaard, Anni B.; Paulander, Wilhelm; Skibsted, Leif H.

2015-01-01

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals. PMID:26150471

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures.

PubMed

Wang, Ying; Hougaard, Anni B; Paulander, Wilhelm; Skibsted, Leif H; Ingmer, Hanne; Andersen, Mogens L

2015-09-01

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A novel point mutation in CD18 causing the expression of dysfunctional CD11/CD18 leucocyte integrins in a patient with leucocyte adhesion deficiency (LAD)

PubMed Central

Mathew, E C; Shaw, J M; Bonilla, F A; Law, S K A; Wright, D A

2000-01-01

Leucocyte adhesion deficiency type 1 (LAD-1) is characterized by the incapacity of leucocytes to carry out their adhesion functions via their CD11/CD18 antigens, which are also referred to as the leucocyte integrins. The patients generally suffer from poor wound healing and recurrent bacterial and fungal infections. In severe cases, the infections are often systemic and life-threatening. A LAD patient (AW) of moderate phenotype has been identified but, unlike most other cases, the level of CD11/CD18 antigens on her leucocytes are uncharacteristically high for a LAD patient. Molecular analysis revealed that she is a compound heterozygote for CD18 mutations. She has inherited a D231H mutation from her father and a G284S mutation from her mother. By transfection studies, it was established that the G284S mutation does not support CD11/CD18 antigen expression on the cell surface. In contrast, the D231H mutation does not affect CD18 forming integrin heterodimers with the CD11 antigens on the cell surface. However, the expressed integrins with the D231H mutation are not adhesive to ligands. PMID:10886250

[Biofilm: set-up and organization of a bacterial community].

PubMed

Filloux, Alain; Vallet, Isabelle

2003-01-01

Bacterial attachment on various surfaces mostly takes place in the form of specialised bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surface, resulting in an organised structure. In this review we have been using Pseudomonas aeruginosa as a model microorganism to describe the series of events that occurred during this developmental process. P. aeruginosa is an opportunistic pathogen that has a wide variety of hosts and infectious sites. In addition to biofilm formation in certain tissues, inert surfaces, such as catheters, are also target for bacterial biofilm development. The use of convenient genetic screens has made possible the identification of numerous biofilm-defective mutants, which have been characterised further. These studies have allowed the proposal for a global model, in which key events are described for the different stages of biofilm formation. Briefly, flagellar mobility is crucial for approaching the surface, whereas type IV pili motility is preponderant for surface colonisation and microcolonies formation. These microcolonies are finally packed together and buried in an exopolysaccharide matrix to form the differentiated bio-film. It is obvious that the different stages of biofilm formation also involved perception of environmental stimuli. These stimuli, and their associated complex regulatory networks, have still to be fully characterised to understand the bacterial strategy, which initiates biofilm formation. One such regulatory system, called Quorum sensing, is one of the key player in the initial differentiation of biofilm. Finally, a better understanding, at the molecular level, of biofilm establishment and persistence should help for the design of antimicrobials that prevent bacterial infections.

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages

PubMed Central

Vargas García, Cynthia E.; Petrova, Mariya; Claes, Ingmar J. J.; De Boeck, Ilke; Verhoeven, Tine L. A.; Dilissen, Ellen; von Ossowski, Ingemar; Palva, Airi; Bullens, Dominique M.; Vanderleyden, Jos

2015-01-01

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili. PMID:25576613

Inactivation of pathogenic bacteria inoculated onto a Bacto™ agar model surface using TiO2-UVC photocatalysis, UVC and chlorine treatments.

PubMed

Yoo, S; Ghafoor, K; Kim, S; Sun, Y W; Kim, J U; Yang, K; Lee, D-U; Shahbaz, H M; Park, J

2015-09-01

The aim of this study was to study inactivation of different pathogenic bacteria on agar model surface using TiO2-UV photocatalysis (TUVP). A unified food surface model was simulated using Bacto(™) agar, a routinely used microbial medium. The foodborne pathogenic bacteria Escherichia coli K12 (as a surrogate for E. coli O157:H7), Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes were inoculated onto the agar surface, followed by investigation of TUVP-assisted inactivation and morphological changes in bacterial cells. The TUVP process showed higher bacterial inactivation, particularly for Gram-negative bacteria, than UVC alone and a control (dark reaction). A TUVP treatment of 17·2 mW cm(-2) (30% lower than the UVC light intensity) reduced the microbial load on the agar surface by 4·5-6·0 log CFU cm(-2). UVC treatment of 23·7 mW cm(-2) caused 3·0-5·3 log CFU cm(-2) reduction. The use of agar model surface is effective for investigation of bacterial disinfection and TUVP is a promising nonthermal technique. The results showing effects of photocatalysis and other treatments for inactivation of bacterial pathogens on model surface can be useful for applying such processes for disinfection of fruit, vegetables and other similar surfaces. © 2015 The Society for Applied Microbiology.

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

PubMed Central

2010-01-01

Background Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. Results Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling. Conclusions We describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches. PMID:20626840

Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea.

