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.

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Bacterial copper storage proteins.

PubMed

Dennison, Christopher; David, Sholto; Lee, Jaeick

2018-03-30

Copper is essential for most organisms as a cofactor for key enzymes involved in fundamental processes such as respiration and photosynthesis. However, copper also has toxic effects in cells, which is why eukaryotes and prokaryotes have evolved mechanisms for safe copper handling. A new family of bacterial proteins uses a Cys-rich four-helix bundle to safely store large quantities of Cu(I). The work leading to the discovery of these proteins, their properties and physiological functions, and how their presence potentially impacts the current views of bacterial copper handling and use are discussed in this review. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts.

PubMed

Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

2017-09-03

In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

Plant immunity: a lesson from pathogenic bacterial effector proteins.

PubMed

Cui, Haitao; Xiang, Tingting; Zhou, Jian-Min

2009-10-01

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant-pathogen interactions.

Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

PubMed Central

Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

2013-01-01

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

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.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

PubMed Central

Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

2017-01-01

ABSTRACT In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between −5.6 °C and −6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs). PMID:28121482

Crystalline Bacterial Surface Layer (S-Layer) Opens Golden Opportunities for Nanobiotechnology in Textiles.

PubMed

Asadi, Narges; Chand, Nima; Rassa, Mehdi

2015-12-01

This study focuses on the successful recrystallization of bacterial S-layer arrays of the Lactobacillus acidophilus ATCC 4356 at textile surfaces to create a novel method and material. Optimum bacterial growth was obtained at approximately 45 °C, pH 5.0, and 14 h pi. The cells were resuspended in guanidine hydrochloride and the 43 kDa S-protein was dialyzed and purified. The optimum reassembly on the polypropylene fabric surface in terms of scanning electron microscopy (SEM), reflectance, and uniformity (spectrophotometry) was obtained at 30 °C, pH 5.0 for 30 minutes in the presence of 2 gr/l (liquor ratio; 1:40) of the S-protein. Overall, our data showed that the functional aspects and specialty applications of the fabric would be very attractive for the textile and related sciences, and result in advanced technical textiles.

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.

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

PubMed Central

Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

2014-01-01

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

An internal thioester in a pathogen surface protein mediates covalent host binding

PubMed Central

Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M; Bergmann, Rene; Saalbach, Gerhard; Kan, Su-Yin; Miller, Ona K; Weckener, Miriam; Jackson, Rosemary J; Shirran, Sally L; Botting, Catherine H; Florence, Gordon J; Rohde, Manfred; Banfield, Mark J; Schwarz-Linek, Ulrich

2015-01-01

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions. DOI: http://dx.doi.org/10.7554/eLife.06638.001 PMID:26032562

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase

PubMed Central

Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M.; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

2012-01-01

Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes. PMID:22778261

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

Bacterial migration along solid surfaces.

PubMed Central

Harkes, G; Dankert, J; Feijen, J

1992-01-01

An in vitro system was developed to study the migration of uropathogenic Escherichia coli strains. In this system an aqueous agar gel is placed against a solid surface, allowing the bacteria to migrate along the gel/solid surface interface. Bacterial strains as well as solid surfaces were characterized by means of water contact angle and zeta potential measurements. When glass was used as the solid surface, significantly different migration times for the strains investigated were observed. Relationships among the observed migration times of six strains, their contact angles, and their zeta potentials were found. Relatively hydrophobic strains exhibited migration times shorter than those of hydrophilic strains. For highly negatively charged strains shorter migration times were found than were found for less negatively charged strains. When the fastest-migrating strain with respect to glass was allowed to migrate along solid surfaces differing in hydrophobicity and charge, no differences in migration times were found. Our findings indicate that strategies to prevent catheter-associated bacteriuria should be based on inhibition of bacterial growth rather than on modifying the physicochemical character of the catheter surface. PMID:1622217

Surface Proteins of Gram-Positive Pathogens: Using Crystallography to Uncover Novel Features in Drug and Vaccine Candidates

NASA Astrophysics Data System (ADS)

Baker, Edward N.; Proft, Thomas; Kang, Haejoo

Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

NASA Astrophysics Data System (ADS)

Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

2015-10-01

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract

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.

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.

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics.

PubMed

Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L; Monteoliva, Lucía; Gil, Concha

2018-01-01

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis . These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV.

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics

PubMed Central

Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L.; Monteoliva, Lucía; Gil, Concha

2018-01-01

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis. These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV. PMID:29867878

Characterization and use of crystalline bacterial cell surface layers

NASA Astrophysics Data System (ADS)

Sleytr, Uwe B.; Sára, Margit; Pum, Dietmar; Schuster, Bernhard

2001-10-01

Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air-water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.

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

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.

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

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

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.

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.

Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

PubMed Central

Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

2018-01-01

The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

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

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

PubMed

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-08-12

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

PubMed Central

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-01-01

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

Bacterial plaque colonization around dental implant surfaces.

PubMed

Covani, Ugo; Marconcini, Simone; Crespi, Roberto; Barone, Antonio

2006-09-01

To examine the distribution of bacteria into the internal and external surfaces of failed implants using histologic analysis. There were 10 failed pure titanium and 5 failed hydroxyapatite-coated titanium implants consecutively removed various years after their placement. Criteria for fixture removal were peri-implant radiolucency and clinical mobility. The mobile fixtures were retrieved with the patients under local anesthesia. Fixtures were removed maintaining the abutments with the aim to observe the bacterial infiltration at the level of abutment/implant interface and on the implant surface. A thin radiolucent space was always present around all the failed implants. The abutments screws were tightly secured in all clinical cases. The bacterial cells were composed of cocci and filaments, which were adherent to the implant surface with an orientation perpendicular to the long axis of the implant. All the specimens included in this study showed bacteria at the level of implant/abutment interface. Histologic analysis at the level of abutment/implant interface in 2-stage implants identified heavy bacterial colonization. These findings appear to support those studies showing bacteria penetration at the level of the micro-gap, which can legitimate the hypothesis that the micro-gap at the bone level could present a risk for bone loss caused by bacterial colonization.

Engineered fluorescent proteins illuminate the bacterial periplasm

PubMed Central

Dammeyer, Thorben; Tinnefeld, Philip

2012-01-01

The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins. PMID:24688673

Protein deposition and its effect on bacterial adhesion to contact lenses.

PubMed

Omali, Negar Babaei; Zhu, Hua; Zhao, Zhenjun; Willcox, Mark D P

2013-06-01

Bacterial adhesion to contact lenses is believed to be the initial step for the development of several adverse reactions that occur during lens wear such as microbial keratitis. This study examined the effect of combinations of proteins on the adhesion of bacteria to contact lenses. Unworn balafilcon A and senofilcon A lenses were soaked in commercially available pure protein mixtures to achieve the same amount of various proteins as found ex vivo. These lenses were then exposed to Pseudomonas aeruginosa and Staphylococcus aureus. Following incubation, the numbers of P. aeruginosa or S. aureus that adhered to the lenses were measured. The possible effect of proteins on bacterial growth was investigated by incubating bacteria in medium containing protein. Although there was a significant (p < 0.003) increase in the total or viable counts of one strain of S. aureus (031) on balafilcon A lenses soaked in the lysozyme/lactoferrin combination, the protein adhered to lenses did not alter the adhesion of any other strains of P. aeruginosa or S. aureus (p > 0.05). Growth of S. aureus 031 (p < 0.0001) but not of P. aeruginosa 6294 was stimulated by addition of lysozyme/lactoferrin combination (2.8/0.5 mg/mL). Addition of lipocalin did not affect the growth of any strains tested (p > 0.05). Adsorption of amounts of lysozyme and lactoferrin or lipocalin equivalent to those extracted from worn contact lenses did not affect the adhesion of most strains of S. aureus or P. aeruginosa to lens surfaces.

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.

Computational Analysis of Uncharacterized Proteins of Environmental Bacterial Genome

NASA Astrophysics Data System (ADS)

Coxe, K. J.; Kumar, M.

2017-12-01

Betaproteobacteria strain CB is a gram-negative bacterium in the phylum Proteobacteria and are found naturally in soil and water. In this complex environment, bacteria play a key role in efficiently eliminating the organic material and other pollutants from wastewater. To investigate the process of pollutant removal from wastewater using bacteria, it is important to characterize the proteins encoded by the bacterial genome. Our study combines a number of bioinformatics tools to predict the function of unassigned proteins in the bacterial genome. The genome of Betaproteobacteria strain CB contains 2,112 proteins in which function of 508 proteins are unknown, termed as uncharacterized proteins (UPs). The localization of the UPs with in the cell was determined and the structure of 38 UPs was accurately predicted. These UPs were predicted to belong to various classes of proteins such as enzymes, transporters, binding proteins, signal peptides, transmembrane proteins and other proteins. The outcome of this work will help better understand wastewater treatment mechanism.

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

PubMed Central

Medina, Eva; van Rooijen, Willemien J.; Spaan, András N.; van Kessel, Kok P. M.; Höök, Magnus; Rooijakkers, Suzan H. M.

2013-01-01

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein. PMID:24348255

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.

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.

Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response.

PubMed

Li, Hui; Zhu, Qing-Feng; Peng, Xuan-Xian; Peng, Bo

2017-01-03

The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity.

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.

Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA.

PubMed Central

Yother, J; White, J M

1994-01-01

Pneumococcal surface protein A (PspA) of Streptococcus pneumoniae has been found to utilize a novel mechanism for anchoring to the bacterial cell surface. In contrast to that of surface proteins from other gram-positive bacteria, PspA anchoring required choline-mediated interactions between the membrane-associated lipoteichoic acid and the C-terminal repeat region of PspA. Release of PspA from the cell surface could be effected by deletion of 5 of the 10 C-terminal repeat units, by high concentrations of choline, or by growth in choline-deficient medium. Other pneumococcal proteins, including autolysin, which has a similar C-terminal repeat region, were not released by these treatments. The attachment mechanism utilized by PspA thus appears to be uniquely adapted to exploit the unusual structure of the pneumococcal cell surface. Further, it has provided the means for rapid and simple isolation of immunogenic PspA from S. pneumoniae. Images PMID:7910604

Studies on Bacterial Proteins Corona Interaction with Saponin Imprinted ZnO Nanohoneycombs and Their Toxic Responses.

PubMed

Sharma, Deepali; Ashaduzzaman, Md; Golabi, Mohsen; Shriwastav, Amritanshu; Bisetty, Krishna; Tiwari, Ashutosh

2015-11-04

Molecular imprinting generates robust, efficient, and highly mesoporous surfaces for biointeractions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understand the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesized saponin imprinted zinc oxide nanohoneycombs (SIZnO NHs), average size 80-125 nm, surface area 20.27 m(2)/g, average pore density 0.23 pore/nm and number-average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential antifungal and antibacterial agents have been studied on Sclerotium rolfsii (S. rolfsii), Pythium debarynum (P. debarynum) and Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), respectively. SIZnO NHs exhibited the highest antibacterial (∼50%) and antifungal (∼40%) activity against Gram-negative bacteria (E. coli) and fungus (P. debarynum), respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalized into the cell. The molecular docking studies were carried out using binding proteins present in the gram negative bacteria (lipopolysaccharide and lipocalin Blc) and gram positive bacteria (Staphylococcal Protein A, SpA). It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies were higher in the case of binding proteins present in gram negative bacteria as compared to that of gram positive bacteria. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalization in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good

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.

Learning about protein solubility from bacterial inclusion bodies

PubMed Central

Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

2009-01-01

The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed. PMID:19133126

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

PubMed

Gunasinghe, Sachith D; Shiota, Takuya; Stubenrauch, Christopher J; Schulze, Keith E; Webb, Chaille T; Fulcher, Alex J; Dunstan, Rhys A; Hay, Iain D; Naderer, Thomas; Whelan, Donna R; Bell, Toby D M; Elgass, Kirstin D; Strugnell, Richard A; Lithgow, Trevor

2018-05-29

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

PubMed Central

Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

2009-01-01

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

Bacterial collagen-like proteins that form triple-helical structures

PubMed Central

Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

2014-01-01

A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

Toward selective elicitation of TH1-controlled vaccination responses: vaccine applications of bacterial surface layer proteins.

PubMed

Jahn-Schmid, B; Messner, P; Unger, F M; Sleytr, U B; Scheiner, O; Kraft, D

1996-01-26

Bacterial surface layer proteins have been utilized as combined vaccine carrier/adjuvants and offer a number of advantages in these applications. The crystalline protein arrays contain functional groups in precisely defined orientations for coupling of haptens. Conventional applications of S-layer vaccines do not cause observable trauma or side effects. Depending on the nature of the S-layer preparations, antigenic conjugates will induce immune responses of a predominantly cellular or predominantly humoral nature. Immune responses to S-layer-hapten conjugates are also observed following oral/nasal application. In the present contribution, the status of investigations with S-layer conjugates in three main immunological projects is reviewed. In a project aimed at immunotherapy of cancer, conjugates of S-layer with small, tumor-associated oligosaccharides have been found to elicit hapten-specific DTH responses. An enlarged program of chemical synthesis has now been initiated to prepare a complete set of mucin-derived, tumor-associated oligosaccharides and their chemically modified analogues for elicitation of cell-mediated immune responses to certain tumors in humans. In another application, oligosaccharides derived from capsules of Streptococcus pneumoniae type 8 have been linked to S-layer proteins and have been found to elicit protective antibody responses in animals. Most recently, allergen S-layer conjugates have been prepared with the intention to suppress the TH2-directed, IgE-mediated allergic responses to Bet nu 1, the major allergen of birch pollen. In the former two applications, the S-layer vaccine technology appears to offer the versatility needed to direct vaccination responses toward predominant control by TH1 or TH2 lymphocytes to meet the different therapeutic or prophylactic requirements in each case. In the third application, work has progressed to a preliminary stage only.

C-reactive Protein Versus Neutrophil/lymphocyte Ratio in Differentiating Bacterial and Non-bacterial Pneumonia in Children.

PubMed

Gauchan, E; Adhikari, S

2016-09-01

Pneumonia is a leading cause of childhood mortality in a low resource country. Simple laboratory markers can help differentiate between bacterial and non-bacterial pneumonias for appropriate management. In children aged one to 60 months with features of lower respiratory infection, C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) were used to differentiate between bacterial and non-bacterial pneumonias. The cutoff values for detecting bacterial pneumonias were evaluated by statistical tools. Bacterial pneumonia was diagnosed in 285 (43.6%) children out of 654 studied. At a cut-off value of 36 mg/L CRP was predictive of bacterial pneumonias with sensitivity and specificity of 61.8% and 91.3% respectively while the sensitivity and specificity for predicting bacterial pneumonia using NLR was 45.6% and 64% respectively with 1.28 used as a cut-off. Our study shows that CRP is superior to NLR in differentiating bacterial from non-bacterial pneumonias in children.

Targeted Proteomics and Absolute Protein Quantification for the Construction of a Stoichiometric Host-Pathogen Surface Density Model*

PubMed Central

Sjöholm, Kristoffer; Kilsgård, Ola; Teleman, Johan; Happonen, Lotta; Malmström, Lars; Malmström, Johan

2017-01-01

Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modeling of host-pathogen interactions in the disease state represents a promising strategy to define molecular events of importance for the transition from superficial to invasive infectious diseases. Here we used the Gram-positive bacterium Streptococcus pyogenes as a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between the S. pyogenes surface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labeled reference peptides and selected reaction monitoring mass spectrometry analysis of a wild-type strain and an M-protein deficient mutant strain, to generate absolutely quantified protein stoichiometry ratios between S. pyogenes and interacting plasma proteins. The stoichiometry ratios in combination with a novel targeted mass spectrometry method to measure cell numbers enabled the construction of a stoichiometric surface density model using protein structures available from the protein data bank. The model outlines the topology and density of the host-pathogen protein interaction network on the S. pyogenes bacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein from S. pyogenes introduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model of S. pyogenes in human blood plasma represents a scalable framework that can continuously be refined with the emergence of new results. Future integration of new results will improve the understanding of protein-protein interactions and their importance for bacterial virulence. Furthermore, we anticipate that the general properties of the developed workflow will facilitate the production of stoichiometric surface density models for other types of host

Targeted Proteomics and Absolute Protein Quantification for the Construction of a Stoichiometric Host-Pathogen Surface Density Model.

PubMed

Sjöholm, Kristoffer; Kilsgård, Ola; Teleman, Johan; Happonen, Lotta; Malmström, Lars; Malmström, Johan

2017-04-01

Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modeling of host-pathogen interactions in the disease state represents a promising strategy to define molecular events of importance for the transition from superficial to invasive infectious diseases. Here we used the Gram-positive bacterium Streptococcus pyogenes as a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between the S. pyogenes surface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labeled reference peptides and selected reaction monitoring mass spectrometry analysis of a wild-type strain and an M-protein deficient mutant strain, to generate absolutely quantified protein stoichiometry ratios between S. pyogenes and interacting plasma proteins. The stoichiometry ratios in combination with a novel targeted mass spectrometry method to measure cell numbers enabled the construction of a stoichiometric surface density model using protein structures available from the protein data bank. The model outlines the topology and density of the host-pathogen protein interaction network on the S. pyogenes bacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein from S. pyogenes introduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model of S. pyogenes in human blood plasma represents a scalable framework that can continuously be refined with the emergence of new results. Future integration of new results will improve the understanding of protein-protein interactions and their importance for bacterial virulence. Furthermore, we anticipate that the general properties of the developed workflow will facilitate the production of stoichiometric surface density models for other types of host

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.

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

PubMed Central

Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2010-01-01

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

Factors affecting the rate of breakdown of bacterial protein in rumen fluid.

PubMed

Wallace, R J; McPherson, C A

1987-09-01

1. The cellular proteins of Butyrivibrio fibrisolvens, Lactobacillus casei, Megasphaera elsdenii, Selenomonas ruminantium and Streptococcus bovis were labelled by growth in the presence of L-[14C]leucine, and the breakdown of labelled protein was measured in incubations of these bacteria with rumen fluid to which unlabelled 5 mM-L-leucine was added. The rate of protein breakdown was estimated from the rate of release of radioactivity into acid-soluble material. 2. Protein breakdown occurred at different rates in different species. The mean rates for B. fibrisolvens, L. casei, M. elsdenii, Sel. ruminantium and Str. bovis were 28.6, 18.1, 17.7, 10.5 and 5.3%/h respectively in samples of strained rumen fluid (SRF) with different protozoal populations. Rates of 3%/h or less were found in SRF from ciliate-free sheep or in faunated SRF from which protozoa had been removed by centrifugation. Further removal of mixed rumen bacteria had little effect. Suspensions of washed protozoa degraded bacterial protein at rates which were of the same order as those found in SRF. 3. The rate of breakdown of bacterial protein in different samples of SRF tended to increase as the numbers of small entodiniomorphid protozoa increased. The numbers of larger entodiniomorphs and holotrichs had no obvious influence on this rate. 4. Autoclaved and u.v.-treated bacteria were generally no different from live bacteria in their susceptibility to breakdown in SRF from faunated sheep, indicating that endogenous protein turnover was not a significant cause of bacterial protein catabolism. 5. The rate of bacterial protein breakdown was unrelated to the proteolytic activity of SRF. 6. It was concluded that predation by small protozoa is by far the most important cause of bacterial protein turnover in the rumen, with autolysis, other lytic factors and endogenous proteolysis being of minor importance.