PubMed

Sánchez, Joaquín; Holmgren, Jan

2005-08-01

Recent work has provided new insights into the pathogenesis of the potentially life-threatening diarrheas caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC): a new mechanism (post-translational degradation), which is involved in the control of cholera toxin expression, has been discovered. Recent evidence also suggests that vibrios upregulate cholera toxin expression in response to intestinal fluid components, and enterotoxin-carrying bacterial outer membrane vesicles might have a function in ETEC pathogenesis. An important role of the environment is supported by the correlation between cholera incidence and elevated sea surface temperature, which supports the notion that the zooplankton is a V. cholerae reservoir. Additionally, environmental lytic cholera phages could influence cholera seasonality by 'terminating' the seasonal epidemic. Finally, the strong herd immunity elicited by an oral cholera vaccine indicates that cholera vaccination could have a significant public health impact.

Isolation, screening, and characterization of surface-active agent-producing, oil-degrading marine bacteria of Mumbai Harbor.

PubMed

Mohanram, Rajamani; Jagtap, Chandrakant; Kumar, Pradeep

2016-04-15

Diverse marine bacterial species predominantly found in oil-polluted seawater produce diverse surface-active agents. Surface-active agents produced by bacteria are classified into two groups based on their molecular weights, namely biosurfactants and bioemulsifiers. In this study, surface-active agent-producing, oil-degrading marine bacteria were isolated using a modified Bushnell-Haas medium with high-speed diesel as a carbon source from three oil-polluted sites of Mumbai Harbor. Surface-active agent-producing bacterial strains were screened using nine widely used methods. The nineteen bacterial strains showed positive results for more than four surface-active agent screening methods; further, these strains were characterized using biochemical and nucleic acid sequencing methods. Based on the results, the organisms belonged to the genera Acinetobacter, Alcanivorax, Bacillus, Comamonas, Chryseomicrobium, Halomonas, Marinobacter, Nesterenkonia, Pseudomonas, and Serratia. The present study confirmed the prevalence of surface-active agent-producing bacteria in the oil-polluted waters of Mumbai Harbor. Copyright © 2016 Elsevier Ltd. All rights reserved.

An in vitro bacterial adhesion assessment of surface-modified medical-grade PVC.

PubMed

Asadinezhad, Ahmad; Novák, Igor; Lehocký, Marián; Sedlarík, Vladimir; Vesel, Alenka; Junkar, Ita; Sáha, Petr; Chodák, Ivan

2010-06-01

Medical-grade polyvinyl chloride was surface modified by a multistep physicochemical approach to improve bacterial adhesion prevention properties. This was fulfilled via surface activation by diffuse coplanar surface barrier discharge plasma followed by radical graft copolymerization of acrylic acid through surface-initiated pathway to render a structured high density brush. Three known antibacterial agents, bronopol, benzalkonium chloride, and chlorhexidine, were then individually coated onto functionalized surface to induce biological properties. Various modern surface probe techniques were employed to explore the effects of the modification steps. In vitro bacterial adhesion and biofilm formation assay was performed. Escherichia coli strain was found to be more susceptible to modifications rather than Staphylococcus aureus as up to 85% reduction in adherence degree of the former was observed upon treating with above antibacterial agents, while only chlorhexidine could retard the adhesion of the latter by 50%. Also, plasma treated and graft copolymerized samples were remarkably effective to diminish the adherence of E. coli. Copyright 2010 Elsevier B.V. All rights reserved.

Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium.

PubMed

Murakami, Asuka; Arimoto, Takafumi; Suzuki, Dai; Iwai-Yoshida, Misato; Otsuka, Fukunaga; Shibata, Yo; Igarashi, Takeshi; Kamijo, Ryutaro; Miyazaki, Takashi

2012-04-01

Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier

PubMed Central

Pawar, Rahul D; Castrezana-Lopez, Liliana; Allam, Ramanjaneyulu; Kulkarni, Onkar P; Segerer, Stephan; Radomska, Ewa; Meyer, Tobias N; Schwesinger, Catherine-Meyer; Akis, Nese; Gröne, Hermann-Josef; Anders, Hans-Joachim

2009-01-01

What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRLlpr/lpr mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)-6, IL-12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non-nephritic mice. This was associated with down-regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRLlpr/lpr mice. Bacterial lipopeptide activates Toll-like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)-γ induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRLlpr/lpr mice. PMID:19175801

The combination of decoy receptor 3 and soluble triggering receptor expressed on myeloid cells-1 for the diagnosis of nosocomial bacterial meningitis.

PubMed

Liu, Yong-Juan; Shao, Li-Hua; Zhang, Jian; Fu, Shan-Ji; Wang, Gang; Chen, Feng-Zhe; Zheng, Feng; Ma, Rui-Ping; Liu, Hai-Hong; Dong, Xiao-Meng; Ma, Li-Xian

2015-03-23

Early diagnosis and appropriate antibiotic treatment can significantly reduce mortality of nosocomial bacterial meningitis. However, it is a challenge for clinicians to make an accurate and rapid diagnosis of bacterial meningitis. This study aimed at determining whether combined biomarkers can provide a useful tool for the diagnosis of bacterial meningitis. A retrospective study was carried out. Cerebrospinal fluid (CSF) levels of decoy receptor 3 (DcR3) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) were detected by enzyme-linked immunosorbent assay (ELISA). The patients with bacterial meningitis had significantly elevated levels of the above mentioned biomarkers. The two biomarkers were all risk factors with bacterial meningitis. The biomarkers were constructed into a "bioscore". The discriminative performance of the bioscore was better than that of each biomarker, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.842 (95% confidence intervals (CI) 0.770-0.914; p< 0.001). Combined measurement of CSF DcR3 and sTREM-1 concentrations improved the prediction of nosocomial bacterial meningitis. The combined strategy is of interest and the validation of that improvement needs further studies.

Electrical response of culture media during bacterial growth on a paper-based device

NASA Astrophysics Data System (ADS)

Srimongkon, Tithimanan; Buerkle, Marius; Nakamura, Akira; Enomae, Toshiharu; Ushijima, Hirobumi; Fukuda, Nobuko

2017-05-01

In this work, we evaluated the feasibility of a paper-based bacterial detection system. The paper served as a substrate for the measurement electrodes and the culture medium. Using a printing technique, we patterned gold electrodes onto the paper substrate and applied Luria broth (LB) agar gel as a culture medium on top of the electrodes. As the first step towards the development of a bacterial detection system, we determined changes in the surface potential during bacterial growth and monitored these changes over 24 h. This allowed us to correlate changes in the surface potential with the different growth phases of the bacteria.

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

PubMed Central

Heithoff, D M; Sinsheimer, R L; Low, D A; Mahan, M J

2000-01-01

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials. PMID:10874736

Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression.

PubMed

Lee, Wilson D; Flynn, Andrew N; LeBlanc, Justin M; Merrill, John K; Dick, Paul; Morck, Douglas W; Buret, Andre G

2004-01-01

The pathology of bacterial pneumonia, such as seen in the bovine lung infected with Mannheimia haemolytica, is due to pathogen virulence factors and to inflammation initiated by the host. Tilmicosin is a macrolide effective in treating bacterial pneumonia and recent findings suggest that this antibiotic may provide anti-inflammatory benefits by inducing polymorphonuclear neutrophilic leukocyte (PMN) apoptosis. Using an in vitro bovine system, we examined the cell-specificity of tilmicosin, characterized the changes in spontaneous leukotriene B4 (LTB4) synthesis by PMN exposed to the macrolide, and assessed its effects on PMN Fas expression. Previous findings demonstrated that tilmicosin is able to induce PMN apoptosis. These results were confirmed in this study by the Annexin-V staining of externalized phosphatidylserine and the analysis with flow cytometry. The cell-specificity of tilmicosin was assessed by quantification of apoptosis in bovine PMN, mononuclear leukocytes, monocyte-derived macrophages, endothelial cells, epithelial cells, and fibroblasts cultured with the macrolide. The effect of tilmicosin on spontaneous LTB4 production by PMN was evaluated via an enzyme-linked immunosorbent assay. Finally, the mechanisms of tilmicosin-induced PMN apoptosis were examined by assessing the effects of tilmicosin on surface Fas expression on PMN. Tilmicosin-induced apoptosis was found to be at least partially cell-specific, as PMN were the only cell type tested to die via apoptosis in response to incubation with tilmicosin. PMN incubated with tilmicosin under conditions that induce apoptosis spontaneously produced less LTB4, but did not exhibit altered Fas expression. In conclusion, tilmicosin-induced apoptosis is specific to PMN, inhibits spontaneous LTB4 production, and occurs through a pathway independent of Fas upregulation.

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

PubMed

Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

2016-08-11

Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Plasma surface modification of rigid contact lenses decreases bacterial adhesion.

PubMed

Wang, Yingming; Qian, Xuefeng; Zhang, Xiaofeng; Xia, Wei; Zhong, Lei; Sun, Zhengtai; Xia, Jing

2013-11-01

Contact lens safety is an important topic in clinical studies. Corneal infections usually occur because of the use of bacteria-carrying contact lenses. The current study investigated the impact of plasma surface modification on bacterial adherence to rigid contact lenses made of fluorosilicone acrylate materials. Boston XO and XO2 contact lenses were modified using plasma technology (XO-P and XO2-P groups). Untreated lenses were used as controls. Plasma-treated and control lenses were incubated in solutions containing Staphylococcus aureus or Pseudomonas aeruginosa. MTT colorimetry, colony-forming unit counting method, and scanning electron microscopy were used to measure bacterial adhesion. MTT colorimetry measurements showed that the optical density (OD) values of XO-P and XO2-P were significantly lower than those of XO and XO2, respectively, after incubation with S. aureus (P < 0.01). The OD value of XO-P was also much lower than that of XO after incubation with P. aeruginosa (P < 0.01). Colony-forming unit counting revealed that a significantly lower number of bacterial colonies attached to the XO-P versus XO lenses and to the XO2-P versus XO2 lenses incubated with S. aureus (P < 0.01). Fewer bacterial colonies attached to the XO-P versus XO lenses incubated with P. aeruginosa (P < 0.01). Further, scanning electron microscopy suggested different bacterial adhesion morphology on plasma-treated versus control lenses. Plasma surface modification can significantly decrease bacterial adhesion to fluorosilicone acrylate contact lenses. This study provides important evidence of a unique benefit of plasma technology in contact lens surface modification.