Competition of bovine serum albumin adsorption and bacterial adhesion onto surface-grafted ODT: in situ study by vibrational SFG and fluorescence confocal microscopy.

PubMed

Bulard, Emilie; Fontaine-Aupart, Marie-Pierre; Dubost, Henri; Zheng, Wanquan; Bellon-Fontaine, Marie-Noëlle; Herry, Jean-Marie; Bourguignon, Bernard

2012-12-11

The interaction of hydrophilic and hydrophobic ovococcoid bacteria and bovine serum albumin (BSA) proteins with a well ordered surface of octadecanethiol (ODT) self assembled monolayer (SAM) has been studied in different situations where proteins were either preadsorbed on ODT or adsorbed simultaneously with bacterial adhesion as in life conditions. The two situations lead to very different antimicrobial behavior. Bacterial adhesion on preadsorbed BSA is very limited, while the simultaneous exposure of ODT SAM to proteins and bacteria lead to a markedly weaker antimicrobial effect. The combination of sum frequency generation spectroscopy and fluorescence confocal microscopy experiments allow one to draw conclusions on the factors that govern the ODT SAM or BSA film interaction with bacteria at the molecular level. On the hydrophobic ODT surface, interaction with hydrophobic or hydrophilic biomolecules results in opposite effects on the SAM, namely, a flattening or a raise of the terminal methyl groups of ODT. On an amphiphilic BSA layer, the bacterial adhesion strength is weakened by the negative charges carried by both BSA and bacteria. Surprisingly, preadsorbed BSA that cover part of the bacteria cell walls increase the adhesion strength to the BSA film and reduce hydrophobic interactions with the ODT SAM. Finally, bacterial adhesion on a BSA film is shown to modify the BSA proteins in some way that change their interaction with the ODT SAM. The antimicrobial effect is much stronger in the case of a preadsorbed BSA layer than when BSA and bacteria are in competition to colonize the ODT SAM surface.

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

PubMed

Arnfinnsdottir, Nina Bjørk; Ottesen, Vegar; Lale, Rahmi; Sletmoen, Marit

2015-01-01

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

PubMed Central

Canova, Marc J.; Molle, Virginie

2014-01-01

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

Bacterial serine/threonine protein kinases in host-pathogen interactions.

PubMed

Canova, Marc J; Molle, Virginie

2014-04-04

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

Hydration dynamics promote bacterial coexistence on rough surfaces

PubMed Central

Wang, Gang; Or, Dani

2013-01-01

Identification of mechanisms that promote and maintain the immense microbial diversity found in soil is a central challenge for contemporary microbial ecology. Quantitative tools for systematic integration of complex biophysical and trophic processes at spatial scales, relevant for individual cell interactions, are essential for making progress. We report a modeling study of competing bacterial populations cohabiting soil surfaces subjected to highly dynamic hydration conditions. The model explicitly tracks growth, motion and life histories of individual bacterial cells on surfaces spanning dynamic aqueous networks that shape heterogeneous nutrient fields. The range of hydration conditions that confer physical advantages for rapidly growing species and support competitive exclusion is surprisingly narrow. The rapid fragmentation of soil aqueous phase under most natural conditions suppresses bacterial growth and cell dispersion, thereby balancing conditions experienced by competing populations with diverse physiological traits. In addition, hydration fluctuations intensify localized interactions that promote coexistence through disproportional effects within densely populated regions during dry periods. Consequently, bacterial population dynamics is affected well beyond responses predicted from equivalent and uniform hydration conditions. New insights on hydration dynamics could be considered in future designs of soil bioremediation activities, affect longevity of dry food products, and advance basic understanding of bacterial diversity dynamics and its role in global biogeochemical cycles. PMID:23051694

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

PubMed

Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

2016-12-01

Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media. Copyright © 2016 Elsevier B.V. All rights reserved.

Physical stress and bacterial colonization

PubMed Central

Otto, Michael

2014-01-01

Bacterial surface colonizers are subject to a variety of physical stresses. During the colonization of human epithelia such as on the skin or the intestinal mucosa, bacteria mainly have to withstand the mechanical stress of being removed by fluid flow, scraping, or epithelial turnover. To that end, they express a series of molecules to establish firm attachment to the epithelial surface, such as fibrillar protrusions (pili) and surface-anchored proteins that bind to human matrix proteins. In addition, some bacteria – in particular gut and urinary tract pathogens – use internalization by epithelial cells and other methods such as directed inhibition of epithelial turnover to ascertain continued association with the epithelial layer. Furthermore, many bacteria produce multi-layered agglomerations called biofilms with a sticky extracellular matrix, providing additional protection from removal. This review will give an overview over the mechanisms human bacterial colonizers have to withstand physical stresses with a focus on bacterial adhesion. PMID:25212723

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...

Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters.

PubMed

Barr, Jeremy J; Auro, Rita; Sam-Soon, Nicholas; Kassegne, Sam; Peters, Gregory; Bonilla, Natasha; Hatay, Mark; Mourtada, Sarah; Bailey, Barbara; Youle, Merry; Felts, Ben; Baljon, Arlette; Nulton, Jim; Salamon, Peter; Rohwer, Forest

2015-11-03

Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4∆hoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4∆hoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4∆hoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage-host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome.

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

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

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

Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

PubMed

Kerk, David; Uhrig, R Glen; Moorhead, Greg B

2013-01-01

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

PubMed Central

Wentzel, Alexander; Christmann, Andreas; Adams, Thorsten; Kolmar, Harald

2001-01-01

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REIv were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications. PMID:11717287

'Drugs from bugs': bacterial effector proteins as promising biological (immune-) therapeutics.

PubMed

Rüter, Christian; Hardwidge, Philip R

2014-02-01

Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example, many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit 'microbial knowledge' to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases. In this review, we focus on bacterial effector and virulence proteins capable of modulating and suppressing distinct signaling pathways with potentially desirable immune-modulating effects for treating unrelated inflammatory diseases. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

USDA-ARS?s Scientific Manuscript database

Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

Surface Mediated Protein Disaggregation

NASA Astrophysics Data System (ADS)

Radhakrishna, Mithun; Kumar, Sanat K.

2014-03-01

Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

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...

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.

Salivary protein adsorption and Streptococccus gordonii adhesion to dental material surfaces.

PubMed

Schweikl, Helmut; Hiller, Karl-Anton; Carl, Ulrich; Schweiger, Rainer; Eidt, Andreas; Ruhl, Stefan; Müller, Rainer; Schmalz, Gottfried

2013-10-01

The initial adhesion of microorganisms to clinically used dental biomaterials is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polytetrafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer cement as well as cobalt-chromium-molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1. Wettability of material surfaces precoated with salivary proteins or left in phosphate-buffered saline was determined by the measurement of water contact angles. Amounts of adsorbed proteins were determined directly on material surfaces after biotinylation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The same technique was used to analyze for the binding of biotinylated bacteria to material surfaces. The highest amount of proteins (0.18μg/cm(2)) adsorbed to hydrophobic PTFE samples, and the lowest amount (0.025μg/cm(2)) was detected on silicone. The highest number of S. gordonii (3.2×10(4)CFU/mm(2)) adhered to the hydrophilic glassionomer cement surface coated with salivary proteins, and the lowest number (4×10(3)CFU/mm(2)) was found on the hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the number of attached microorganisms were weakly negatively correlated. No such correlation between hydrophobicity and the number of bacteria was detected when surfaces were coated with salivary proteins. Functional groups added by the adsorption of specific salivary proteins to material surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a physical property. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Bacterial adhesion capacity on food service contact surfaces.

PubMed

Fink, Rok; Okanovič, Denis; Dražič, Goran; Abram, Anže; Oder, Martina; Jevšnik, Mojca; Bohinc, Klemen

2017-06-01

The aim of this study was to analyse the adhesion of E. coli, P. aeruginosa and S. aureus on food contact materials, such as polyethylene terephthalate, silicone, aluminium, Teflon and glass. Surface roughness, streaming potential and contact angle were measured. Bacterial properties by contact angle and specific charge density were characterised. The bacterial adhesion analysis using staining method and scanning electron microscopy showed the lowest adhesion on smooth aluminium and hydrophobic Teflon for most of the bacteria. However, our study indicates that hydrophobic bacteria with high specific charge density attach to those surfaces more intensively. In food services, safety could be increased by selecting material with low adhesion to prevent cross contamination.

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.