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli.

PubMed

McCracken, Vance J; Chun, Taehoon; Baldeón, Manuel E; Ahrné, Siv; Molin, Göran; Mackie, Roderick I; Gaskins, H Rex

2002-09-01

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.

Development of the primary bacterial microfouling layer on antifouling and fouling release coatings in temperate and tropical environments in Eastern Australia.

PubMed

Molino, Paul J; Childs, Samantha; Eason Hubbard, Maeve R; Carey, Janet M; Burgman, Mark A; Wetherbee, Richard

2009-01-01

The role played by bacteria during the pioneering stages of colonisation on marine coatings was investigated over three distinct seasons in both tropical and temperate environments. Novel methods were developed to facilitate the study of the adhered bacterial population on the test coatings in their native, hydrated state. The approach eliminated destructive sample preparation techniques, including sample dehydration and/or removal from the substratum surface prior to analysis. Bacterial colonisation during initial biofilm formation was evaluated on two antifouling paints, Intersmooth 360 and Super Yacht 800, and a fouling release coating, Intersleek 700. Bacterial colonisation was quantified on all three coating surfaces. Intersleek 700 displayed the quickest colonisation by bacteria, resulting in major modification of the substratum surface within 2-4 days following immersion in the ocean. Whilst fouling accumulated more quickly on Intersleek 700, by 16 days all three coatings were fouled significantly. Bacterial fouling was correlated to both location and season, with fouling occurring at a more rapid rate at the Cairns location, as well as during the summer months, when higher water temperatures were recorded. Successful colonisation of all coatings by bacteria soon after immersion modifies the characteristics of the surfaces at the hull/water interface, and subsequent settlement by higher biofouling organisms must be moderated by these modified surfaces.

Bacterial attachment on titanium surfaces is dependent on topography and chemical changes induced by nonthermal atmospheric pressure plasma.

PubMed

Jeong, Won-Seok; Kwon, Jae-Sung; Lee, Jung-Hwan; Uhm, Soo-Hyuk; Ha Choi, Eun; Kim, Kwang-Mahn

2017-07-26

Here, we investigated the antibacterial effects of chemical changes induced by nonthermal atmospheric pressure plasma (NTAPP) on smooth and rough Ti. The morphologies of smooth and rough surfaces of Ti were examined using scanning electron microscopy (SEM). Both Ti specimens were then treated for 10 min by NTAPP with nitrogen gas. The surface roughness, chemistry, and wettability were examined by optical profilometry, x-ray photoelectron spectroscopy, and water contact angle analysis, respectively. Bacterial attachment was measured by determining the number of colony forming units and by SEM analysis. The rough Ti showed irregular micropits, whereas smooth Ti had a relatively regular pattern on the surface. There were no differences in morphology between samples before and after NTAPP treatment. NTAPP treatment resulted in changes from hydrophobic to hydrophilic properties on rough and smooth Ti; rough Ti showed relatively higher hydrophilicity. Before NTAPP treatment, Streptococcus sanguinis (S. sanguinis) showed greater attachment on rough Ti, and after NTAPP treatment, there was a significant reduction in bacterial attachment. Moreover, the bacterial attachment rate was significantly lower on rough Ti, and the structure of S. sanguinis colonies were significantly changed on NTAPP-treated Ti. NTAPP treatment inhibited bacterial attachment surrounding titanium implants, regardless of surface topography. Therefore, NTAPP treatment on Ti is a next-generation tool for antibacterial applications in the orthopaedic and dental fields.

Possible ecological role of pseudopterosins G and P-U and seco-pseudopterosins J and K from the gorgonian Pseudopterogorgia elisabethae from Providencia Island (SW Caribbean) in regulating microbial surface communities.

PubMed

Correa, Hebelin; Zorro, Pamela; Arevalo-Ferro, Catalina; Puyana, Monica; Duque, Carmenza

2012-09-01

The gorgonian Pseudopterogorgia elisabethae collected at Providencia Island (Colombia) has an unfouled surface, free of obvious algal and invertebrate growth. This gorgonian produces significant amounts of the glycosilated diterpenes pseudopterosins and seco-pseudopterosins (Ps and seco-Ps). Our previous experiments have shown activity of these compounds against eukaryotic (human cancer cell lines and Candida albicans) and prokaryotic cells (Staphylococcus aureus and Enterococcus faecalis). However, the potential role of pseudopterosins on the regulation of the fouling process is still under study. We evaluated the activity of these compounds against bacteria isolated from heavily fouled marine surfaces as an indicator of antifouling activity. Additionally, we assessed their activity against bacteria isolated from P. elisabethae to determine whether potentially they play a role in preventing surface bacterial colonization, thus impairing presumptively the establishment of further successional stages of fouling communities. Results showed that Ps and seco-Ps seem to modulate bacterial growth (controlling Gram-positive bacterial growth and inducing Gram-negative bacterial associations). We thus hypothesized that Ps and seco-Ps may play a role in controlling microbial fouling communities on the surface of this gorgonian. By using bTEFAP and FISH we showed that the most abundant bacteria present in the microbial communities associated with P. elisabethae are Gram-negative bacteria, with Proteobacteria and Gammaproteobacteria the most representative. To evaluate whether Ps and seco-Ps have a direct effect on the structure of the bacterial community associated with P. elisabethae, we tested these compounds against culturable bacteria associated with the surface of P. elisabethae, finding remarkable selectivity against Gram-positive bacteria. The evidence presented here suggests that Ps and seco-Ps might have a role in the selection of organisms associated with the gorgonian surface and in the regulation of the associated bacterial community composition.

RNA interference of tubulin genes has lethal effects in Mythimna separate.

PubMed

Wang, Jin-da; Wang, Ya-Ru; Wang, Yong-Zhi; Wang, Wei-Zhong; Wang, Rong; Gao, San-Ji

2018-05-23

RNAi (RNA interference) is a technology for silencing expression of target genes via sequence-specific double-stranded RNA (dsRNA). Recently, dietary introduction of bacterially expressed dsRNA has shown great potential in the field of pest management. Identification of potential candidate genes for RNAi is the first step in this application. The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae) is a polyphagous, migratory pest, and outbreaks have led to severe crop damage in China. In the present study, two tubulin genes were chosen as target genes because of their crucial role in insect development. Both Msα-tubulin and Msβ-tubulin genes are expressed across all life stages and are highly expressed in the head and epidermis. Feeding of bacterially expressed dsRNA of Msα-tubulin and Msβ-tubulin to third-instar larvae knocked down target mRNAs. A lethal phenotype was observed with knockdown of Msα-tubulin and Msβ-tubulin concurrent with reduction in body weight. Bacterially expressed dsRNA can be used to control M. separata, and tubulin genes could be effective candidate genes for an RNAi-based control strategy of this pest. Copyright © 2017. Published by Elsevier B.V.

Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

PubMed Central

Chella Krishnan, Karthickeyan; Mukundan, Santhosh; Landero Figueroa, Julio A.; Caruso, Joseph A.

2014-01-01

Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues 47Cys and 195His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections. PMID:24799625

Discovery of J Chain in African Lungfish (Protopterus dolloi, Sarcopterygii) Using High Throughput Transcriptome Sequencing: Implications in Mucosal Immunity

PubMed Central

Tacchi, Luca; Larragoite, Erin; Salinas, Irene

2013-01-01

J chain is a small polypeptide responsible for immunoglobulin (Ig) polymerization and transport of Igs across mucosal surfaces in higher vertebrates. We identified a J chain in dipnoid fish, the African lungfish (Protopterus dolloi) by high throughput sequencing of the transcriptome. P. dolloi J chain is 161 aa long and contains six of the eight Cys residues present in mammalian J chain. Phylogenetic studies place the lungfish J chain closer to tetrapod J chain than to the coelacanth or nurse shark sequences. J chain expression occurs in all P. dolloi immune tissues examined and it increases in the gut and kidney in response to an experimental bacterial infection. Double fluorescent in-situ hybridization shows that 88.5% of IgM+ cells in the gut co-express J chain, a significantly higher percentage than in the pre-pyloric spleen. Importantly, J chain expression is not restricted to the B-cell compartment since gut epithelial cells also express J chain. These results improve our current view of J chain from a phylogenetic perspective. PMID:23967082

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

PubMed

Chourashi, Rhishita; Das, Suman; Dhar, Debarpan; Okamoto, Keinosuke; Mukhopadhyay, Asish K; Chatterjee, Nabendu Sekhar

2018-05-01

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

USDA-ARS?s Scientific Manuscript database

Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

NASA Astrophysics Data System (ADS)

Nomura, Toshiyuki; Fujisawa, Eri; Itoh, Shikibu; Konishi, Yasuhiro

2016-06-01

The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells ( Escherichia coli and Lactococcus lactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.

New Weapons to Fight Old Enemies: Novel Strategies for the (Bio)control of Bacterial Biofilms in the Food Industry.

PubMed

Coughlan, Laura M; Cotter, Paul D; Hill, Colin; Alvarez-Ordóñez, Avelino

2016-01-01

Biofilms are microbial communities characterized by their adhesion to solid surfaces and the production of a matrix of exopolymeric substances, consisting of polysaccharides, proteins, DNA and lipids, which surround the microorganisms lending structural integrity and a unique biochemical profile to the biofilm. Biofilm formation enhances the ability of the producer/s to persist in a given environment. Pathogenic and spoilage bacterial species capable of forming biofilms are a significant problem for the healthcare and food industries, as their biofilm-forming ability protects them from common cleaning processes and allows them to remain in the environment post-sanitation. In the food industry, persistent bacteria colonize the inside of mixing tanks, vats and tubing, compromising food safety and quality. Strategies to overcome bacterial persistence through inhibition of biofilm formation or removal of mature biofilms are therefore necessary. Current biofilm control strategies employed in the food industry (cleaning and disinfection, material selection and surface preconditioning, plasma treatment, ultrasonication, etc.), although effective to a certain point, fall short of biofilm control. Efforts have been explored, mainly with a view to their application in pharmaceutical and healthcare settings, which focus on targeting molecular determinants regulating biofilm formation. Their application to the food industry would greatly aid efforts to eradicate undesirable bacteria from food processing environments and, ultimately, from food products. These approaches, in contrast to bactericidal approaches, exert less selective pressure which in turn would reduce the likelihood of resistance development. A particularly interesting strategy targets quorum sensing systems, which regulate gene expression in response to fluctuations in cell-population density governing essential cellular processes including biofilm formation. This review article discusses the problems associated with bacterial biofilms in the food industry and summarizes the recent strategies explored to inhibit biofilm formation, with special focus on those targeting quorum sensing.