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

NASA Astrophysics Data System (ADS)

Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

2014-10-01

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

Development of antifouling surfaces to reduce bacterial attachment

NASA Astrophysics Data System (ADS)

Graham, Mary Viola

Bacteria are exceptionally good at adhering to surfaces and forming complex structures known as biofilms. This process, known as biofouling, can cause problems for infrastructure (eg, clogging and damaging pipes), for the food industry (eg, contamination of processing surfaces and equipment, and for the medical industry (eg, contamination of indwelling medical devices). Accordingly, multiple strategies have been explored to combat biofouling, including chemical modification of surfaces, development of antibiotic coatings, and more recently, the use of engineered surface topography. When designed properly, engineered surface topographies can significantly reduce bacterial surface attachment, ultimately limiting surface colonization. In this work, we hypothesized that the morphology, size, spacing, and surface pre-treatment of topographical features should directly correlate with the size and shape of target organisms, in order to reduce biofouling. Topographical features with size and spacing from 0.25 to 2 mum were fabricated in silicone elastomer and tested against rod shaped bacteria with an average size of 0.5 x 2 mum and spherical bacteria (cocci) ranging from 0.5 - 1 μm in diameter. Antifouling properties of the different topographical features were tested in both static and flow-based assays, and under oxygen plasma-treated (hydrophilic) and untreated (hydrophobic) surface conditions. We found that surface pre-treatment universally affects the ability bacteria to attach to surfaces, while surface topography limits attachment in a manner dependent on the bacterial size/shape and the size/spacing of the topography.

Connecting the dots between bacterial biofilms and ice cream.

PubMed

Stanley-Wall, Nicola R; MacPhee, Cait E

2015-12-18

Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.

Connecting the dots between bacterial biofilms and ice cream

NASA Astrophysics Data System (ADS)

Stanley-Wall, Nicola R.; MacPhee, Cait E.

2015-12-01

Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.

Protein-Nanoparticle Interactions: Improving Immobilized Lytic Enzyme Activity and Surface Energy Effects

NASA Astrophysics Data System (ADS)

Downs, Emily Elizabeth

-particle interactions to protein-protein interactions and were thicker with greater surface energy, which resulted in the recovery of secondary structure in the outermost layer. To help understand the impact of protein structure on nano-bio conjugate interactions, a listeria specific protein was used. This system was chosen as it has applications in the food industry in preventing bacterial contamination. The insertion of an amino acid linker between the enzymatic and binding domain of the protein improved the flexibility between domains, leading to increased adsorption, and improved activity in both cell-wall and plating assays. Additionally, linker modified protein incorporated into the silica-polymer nanocomposite showed significant activity in a real-world example of contaminated lettuce. This thesis study has isolated the impact of surface energy and protein flexibility on protein adsorption and structure. Particle surface energy affects adsorbed protein concentration and conformation. Coupled with protein surface charge, surface energy was also found to dictate multilayer thickness. The conformational flexibility of the protein was shown to help in controlling not only protein adsorption concentration but also in retaining protein activity after immobilization. Also, a controllable synthesis method for particles with adjustable surface energy, an ideal platform for studying protein-particle interactions, has been established.

Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

PubMed

Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E; Spiciarich, David R; Evans, David J; Bertozzi, Carolyn R; Fleiszig, Suzanne M J

2017-06-01

Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N -azidoacetylgalactosamine (Ac 4 GalNAz). Ac 4 GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88 -/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas ( P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88 -/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. © FASEB.

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.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells.

PubMed

Peternel, Spela; Komel, Radovan

2010-09-10

In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process.To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared.During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation.During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity.High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

Channel crossing: how are proteins shipped across the bacterial plasma membrane?

PubMed

Collinson, Ian; Corey, Robin A; Allen, William J

2015-10-05

The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation--the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins. © 2015 The Authors.

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.

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

Bacterial anoxygenic photosynthesis on plant leaf surfaces.

PubMed

Atamna-Ismaeel, Nof; Finkel, Omri; Glaser, Fabian; von Mering, Christian; Vorholt, Julia A; Koblížek, Michal; Belkin, Shimshon; Béjà, Oded

2012-04-01

The aerial surface of plants, the phyllosphere, is colonized by numerous bacteria displaying diverse metabolic properties that enable their survival in this specific habitat. Recently, we reported on the presence of microbial rhodopsin harbouring bacteria on the top of leaf surfaces. Here, we report on the presence of additional bacterial populations capable of harvesting light as a means of supplementing their metabolic requirements. An analysis of six phyllosphere metagenomes revealed the presence of a diverse community of anoxygenic phototrophic bacteria, including the previously reported methylobacteria, as well as other known and unknown phototrophs. The presence of anoxygenic phototrophic bacteria was also confirmed in situ by infrared epifluorescence microscopy. The microscopic enumeration correlated with estimates based on metagenomic analyses, confirming both the presence and high abundance of these microorganisms in the phyllosphere. Our data suggest that the phyllosphere contains a phylogenetically diverse assemblage of phototrophic species, including some yet undescribed bacterial clades that appear to be phyllosphere-unique. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

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

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production.

PubMed

Servais, P

1995-03-01

In aquatic ecosystems, [(3)H]thymidine incorporation into bacterial DNA and [(3)H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2-77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91-1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.

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 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

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

Influence of day and night wear on surface properties of silicone hydrogel contact lenses and bacterial adhesion.

PubMed

Vermeltfoort, Pit B J; Rustema-Abbing, Minie; de Vries, Joop; Bruinsma, Gerda M; Busscher, Henk J; van der Linden, Matthijs L; Hooymans, Johanna M M; van der Mei, Henny C

2006-06-01

The aim of this study was to determine the effect of continuous wear on physicochemical surface properties of silicone hydrogel (S-H) lenses and their susceptibility to bacterial adhesion. In this study, volunteers wore 2 pairs of either "lotrafilcon A" or "balafilcon A" S-H contact lenses. The first pair was worn continuously for a week and the second pair for 4 weeks. One lens of each pair was used for surface characterization and the other one for bacterial adhesion experiments. Lens surfaces were characterized by examination of their wettability, roughness, elemental composition, and proteins attached to their surfaces. Adhesion of Staphylococcus aureus 835 and Pseudomonas aeruginosa #3 to a lens was studied using a parallel plate flow chamber. Before use, the lotrafilcon A lens was rougher than the balafilcon A lens and had a lower water contact angle and a higher affinity for S. aureus 835. After wear, both lens types had similar water contact angles, whereas the differences in elemental surface composition decreased as well. S. aureus 835 adhered in higher numbers to worn balafilcon A lenses, whereas the opposite was seen for P. aeruginosa #3. The initial deposition rates of both bacterial strains to lotrafilcon A lenses decreased by wearing and were found to correlate significant (P < 0.001) with the surface roughness of worn lenses. In this study, the differences in surface properties between 2 types of S-H lenses were found to change after 1 week of continuous wear. Generally, bacteria adhered in lower numbers and less tenaciously to worn lenses, except S. aureus 835, adhering in higher numbers to worn balafilcon A lenses.

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

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.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

PubMed Central

2010-01-01

Background In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells. PMID:20831775

Structure, dynamics and biophysics of the cytoplasmic protein–protein complexes of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system

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

Clore, G. Marius; Venditti, Vincenzo

2013-10-01

The bacterial phosphotransferase system (PTS) couples phosphoryl transfer, via a series of bimolecular protein–protein interactions, to sugar transport across the membrane. The multitude of complexes in the PTS provides a paradigm for studying protein interactions, and for understanding how the same binding surface can specifically recognize a diverse array of targets. Fifteen years of work aimed at solving the solution structures of all soluble protein–protein complexes of the PTS has served as a test bed for developing NMR and integrated hybrid approaches to study larger complexes in solution and to probe transient, spectroscopically invisible states, including encounter complexes. We reviewmore » these approaches, highlighting the problems that can be tackled with these methods, and summarize the current findings on protein interactions.« less

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

PubMed Central

Mitra, Avishek; Speer, Alexander; Lin, Kan; Ehrt, Sabine

2017-01-01

ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. PMID:28119467

Bacterial growth on a superhydrophobic surface containing silver nanoparticles

NASA Astrophysics Data System (ADS)

Heinonen, S.; Nikkanen, J.-P.; Laakso, J.; Raulio, M.; Priha, O.; Levänen, E.

2013-12-01

The antibacterial effect of silver can be exploited in the food and beverage industry and medicinal applications to reduce biofouling of surfaces. Very small amount of silver ions are enough to destructively affect the metabolism of bacteria. Moreover, superhydrophobic properties could reduce bacterial adhesion to the surface. In this study we fabricated superhydrophobic surfaces that contained nanosized silver particles. The superhydrophobic surfaces were manufactured onto stainless steel as combination of ceramic nanotopography and hydrophobication by fluorosilane. Silver nanoparticles were precipitated onto the surface by a chemical method. The dissolution of silver from the surface was tested in an aqueous environment under pH values of 1, 3, 5, 7, 9, 11 and 13. The pH value was adjusted with nitric acid and ammonia. It was found that dissolution rate of silver increased as the pH of the solution altered from the pH of de-ionized water to lower and higher pH values but dissolution occurred also in de-ionized water. The antimicrobial potential of this coating was investigated using bacterial strains isolated from the brewery equipment surfaces. The results showed that the number of bacteria adhering onto steel surface was significantly reduced (88%) on the superhydrophobic silver containing coating.

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

PubMed

Albillos, Agustín; de-la-Hera, Antonio; Alvarez-Mon, Melchor

2004-05-15

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

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.