New Weapons to Fight Old Enemies: Novel Strategies for the (Bio)control of Bacterial Biofilms in the Food Industry

PubMed Central

Coughlan, Laura M.; Cotter, Paul D.; Hill, Colin; Alvarez-Ordóñez, Avelino

2016-01-01

Biofilms are microbial communities characterized by their adhesion to solid surfaces and the production of a matrix of exopolymeric substances, consisting of polysaccharides, proteins, DNA and lipids, which surround the microorganisms lending structural integrity and a unique biochemical profile to the biofilm. Biofilm formation enhances the ability of the producer/s to persist in a given environment. Pathogenic and spoilage bacterial species capable of forming biofilms are a significant problem for the healthcare and food industries, as their biofilm-forming ability protects them from common cleaning processes and allows them to remain in the environment post-sanitation. In the food industry, persistent bacteria colonize the inside of mixing tanks, vats and tubing, compromising food safety and quality. Strategies to overcome bacterial persistence through inhibition of biofilm formation or removal of mature biofilms are therefore necessary. Current biofilm control strategies employed in the food industry (cleaning and disinfection, material selection and surface preconditioning, plasma treatment, ultrasonication, etc.), although effective to a certain point, fall short of biofilm control. Efforts have been explored, mainly with a view to their application in pharmaceutical and healthcare settings, which focus on targeting molecular determinants regulating biofilm formation. Their application to the food industry would greatly aid efforts to eradicate undesirable bacteria from food processing environments and, ultimately, from food products. These approaches, in contrast to bactericidal approaches, exert less selective pressure which in turn would reduce the likelihood of resistance development. A particularly interesting strategy targets quorum sensing systems, which regulate gene expression in response to fluctuations in cell-population density governing essential cellular processes including biofilm formation. This review article discusses the problems associated with bacterial biofilms in the food industry and summarizes the recent strategies explored to inhibit biofilm formation, with special focus on those targeting quorum sensing. PMID:27803696

Bacterial secondary production on vascular plant detritus: relationships to detritus composition and degradation rate.

PubMed Central

Moran, M A; Hodson, R E

1989-01-01

Bacterial production at the expense of vascular plant detritus was measured for three emergent plant species (Juncus effusus, Panicum hemitomon, and Typha latifolia) degrading in the littoral zone of a thermally impacted lake. Bacterial secondary production, measured as tritiated thymidine incorporation into DNA, ranged from 0.01 to 0.81 microgram of bacterial C mg of detritus-1 day-1. The three plant species differed with respect to the amount of bacterial productivity they supported per milligram of detritus, in accordance with the predicted biodegradability of the plant material based on initial nitrogen content, lignin content, and C/N ratio. Bacterial production also varied throughout the 22 weeks of in situ decomposition and was positively related to the nitrogen content and lignin content of the remaining detritus, as well as to the temperature of the lake water. Over time, production was negatively related to the C/N ratio and cellulose content of the degrading plant material. Bacterial production on degrading plant material was also calculated on the basis of plant surface area and ranged from 0.17 to 1.98 micrograms of bacterial C cm-2 day-1. Surface area-based calculations did not correlate well with either initial plant composition or changing composition of the remaining detritus during decomposition. The rate of bacterial detritus degradation, calculated from measured production of surface-attached bacteria, was much lower than the actual rate of weight loss of plant material. This discrepancy may be attributable to the importance of nonbacterial organisms in the degradation and loss of plant material from litterbags or to the microbially mediated solubilization of particulate material prior to bacterial utilization, or both. PMID:2802603

Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium.

PubMed

He, Xiaochuan; Chen, Wenli; Huang, Qiaoyun

2012-09-01

Monkey metallothionein α domain tandem repeats (4mMTα), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC-10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMTα-EGFP fusion, was constructed and used to target 4mMTα and EGFP on the surface of Pseudomonas putida X4 (CCTCC-209319). The surface location of the INAXN-4mMTα-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMTα on the engineered strains was four times higher than that of the wild-type X4. The Cd²⁺ accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu²⁺ and Zn²⁺. Moreover, the surface-engineered strains could effectively bind Cd²⁺ under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMTα-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMTα-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants.

Bacteria use type IV pili to walk upright and detach from surfaces.

PubMed

Gibiansky, Maxsim L; Conrad, Jacinta C; Jin, Fan; Gordon, Vernita D; Motto, Dominick A; Mathewson, Margie A; Stopka, Wiktor G; Zelasko, Daria C; Shrout, Joshua D; Wong, Gerard C L

2010-10-08

Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili-driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and "walked" with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.

Comparison of bacterial counts in expressed breast milk following standard or strict infection control regimens in neonatal intensive care units: compliance of mothers does matter.

PubMed

Haiden, N; Pimpel, B; Assadian, O; Binder, C; Kreissl, A; Repa, A; Thanhäuser, M; Roberts, C D; Berger, A

2016-03-01

Bacterial counts in 1466 expressed breast milk (EBM) samples from women following one of two infection control regimens (standard vs strict) were investigated. Overall, 12% of samples yielded Gram-negative bacteria, with no significant differences between the standard [11.9% (94/788)] and strict [12.1% (82/678)] regimens (P = 0.92). Significantly more samples were contaminated when expressed at home (standard regimen home/hospital: 17.9% vs 6.1%; strict regimen home/hospital: 19.6% vs 3.4%; P < 0.001). Bacterial contamination of EBM was not associated with the regimen, but was associated with the location of breast milk expression. Attempts to improve personal hygiene during milk collection seem to be of limited value. Good hygiene of collection and storage equipment is likely to be the most important way to ensure the microbiological quality of EBM. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

PubMed

Nishino, Kunihiko

2018-01-01

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM

PubMed Central

Johnson, Brant; Selle, Kurt; O’Flaherty, Sarah; Goh, Yong Jun

2013-01-01

Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers. PMID:24002751

SURFACE FINISHES ON STAINLESS STEEL REDUCE BACTERIAL ATTACHMENT AND EARLY BIOFILM FORMATION: SCANNING ELECTRON AND ATOMIC FORCE MICROSCOPY STUDY

EPA Science Inventory

Three common finishing treatments of stainless steel that are used for equipment during poultry processing were tested for resistance to bacterial contamination. Methods were developed to measure attached bacteria and to identify factors that make surface finishes susceptible or ...

Bacterial Cleanability of Various Types of Eating Surfaces.

ERIC Educational Resources Information Center

Ridenour, Gerald M.; Armbruster, E. H.

1953-01-01

Presents a study of the capability of commercial dishwashers to remove bacteria from various kinds of service plates. Gives an account of preliminary research on the bacterial cleanability of eating surfaces of different materials by two radiological procedures--(1) radiological count, and (2) autoradiographic measurement. Among the factors…

A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geologic settings

USGS Publications Warehouse

Borrok, D.; Turner, B.F.; Fein, J.B.

2005-01-01

Adsorption onto bacterial cell walls can significantly affect the speciation and mobility of aqueous metal cations in many geologic settings. However, a unified thermodynamic framework for describing bacterial adsorption reactions does not exist. This problem originates from the numerous approaches that have been chosen for modeling bacterial surface protonation reactions. In this study, we compile all currently available potentiometric titration datasets for individual bacterial species, bacterial consortia, and bacterial cell wall components. Using a consistent, four discrete site, non-electrostatic surface complexation model, we determine total functional group site densities for all suitable datasets, and present an averaged set of 'universal' thermodynamic proton binding and site density parameters for modeling bacterial adsorption reactions in geologic systems. Modeling results demonstrate that the total concentrations of proton-active functional group sites for the 36 bacterial species and consortia tested are remarkably similar, averaging 3.2 ?? 1.0 (1??) ?? 10-4 moles/wet gram. Examination of the uncertainties involved in the development of proton-binding modeling parameters suggests that ignoring factors such as bacterial species, ionic strength, temperature, and growth conditions introduces relatively small error compared to the unavoidable uncertainty associated with the determination of cell abundances in realistic geologic systems. Hence, we propose that reasonable estimates of the extent of bacterial cell wall deprotonation can be made using averaged thermodynamic modeling parameters from all of the experiments that are considered in this study, regardless of bacterial species used, ionic strength, temperature, or growth condition of the experiment. The average site densities for the four discrete sites are 1.1 ?? 0.7 ?? 10-4, 9.1 ?? 3.8 ?? 10-5, 5.3 ?? 2.1 ?? 10-5, and 6.6 ?? 3.0 ?? 10-5 moles/wet gram bacteria for the sites with pKa values of 3.1, 4.7, 6.6, and 9.0, respectively. It is our hope that this thermodynamic framework for modeling bacteria-proton binding reactions will also provide the basis for the development of an internally consistent set of bacteria-metal binding constants. 'Universal' constants for bacteria-metal binding reactions can then be used in conjunction with equilibrium constants for other important metal adsorption and complexation reactions to calculate the overall distribution of metals in realistic geologic systems.

Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat.