The presence of INA proteins on the surface of single cells of Pseudomonas syringae R10.79 isolated from rain

NASA Astrophysics Data System (ADS)

Šantl-Temkiv, Tina; Ling, Meilee; Holm, Stine; Finster, Kai; Boesen, Thomas

2016-04-01

One of the important open questions in atmospheric ice nucleation is the impact of bioaerosols on the ice content of mix phase clouds (DeMott and Prenni 2010). Biogenic ice nuclei have a unique capacity of facilitating ice formation at temperatures between -1 and -10 °C. The model biogenic ice nuclei are produced by a few species of plant-surface bacteria, such as Pseudomonas syringae, that are commonly transported through the atmosphere. These bacterial species have highly specialized proteins, the so-called ice nucleation active (INA) proteins, which are exposed at the outer membrane surface of the cell where they promote ice particle formation. The mechanisms behind the onset of INA protein synthesis in single bacterial cells are not well understood. We performed a laboratory study in order to (i) investigate the presence of INA proteins on single bacterial cells and (ii) understand the conditions that induce INA protein production. We previously isolated an INA-positive strain of Pseudomonas syringae from rain samples collected in Denmark. Bacterial cells initiated ice nucleation activity at temperatures ≤-2°C and the cell fragments at temperatures ≤-8°C (Šantl-Temkiv et al 2015). We determined the amino-acid sequence of the INA protein and used the sequence to produce custom-made antibodies (GenScript, Germany). These antibodies were used to specifically stain and visualize the INA protein on the surfaces of single cells, which can then be quantified by a technique called flow cytometry. The synthesis of INA proteins by individual cells was followed during a batch growth experiment. An unusually high proportion of cells that were adapting to the new conditions prior to growth produced INA proteins (~4.4% of all cells). A smaller fraction of actively growing cells was carrying INA proteins (~1.2 % of all cells). The cells that stopped growing due to unfavorable conditions had the lowest fraction of cells carrying INA proteins (~0.5 % of all cells). To

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.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

PubMed Central

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

PubMed

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

[Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

PubMed

Minamino, Tohru

2015-01-01

The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

Proteome Analysis and Serological Characterization of Surface-Exposed Proteins of Rickettsia heilongjiangensis

PubMed Central

Qi, Yong; Xiong, Xiaolu; Wang, Xile; Duan, Changsong; Jia, Yinjun; Jiao, Jun; Gong, Wenping; Wen, Bohai

2013-01-01

Background Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. Methods R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA). Results Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. Conclusions Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease. PMID:23894656

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

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...

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

PubMed Central

Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

2015-01-01

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

Influences of Mn(II) and V(IV) on Bacterial Surface Chemistry and Metal Reactivity

NASA Astrophysics Data System (ADS)

French, S.; Fakra, S.; Glasauer, S.

2009-05-01

oxygen. Results from NEXAFS spectroscopy revealed that oxygen presence had a strong impact on metal sorption, especially in the case of V(IV) association with membranes when oxygen is present. Bacterial membranes are necessarily dynamic, the membrane components are in a state of constant fluidity. Metal sorption to the cell surface, especially soluble metals which can fully engulf the cell, would limit the mobility of membrane components. Supporting this notion, CN32 cell membranes were observed via spectrofluorometry to become significantly stabilized when exposed to Mn(II) and V(IV) metals under anoxia. Despite stabilizing effects, cells are not adversely affected by metal presence in their growth environments, which is also supported by observations of metal coated cells by transmission electron microscopy (TEM). This supports STXM observations that cells counteract the metal effects on their surfaces by altering their membrane composition, and is enhanced by significant differences in cell membrane protein composition and quantity after SDS-PAGE separation. Our studies reveal several clear patterns in how cells interact with soluble metals in their environments, as well as the often overlooked subsequent effects that those metals, as well as oxygen, have on bacterial membranes.

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

PubMed Central

Gauss, George H.; Reott, Michael A.; Rocha, Edson R.; Young, Mark J.; Douglas, Trevor

2012-01-01

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer. PMID:22020642

Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

PubMed

Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

2013-11-01

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Rapid bacterial diagnostics via surface enhanced Raman microscopy.

PubMed

Premasiri, W R; Sauer-Budge, A F; Lee, J C; Klapperich, C M; Ziegler, L D

2012-06-01

There is a continuing need to develop new techniques for the rapid and specific identification of bacterial pathogens in human body fluids especially given the increasing prevalence of drug resistant strains. Efforts to develop a surface enhanced Raman spectroscopy (SERS) based approach, which encompasses sample preparation, SERS substrates, portable Raman microscopy instrumentation and novel identification software, are described. The progress made in each of these areas in our laboratory is summarized and illustrated by a spiked infectious sample for urinary tract infection (UTI) diagnostics. SERS bacterial spectra exhibit both enhanced sensitivity and specificity allowing the development of an easy to use, portable, optical platform for pathogen detection and identification. SERS of bacterial cells is shown to offer not only reproducible molecular spectroscopic signatures for analytical applications in clinical diagnostics, but also is a new tool for studying biochemical activity in real time at the outer layers of these organisms.

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…

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

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

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

Protein aggregation as bacterial inclusion bodies is reversible.

PubMed

Carrió, M M; Villaverde, A

2001-01-26

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.

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

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins).

PubMed

Pratt, R F

2008-07-01

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

Bacterial plaque retention on oral hard materials: effect of surface roughness, surface composition, and physisorbed polycarboxylate.

PubMed

McConnell, Marla D; Liu, Yu; Nowak, Andrew P; Pilch, Shira; Masters, James G; Composto, Russell J

2010-03-15

Bacterial adhesion to oral hard materials is dependent on various factors, for example, surface roughness and surface composition. In this study, bacteria retention on three oral hard substrates, hydroxyapatite (HAP), enamel, and polished enamel (p-enamel) were investigated. The surface morphology and roughness of the three substrates were measured by scanning probe microscopy. HAP had the roughest surface, followed by enamel and polished enamel. For each individual substrate type, the roughness was shown to increase with scan size up to 50 microm x 50 microm. For HAP and enamel, roughness decreased considerably after formation of a pellicle, while addition of polymer coating to the pellicle layer reduced roughness much less in comparison. Bacterial surface coverage was measured at 30 min, 3 h, and 24 h on both native and surface-modified substrates, which were coated with two different polycarboxylate-based polymers, Gantrez S97 and Carbopol 940. As a result, the polymer coated surfaces had reduced bacteria coverage compared with the native surfaces over all time points and substrates measured. The reduction is the combined effect of electrostatic repulsion and sequestering of Ca(2+) ions at the surface, which plays a key role in the initial adhesion of bacteria to enamel surfaces in models of plaque formation. (c) 2009 Wiley Periodicals, Inc.

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

PubMed

Van Amersfoort, Edwin S; Van Berkel, Theo J C; Kuiper, Johan

2003-07-01

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

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.

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.

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.

Bacterial flagellar capping proteins adopt diverse oligomeric states

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

Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.

2016-09-24

Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins atmore » the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.« less

Light Structures Phototroph, Bacterial and Fungal Communities at the Soil Surface

PubMed Central

Davies, Lawrence O.; Schäfer, Hendrik; Marshall, Samantha; Bramke, Irene; Oliver, Robin G.; Bending, Gary D.

2013-01-01

The upper few millimeters of soil harbour photosynthetic microbial communities that are structurally distinct from those of underlying bulk soil due to the presence of light. Previous studies in arid zones have demonstrated functional importance of these communities in reducing soil erosion, and enhancing carbon and nitrogen fixation. Despite being widely distributed, comparative understanding of the biodiversity of the soil surface and underlying soil is lacking, particularly in temperate zones. We investigated the establishment of soil surface communities on pasture soil in microcosms exposed to light or dark conditions, focusing on changes in phototroph, bacterial and fungal communities at the soil surface (0–3 mm) and bulk soil (3–12 mm) using ribosomal marker gene analyses. Microbial community structure changed with time and structurally similar phototrophic communities were found at the soil surface and in bulk soil in the light exposed microcosms suggesting that light can influence phototroph community structure even in the underlying bulk soil. 454 pyrosequencing showed a significant selection for diazotrophic cyanobacteria such as Nostoc punctiforme and Anabaena spp., in addition to the green alga Scenedesmus obliquus. The soil surface also harboured distinct heterotrophic bacterial and fungal communities in the presence of light, in particular, the selection for the phylum Firmicutes. However, these light driven changes in bacterial community structure did not extend to the underlying soil suggesting a discrete zone of influence, analogous to the rhizosphere. PMID:23894406

Self-organization and positioning of bacterial protein clusters

NASA Astrophysics Data System (ADS)

Murray, Seán M.; Sourjik, Victor

2017-10-01

Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins

PubMed Central

1991-01-01

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one- fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1. PMID:1714460

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities.

PubMed

Mondal, Manas; Chakrabarti, Jaydeb; Ghosh, Mahua

2018-03-01

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer-membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co-occur and are from neighboring gene in Salmonella Typhi-occurrence of homologs of both STY3178 and STY3179 are identified in many Gram-negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra-cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra-cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin. © 2017 Wiley Periodicals, Inc.

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.

Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

PubMed

Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

2015-01-01

It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible

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 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

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.

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.

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.

A role for surface hydrophobicity in protein-protein recognition.

PubMed Central

Young, L.; Jernigan, R. L.; Covell, D. G.

1994-01-01

The role of hydrophobicity as a determinant of protein-protein interactions is examined. Surfaces of apo-protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol-binding protein, and their associated ligands with available X-ray structures for their complexed forms, are scanned to determine clusters of surface-accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co-crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one-third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well-defined loci for possible ligand attachment. PMID:8061602

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

Hierarchical protein export mechanism of the bacterial flagellar type III protein export apparatus.