PubMed

Schoonbeek, Henk-Jan; Wang, Hsi-Hua; Stefanato, Francesca L; Craze, Melanie; Bowden, Sarah; Wallington, Emma; Zipfel, Cyril; Ridout, Christopher J

2015-04-01

Perception of pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (PRRs) is a key component of plant innate immunity. The Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial PAMP elongation factor Tu (EF-Tu) and its derived peptide elf18. Previous work revealed that transgenic expression of AtEFR in Solanaceae confers elf18 responsiveness and broad-spectrum bacterial disease resistance. In this study, we developed a set of bioassays to study the activation of PAMP-triggered immunity (PTI) in wheat. We generated transgenic wheat (Triticum aestivum) plants expressing AtEFR driven by the constitutive rice actin promoter and tested their response to elf18. We show that transgenic expression of AtEFR in wheat confers recognition of elf18, as measured by the induction of immune marker genes and callose deposition. When challenged with the cereal bacterial pathogen Pseudomonas syringae pv. oryzae, transgenic EFR wheat lines had reduced lesion size and bacterial multiplication. These results demonstrate that AtEFR can be transferred successfully from dicot to monocot species, further revealing that immune signalling pathways are conserved across these distant phyla. As novel PRRs are identified, their transfer between plant families represents a useful strategy for enhancing resistance to pathogens in crops. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Surface roughness mediated adhesion forces between borosilicate glass and gram-positive bacteria.

PubMed

Preedy, Emily; Perni, Stefano; Nipiĉ, Damijan; Bohinc, Klemen; Prokopovich, Polina

2014-08-12

It is well-known that a number of surface characteristics affect the extent of adhesion between two adjacent materials. One of such parameters is the surface roughness as surface asperities at the nanoscale level govern the overall adhesive forces. For example, the extent of bacterial adhesion is determined by the surface topography; also, once a bacteria colonizes a surface, proliferation of that species will take place and a biofilm may form, increasing the resistance of bacterial cells to removal. In this study, borosilicate glass was employed with varying surface roughness and coated with bovine serum albumin (BSA) in order to replicate the protein layer that covers orthopedic devices on implantation. As roughness is a scale-dependent process, relevant scan areas were analyzed using atomic force microscope (AFM) to determine Ra; furthermore, appropriate bacterial species were attached to the tip to measure the adhesion forces between cells and substrates. The bacterial species chosen (Staphylococci and Streptococci) are common pathogens associated with a number of implant related infections that are detrimental to the biomedical devices and patients. Correlation between adhesion forces and surface roughness (Ra) was generally better when the surface roughness was measured through scanned areas with size (2 × 2 μm) comparable to bacteria cells. Furthermore, the BSA coating altered the surface roughness without correlation with the initial values of such parameter; therefore, better correlations were found between adhesion forces and BSA-coated surfaces when actual surface roughness was used instead of the initial (nominal) values. It was also found that BSA induced a more hydrophilic and electron donor characteristic to the surfaces; in agreement with increasing adhesion forces of hydrophilic bacteria (as determined through microbial adhesion to solvents test) on BSA-coated substrates.

Evolution of defence cocktails: Antimicrobial peptide combinations reduce mortality and persistent infection.

PubMed

Zanchi, Caroline; Johnston, Paul R; Rolff, Jens

2017-10-01

The simultaneous expression of costly immune effectors such as multiple antimicrobial peptides is a hallmark of innate immunity of multicellular organisms, yet the adaptive advantage remains unresolved. Here, we test current hypotheses on the evolution of such defence cocktails. We use RNAi gene knock-down to explore, the effects of three highly expressed antimicrobial peptides, displaying different degrees of activity in vitro against Staphylococcus aureus, during an infection in the beetle Tenebrio molitor. We find that a defensin confers no survival benefit but reduces bacterial loads. A coleoptericin contributes to host survival without affecting bacterial loads. An attacin has no individual effect. Simultaneous knock-down of the defensin with the other AMPs results in increased mortality and elevated bacterial loads. Contrary to common expectations, the effects on host survival and bacterial load can be independent. The expression of multiple AMPs increases host survival and contributes to the control of persisting infections and tolerance. This is an emerging property that explains the adaptive benefit of defence cocktails. © 2017 John Wiley & Sons Ltd.

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Fight or flight? - Flight increases immune gene expression but does not help to fight an infection.

PubMed

Woestmann, L; Kvist, J; Saastamoinen, M

2017-03-01

Flight represents a key trait in most insects, being energetically extremely demanding, yet often necessary for foraging and reproduction. Additionally, dispersal via flight is especially important for species living in fragmented landscapes. Even though, based on life-history theory, a negative relationship may be expected between flight and immunity, a number of previous studies have indicated flight to induce an increased immune response. In this study, we assessed whether induced immunity (i.e. immune gene expression) in response to 15-min forced flight treatment impacts individual survival of bacterial infection in the Glanville fritillary butterfly (Melitaea cinxia). We were able to confirm previous findings of flight-induced immune gene expression, but still observed substantially stronger effects on both gene expression levels and life span due to bacterial infection compared to flight treatment. Even though gene expression levels of some immunity-related genes were elevated due to flight, these individuals did not show increased survival of bacterial infection, indicating that flight-induced immune activation does not completely protect them from the negative effects of bacterial infection. Finally, an interaction between flight and immune treatment indicated a potential trade-off: flight treatment increased immune gene expression in naïve individuals only, whereas in infected individuals no increase in immune gene expression was induced by flight. Our results suggest that the up-regulation of immune genes upon flight is based on a general stress response rather than reflecting an adaptive response to cope with potential infections during flight or in new habitats. © 2016 The Authors. Journal of Evolutionary Biology Published by John Wiley & Sons ltd on behalf of European Society for Evolutionary Biology.