PubMed

Minamino, Tohru

2018-06-01

The bacterial flagellum is supramolecular motility machinery consisting of the basal body, the hook and the filament. Flagellar proteins are translocated across the cytoplasmic membrane via a type III protein export apparatus, diffuse down the central channel of the growing structure and assemble at the distal end. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. The completion of hook assembly is the most important morphological checkpoint of the sequential flagellar assembly process. When the hook reaches its mature length of about 55 nm in Salmonella enterica, the type III protein export apparatus switches export specificity from proteins required for the structure and assembly of the hook to those responsible for filament assembly, thereby terminating hook assembly and initiating filament assembly. Three flagellar proteins, namely FliK, FlhB and FlhA, are responsible for this substrate specificity switching. Upon completion of the switching event, interactions among FlhA, the cytoplasmic ATPase complex and flagellar type III export chaperones establish the assembly order of the filament at the hook tip. Here, we describe our current understanding of a hierarchical protein export mechanism used in flagellar type III protein export.

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.

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

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.

Viability and virulence of pneumolysin, pneumococcal surface protein A, and pneumolysin/pneumococcal surface protein A mutants in the ear.

PubMed

Schachern, Patricia A; Tsuprun, Vladimir; Goetz, Sarah; Cureoglu, Sebahattin; Juhn, Steven K; Briles, David E; Paparella, Michael M; Ferrieri, Patricia

2013-09-01

Understanding how pneumococcal proteins affect the pathology of the middle ear and inner ear is important for the development of new approaches to prevent otitis media and its complications. To determine the viability and virulence of Streptococcus pneumoniae mutants deficient in pneumolysin (Ply-) and pneumococcal surface protein A (PspA-) in the chinchilla middle ear. Bullae of chinchillas were inoculated bilaterally with wild-type (Wt), Ply-, PspA-, and Ply-/PspA- strains. Bacterial colony-forming units (CFUs) in middle ear effusions were counted at 48 hours. The CFUs of the PspA- group were also counted at 6 to 36 hours after inoculation. Temporal bone histopathological results were compared. Twenty-seven chinchillas in an academic research laboratory. Chinchilla middle ears were inoculated with S pneumoniae to produce sufficient volumes of effusions and noticeable histopathological changes in the ears. The CFU counts in the middle ear effusions and histopathological changes were compared to determine the effect of pneumococcal protein mutations on chinchilla ears. At 48 hours, CFUs in middle ears were increased for the Wt and Ply-/PspA- strains, but Ply- remained near inoculum level. No bacteria were detected in the PspA- group. The CFUs of PspA- decreased over time to a low level at 30 to 36 hours. In vitro, PspA- in Todd-Hewitt broth showed an increase in bacterial growth of 2 logs at 43 hours, indicating PspA- susceptibility to host defenses in vivo. The PspA- and Ply- groups had fewer pathologic findings than the Wt or Ply-/PspA- groups. Histopathological analysis showed significant differences in the number of bacteria in the scala tympani in the Wt group compared with the Ply-, PspA-, and Ply-/PspA- groups. The PspA- strain was the least virulent. The PspA- mutant was much less viable and less virulent in the ear than the Wt, Ply-, and Ply-/PspA- strains. There was no significant attenuation in the viability and virulence of the Ply-/PspA- mutant

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

Bacterial diversity of surface sand samples from the Gobi and Taklamaken deserts.

PubMed

An, Shu; Couteau, Cécile; Luo, Fan; Neveu, Julie; DuBow, Michael S

2013-11-01

Arid regions represent nearly 30 % of the Earth's terrestrial surface, but their microbial biodiversity is not yet well characterized. The surface sands of deserts, a subset of arid regions, are generally subjected to large temperature fluctuations plus high UV light exposure and are low in organic matter. We examined surface sand samples from the Taklamaken (China, three samples) and Gobi (Mongolia, two samples) deserts, using pyrosequencing of PCR-amplified 16S V1/V2 rDNA sequences from total extracted DNA in order to gain an assessment of the bacterial population diversity. In total, 4,088 OTUs (using ≥97 % sequence similarity levels), with Chao1 estimates varying from 1,172 to 2,425 OTUs per sample, were discernable. These could be grouped into 102 families belonging to 15 phyla, with OTUs belonging to the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla being the most abundant. The bacterial population composition was statistically different among the samples, though members from 30 genera were found to be common among the five samples. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments. Our results imply an unexpectedly large bacterial diversity residing in the harsh environment of these two Asian deserts, worthy of further investigation.

Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

PubMed

Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2013-06-14

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

PubMed Central

Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

2016-01-01

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

Protein export through the bacterial flagellar type III export pathway.

PubMed

Minamino, Tohru

2014-08-01

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

PubMed

Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

2011-11-01

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG-Au conjugates.

PubMed

Tatlybaeva, Elena B; Nikiyan, Hike N; Vasilchenko, Alexey S; Deryabin, Dmitri G

2013-01-01

The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A-IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG-Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations.

Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG–Au conjugates

PubMed Central

Tatlybaeva, Elena B; Vasilchenko, Alexey S; Deryabin, Dmitri G

2013-01-01

Summary The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A–IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG–Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations. PMID:24367742

Screening host proteins required for bacterial adherence after H9N2 virus infection.

PubMed

Ma, Li-Li; Sun, Zhen-Hong; Xu, Yu-Lin; Wang, Shu-Juan; Wang, Hui-Ning; Zhang, Hao; Hu, Li-Ping; Sun, Xiao-Mei; Zhu, Lin; Shang, Hong-Qi; Zhu, Rui-Liang; Wei, Kai

2018-01-01

H9N2 subtype low pathogenic avian influenza virus (LPAIV) is distributed worldwide and causes great economic losses in the poultry industry, especially when complicated with other bacterial infections. Tissue damages caused by virus infection provide an opportunity for bacteria invasion, but this mechanism is not sufficient for low pathogenic strains. Moreover, although H9N2 virus infection was demonstrated to promote bacterial infection in several studies, its mechanism remained unclear. In this study, infection experiments in vivo and in vitro demonstrated that the adhesion of Escherichia coli (E. coli) to host cells significantly increased after H9N2 virus infection, and this increase was not caused by pathological damages. Subsequently, we constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 virus infection. A total of 279 significantly differential expressed proteins were detected through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed proteins were enriched in host innate immunity; cell proliferation, differentiation, and apoptosis; and pathogenicity-related signaling pathways. Finally, we screened out several proteins, such as TGF-β1, integrins, cortactin, E-cadherin, vinculin, and fibromodulin, which were probably associated with bacterial adhesion. The study analyzed the mechanism of secondary bacterial infection induced by H9N2 virus infection from a novel perspective, which provided theoretical and data support for investigating the synergistic infection mechanism between the H9N2 virus and bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

PubMed Central

Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

2012-01-01

We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection.

PubMed

Li, Hui; Huang, Xiaoyan; Zeng, Zaohai; Peng, Xuan-Xian; Peng, Bo

2016-09-01

Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility

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

Orans, Jillian; Johnson, Michael D.L.; Coggan, Kimberly A.

Several bacterial pathogens require the 'twitching' motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 {angstrom} resolution crystal structure of the Pseudomonas aeruginosa pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified {beta}-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calciummore » binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts Pseudomonas twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner - by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility.« less

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.

Tick receptor for outer surface protein A from Ixodes ricinus — the first intrinsically disordered protein involved in vector-microbe recognition

NASA Astrophysics Data System (ADS)

Urbanowicz, Anna; Lewandowski, Dominik; Szpotkowski, Kamil; Figlerowicz, Marek

2016-04-01

The tick receptor for outer surface protein A (TROSPA) is the only identified factor involved in tick gut colonization by various Borrelia species. TROSPA is localized in the gut epithelium and can recognize and bind the outer surface bacterial protein OspA via an unknown mechanism. Based on earlier reports and our latest observations, we considered that TROSPA would be the first identified intrinsically disordered protein (IDP) involved in the interaction between a vector and a pathogenic microbe. To verify this hypothesis, we performed structural studies of a TROSPA mutant from Ixodes ricinus using both computational and experimental approaches. Irrespective of the method used, we observed that the secondary structure content of the TROSPA polypeptide chain is low. In addition, the collected SAXS data indicated that this protein is highly extended and exists in solution as a set of numerous conformers. These features are all commonly considered hallmarks of IDPs. Taking advantage of our SAXS data, we created structural models of TROSPA and proposed a putative mechanism for the TROSPA-OspA interaction. The disordered nature of TROSPA may explain the ability of a wide spectrum of Borrelia species to colonize the tick gut.

Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

PubMed Central

Derebe, Mehabaw G; Zlatkov, Clare M; Gattu, Sureka; Ruhn, Kelly A; Vaishnava, Shipra; Diehl, Gretchen E; MacMillan, John B; Williams, Noelle S; Hooper, Lora V

2014-01-01

Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection. DOI: http://dx.doi.org/10.7554/eLife.03206.001 PMID:25073702

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

PubMed

Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

2013-05-15

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

PubMed

Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile

2015-11-23

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.

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 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

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.

Adsorption and mineralization of REE-lanthanum onto bacterial cell surface.

PubMed

Cheng, Yangjian; Zhang, Li; Bian, Xiaojing; Zuo, Hongyang; Dong, Hailiang

2017-07-11

A large number of rare earth element mining and application resulted in a series of problems of soil and water pollution. Environmental remediation of these REE-contaminated sites has become a top priority. This paper explores the use of Bacillus licheniformis to adsorb lanthanum and subsequent mineralization process in contaminated water. The maximum adsorption capacity of lanthanum on bacteria was 113.98 mg/g (dry weight) biomass. X-ray diffraction (XRD) and transmission electron microscopy (TEM) data indicated that adsorbed lanthanum on bacterial cell surface occurred in an amorphous form at the initial stage. Scanning electron microscopy with X-ray energy-dispersive spectroscopy (SEM/EDS) results indicated that lanthanum adsorption was correlated with phosphate. The amorphous material was converted into scorpion-like monazite (LaPO 4 nanoparticles) in a month. The above results provide a method of using bacterial surface as adsorption and nucleation sites to treat REE-contaminated water.

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.

Rapid comparison of properties on protein surface

PubMed Central

Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

2008-01-01

The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM β/α barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure. PMID:18618695

Rapid comparison of properties on protein surface.

PubMed

Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

2008-10-01

The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure.

Clinical Prognosis in Neonatal Bacterial Meningitis: The Role of Cerebrospinal Fluid Protein.

PubMed

Tan, Jintong; Kan, Juan; Qiu, Gang; Zhao, Dongying; Ren, Fang; Luo, Zhongcheng; Zhang, Yongjun

2015-01-01

Neonates are at high risk of meningitis and of resulting neurologic complications. Early recognition of neonates at risk of poor prognosis would be helpful in providing timely management. From January 2008 to June 2014, we enrolled 232 term neonates with bacterial meningitis admitted to 3 neonatology departments in Shanghai, China. The clinical status on the day of discharge from these hospitals or at a postnatal age of 2.5 to 3 months was evaluated using the Glasgow Outcome Scale (GOS). Patients were classified into two outcome groups: good (167 cases, 72.0%, GOS = 5) or poor (65 cases, 28.0%, GOS = 1-4). Neonates with good outcome had less frequent apnea, drowsiness, poor feeding, bulging fontanelle, irritability and more severe jaundice compared to neonates with poor outcome. The good outcome group also had less pneumonia than the poor outcome group. Besides, there were statistically significant differences in hemoglobin, mean platelet volume, platelet distribution width, C-reaction protein, procalcitonin, cerebrospinal fluid (CSF) glucose and CSF protein. Multivariate logistic regression analyses suggested that poor feeding, pneumonia and CSF protein were the predictors of poor outcome. CSF protein content was significantly higher in patients with poor outcome. The best cut-offs for predicting poor outcome were 1,880 mg/L in CSF protein concentration (sensitivity 70.8%, specificity 86.2%). After 2 weeks of treatment, CSF protein remained higher in the poor outcome group. High CSF protein concentration may prognosticate poor outcome in neonates with bacterial meningitis.

Modulators of heterogeneous protein surface water dynamics

NASA Astrophysics Data System (ADS)

Han, Songi

The hydration water that solvates proteins is a major factor in driving or enabling biological events, including protein-protein and protein-ligand interactions. We investigate the role of the protein surface in modulating the hydration water fluctuations on both the picosecond and nanosecond timescale with an emerging experimental NMR technique known as Overhauser Dynamic Nuclear Polarization (ODNP). We carry out site-specific ODNP measurements of the hydration water fluctuations along the surface of Chemotaxis Y (CheY), and correlate the measured fluctuations to hydropathic and topological properties of the CheY surface as derived from molecular dynamics (MD) simulation. Furthermore, we compare hydration water fluctuations measured on the CheY surface to that of other globular proteins, as well as intrinsically disordered proteins, peptides, and liposome surfaces to systematically test characteristic effects of the biomolecular surface on the hydration water dynamics. Our results suggest that the labile (ps) hydration water fluctuations are modulated by the chemical nature of the surface, while the bound (ns) water fluctuations are present on surfaces that feature a rough topology and chemical heterogeneity such as the surface of a folded and structured protein. In collaboration with: Ryan Barnes, Dept of Chemistry and Biochemistry, University of California Santa Barbara

Identification of a virulence-related surface protein XF in piscine Streptococcus agalactiae by pre-absorbed immunoproteomics.

PubMed

Liu, Guangjin; Zhang, Wei; Liu, Yongjie; Yao, Huochun; Lu, Chengping; Xu, Pao

2014-10-26

Since 2009, large-scale Streptococcus agalactiae infections have broken out in cultured tilapia farms in China, resulting in considerable economic losses. Screening of the surface proteins is required to identify virulence factors or protective antigens involved in piscine S.agalactiae infections in tilapia. Pre-absorbed immunoproteomics method (PAIM) is a useful method previously established in our laboratory for identifying bacterial surface proteins. A serine-rich repeat protein family 1 (Srr-1), designated XF, was identified by PAIM in piscine S. agalactiae isolate GD201008-001. To investigate the role of XF in the pathogenesis of piscine S. agalactiae, an isogenic xf mutant strain (Δxf) and a complemented strain (CΔxf) were successfully constructed. The Δxf mutant and CΔxf showed no significant differences in growth characteristics and adherence to HEp-2 cells compared with the wild-type strain. However the 50% lethal dose of Δxf was increased (4-fold) compared with that of the parental strain in a zebrafish infection model. The findings demonstrated that XF is a virulence-related, highly immunoreactive surface protein and is involved in the pathogenicity of S. agalactiae infections in fish.

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.

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.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

PubMed Central

Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

2013-01-01

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

Bacterial expansins and related proteins from the world of microbes

DOE PAGES

Georgelis, Nikolaos; Nikolaidis, Nikolas; Cosgrove, Daniel J.

2015-04-02

The discovery of microbial expansins emerged from studies of the mechanism of plant cell growth and the molecular basis of plant cell wall extensibility. Expansins are wall-loosening proteins that are universal in the plant kingdom and are also found in a small set of phylogenetically diverse bacteria, fungi, and other organisms, most of which colonize plant surfaces. They loosen plant cell walls without detectable lytic activity. Bacterial expansins have attracted considerable attention recently for their potential use in cellulosic biomass conversion for biofuel production, as a means to disaggregate cellulosic structures by nonlytic means (“amorphogenesis”). Evolutionary analysis indicates that microbialmore » expansins originated by multiple horizontal gene transfers from plants. Crystallographic analysis of BsEXLX1, the expansin from Bacillus subtilis, shows that microbial expansins consist of two tightly packed domains: the N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl hydrolase family-45 enzymes but lacks catalytic residues usually required for hydrolysis; the C-terminal domain D2 has a unique β-sandwich fold with three co-linear aromatic residues that bind β-1,4-glucans by hydrophobic interactions. Genetic deletion of expansin in Bacillus and Clavibacter cripples their ability to colonize plant tissues. In this paper, we assess reports that expansin addition enhances cellulose breakdown by cellulase and compare expansins with distantly related proteins named swollenin, cerato-platanin, and loosenin. Finally, we end in a speculative vein about the biological roles of microbial expansins and their potential applications. Advances in this field will be aided by a deeper understanding of how these proteins modify cellulosic structures.« less

Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

PubMed

Hewitt, Sarah H; Filby, Maria H; Hayes, Ed; Kuhn, Lars T; Kalverda, Arnout P; Webb, Michael E; Wilson, Andrew J

2017-01-17

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H, 15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds

NASA Astrophysics Data System (ADS)

Oh, Yoo Jin; Hubauer-Brenner, Michael; Gruber, Hermann J.; Cui, Yidan; Traxler, Lukas; Siligan, Christine; Park, Sungsu; Hinterdorfer, Peter

2016-09-01

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

NASA Astrophysics Data System (ADS)

Roth, Julian; Koch, Matthias; Rohrbach, Alexander

2015-03-01

The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

Procalcitonin and C-reactive protein as markers of bacterial infection in critically ill children at onset of systemic inflammatory response syndrome.

PubMed

Simon, Liliana; Saint-Louis, Patrick; Amre, Devendra K; Lacroix, Jacques; Gauvin, France

2008-07-01

To compare the accuracy of procalcitonin and C-reactive protein as diagnostic markers of bacterial infection in critically ill children at the onset of systemic inflammatory response syndrome (SIRS). Prospective cohort study. Tertiary care, university-affiliated pediatric intensive care unit (PICU). Consecutive patients with SIRS. From June to December 2002, all PICU patients were screened daily to include cases of SIRS. At inclusion (onset of SIRS), procalcitonin and C-reactive protein levels as well as an array of cultures were obtained. Diagnosis of bacterial infection was made a posteriori by an adjudicating process (consensus of experts unaware of the results of procalcitonin and C-reactive protein). Baseline and daily data on severity of illness, organ dysfunction, and outcome were collected. Sixty-four patients were included in the study and were a posteriori divided into the following groups: bacterial SIRS (n = 25) and nonbacterial SIRS (n = 39). Procalcitonin levels were significantly higher in patients with bacterial infection compared with patients without bacterial infection (p = .01). The area under the receiver operating characteristic curve for procalcitonin was greater than that for C-reactive protein (0.71 vs. 0.65, respectively). A positive procalcitonin level (>or=2.5 ng/mL), when added to bedside clinical judgment, increased the likelihood of bacterial infection from 39% to 92%, while a negative C-reactive protein level (<40 mg/L) decreased the probability of bacterial infection from 39% to 2%. Procalcitonin is better than C-reactive protein for differentiating bacterial from nonbacterial SIRS in critically ill children, although the accuracy of both tests is moderate. Diagnostic accuracy could be enhanced by combining these tests with bedside clinical judgment.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

PubMed

Gennaris, Alexandra; Ezraty, Benjamin; Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

2015-12-17

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

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.

Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons.

PubMed Central

Cheetham, M E; Brion, J P; Anderton, B H

1992-01-01

The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1599432

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

PubMed Central

Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

2011-01-01

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

Phylogenetic and Complementation Analysis of a Single-Stranded DNA Binding Protein Family from Lactococcal Phages Indicates a Non-Bacterial Origin

PubMed Central

Mariadassou, Mahendra; Bardowski, Jacek K.; Bidnenko, Elena

2011-01-01

Background The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14bIL67-like proteins) have been identified and characterized structurally and biochemically. Methodology/Principal Findings This study focused on the determination of phylogenetic relationships between Orf14bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli. Conclusions/Significance Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages. PMID:22073223

Surface Bacterial-Spore Assay Using Tb3+/DPA Luminescence

NASA Technical Reports Server (NTRS)

Ponce, Adrian

2007-01-01

Equipment and a method for rapidly assaying solid surfaces for contamination by bacterial spores are undergoing development. The method would yield a total (nonviable plus viable) spore count of a surface within minutes and a viable-spore count in about one hour. In this method, spores would be collected from a surface by use of a transparent polymeric tape coated on one side with a polymeric adhesive that would be permeated with one or more reagent(s) for detection of spores by use of visible luminescence. The sticky side of the tape would be pressed against a surface to be assayed, then the tape with captured spores would be placed in a reader that illuminates the sample with ultraviolet light and counts the green luminescence spots under a microscope to quantify the number of bacterial spores per unit area. The visible luminescence spots seen through the microscope would be counted to determine the concentration of spores on the surface. This method is based on the chemical and physical principles of methods described in several prior NASA Tech Briefs articles, including Live/Dead Spore Assay Using DPA-Triggered Tb Luminescence (NPO-30444), Vol. 27, No. 3 (March 2003), page 7a. To recapitulate: The basic idea is to exploit the observations that (1) dipicolinic acid (DPA) is present naturally only in bacterial spores; and (2) when bound to Tb3+ ions, DPA triggers intense green luminescence of the ions under ultraviolet excitation; (3) DPA can be released from the viable spores by using L-alanine to make them germinate; and (4) by autoclaving, microwaving, or sonicating the sample, one can cause all the spores (non-viable as well as viable) to release their DPA. One candidate material for use as the adhesive in the present method is polydimethysiloxane (PDMS). In one variant of the method for obtaining counts of all (viable and nonviable) spores the PDMS would be doped with TbCl3. After collection of a sample, the spores immobilized on the sticky tape surface

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function

PubMed Central

Clark, Chase M.; Costa, Maria S.

2018-01-01

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies. PMID:29686101

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.

Analysis of Bacterial Detachment from Substratum Surfaces by the Passage of Air-Liquid Interfaces

PubMed Central

Gómez-Suárez, Cristina; Busscher, Henk J.; van der Mei, Henny C.

2001-01-01

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 × 106 cells cm−2 was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s−1), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces.

PubMed

Gómez-Suárez, C; Busscher, H J; van der Mei, H C

2001-06-01

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high

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

Selective dye-labeling of newly synthesized proteins in bacterial cells.

PubMed

Beatty, Kimberly E; Xie, Fang; Wang, Qian; Tirrell, David A

2005-10-19

We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.

Food-safe modification of stainless steel food processing surfaces to reduce bacterial biofilms.

PubMed

Awad, Tarek Samir; Asker, Dalal; Hatton, Benjamin D

2018-06-11

Biofilm formation on stainless steel (SS) surfaces of food processing plants, leading to foodborne illness outbreaks, is enabled by the attachment and confinement within microscale cavities of surface roughness (grooves, scratches). We report Foodsafe Oil-based Slippery Coatings (FOSCs) for food processing surfaces that suppress bacterial adherence and biofilm formation by trapping residual oil lubricant within these surface cavities to block microbial growth. SS surfaces were chemically functionalized with alkylphosphonic acid to preferentially wet a layer of food grade oil. FOSCs reduced the effective surface roughness, the adhesion of organic food residue, and bacteria. FOSCs significantly reduced Pseudomonas aeruginosa biofilm formation on standard roughness SS-316 by 5 log CFU cm-2, and by 3 log CFU cm-2 for mirror-finished SS. FOSCs also enhanced surface cleanability, which we measured by bacterial counts after conventional detergent cleaning. Importantly, both SS grades maintained their anti-biofilm activity after erosion of the oil layer by surface wear with glass beads, which suggests there is a residual volume of oil that remains to block surface cavity defects. These results indicate the potential of such low-cost, scalable approaches to enhance the cleanability of SS food processing surfaces and improve food safety by reducing biofilm growth.

Initial Bacterial Adhesion on Different Yttria-Stabilized Tetragonal Zirconia Implant Surfaces in Vitro

PubMed Central

Karygianni, Lamprini; Jähnig, Andrea; Schienle, Stefanie; Bernsmann, Falk; Adolfsson, Erik; Kohal, Ralf J.; Chevalier, Jérôme; Hellwig, Elmar; Al-Ahmad, Ali

2013-01-01

Bacterial adhesion to implant biomaterials constitutes a virulence factor leading to biofilm formation, infection and treatment failure. The aim of this study was to examine the initial bacterial adhesion on different implant materials in vitro. Four implant biomaterials were incubated with Enterococcus faecalis, Staphylococcus aureus and Candida albicans for 2 h: 3 mol % yttria-stabilized tetragonal zirconia polycrystal surface (B1a), B1a with zirconium oxide (ZrO2) coating (B2a), B1a with zirconia-based composite coating (B1b) and B1a with zirconia-based composite and ZrO2 coatings (B2b). Bovine enamel slabs (BES) served as control. The adherent microorganisms were quantified and visualized using scanning electron microscopy (SEM); DAPI and live/dead staining. The lowest bacterial count of E. faecalis was detected on BES and the highest on B1a. The fewest vital C. albicans strains (42.22%) were detected on B2a surfaces, while most E. faecalis and S. aureus strains (approximately 80%) were vital overall. Compared to BES; coated and uncoated zirconia substrata exhibited no anti-adhesive properties. Further improvement of the material surface characteristics is essential. PMID:28788415

Initial Bacterial Adhesion on Different Yttria-Stabilized Tetragonal Zirconia Implant Surfaces in Vitro.

PubMed

Karygianni, Lamprini; Jähnig, Andrea; Schienle, Stefanie; Bernsmann, Falk; Adolfsson, Erik; Kohal, Ralf J; Chevalier, Jérôme; Hellwig, Elmar; Al-Ahmad, Ali

2013-12-04

Bacterial adhesion to implant biomaterials constitutes a virulence factor leading to biofilm formation, infection and treatment failure. The aim of this study was to examine the initial bacterial adhesion on different implant materials in vitro . Four implant biomaterials were incubated with Enterococcus faecalis , Staphylococcus aureus and Candida albicans for 2 h: 3 mol % yttria-stabilized tetragonal zirconia polycrystal surface (B1a), B1a with zirconium oxide (ZrO₂) coating (B2a), B1a with zirconia-based composite coating (B1b) and B1a with zirconia-based composite and ZrO₂ coatings (B2b). Bovine enamel slabs (BES) served as control. The adherent microorganisms were quantified and visualized using scanning electron microscopy (SEM); DAPI and live/dead staining. The lowest bacterial count of E. faecalis was detected on BES and the highest on B1a. The fewest vital C. albicans strains (42.22%) were detected on B2a surfaces, while most E. faecalis and S. aureus strains (approximately 80%) were vital overall. Compared to BES; coated and uncoated zirconia substrata exhibited no anti-adhesive properties. Further improvement of the material surface characteristics is essential.

Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces

PubMed Central

Xu, Li-Chong; Siedlecki, Christopher A.

2013-01-01

Atomic force microscopy (AFM) was used to directly measure the adhesion forces between three test proteins and low density polyethylene (LDPE) surfaces treated by glow discharge plasma to yield various levels of water wettability. The adhesion of proteins to the LDPE substrates showed a step dependence on the wettability of surfaces as measured by the water contact angle (θ). For LDPE surfaces with θ > ∼60–65°, stronger adhesion forces were observed for bovine serum albumin, fibrinogen and human FXII than for the surfaces with θ < 60°. Smaller adhesion forces were observed for FXII than for the other two proteins on all surfaces although trends were identical. Increasing the contact time from 0 to 50 s for each protein–surface combination increased the adhesion force regardless of surface wettability. Time varying adhesion data was fit to an exponential model and free energies of protein unfolding were calculated. This data, viewed in light of previously published studies, suggests a 2-step model of protein denaturation, an early stage on the order of seconds to minutes where the outer surface of the protein interacts with the substrate and a second stage involving movement of hydrophobic amino acids from the protein core to the protein/surface interface. Impact statement The work described in this manuscript shows a stark transition between protein adherent and protein non-adherent materials in the range of water contact angles 60–65°, consistent with known changes in protein adsorption and activity. Time-dependent changes in adhesion force were used to calculate unfolding energies relating to protein–surface interactions. This analysis provides justification for a 2-step model of protein denaturation on surfaces. PMID:17466368

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

PubMed Central

Hovingh, Elise S.; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H. M.

2017-01-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis. PMID:28742139

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H M; Jongerius, Ilse

2017-07-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Surface Passivation for Single-molecule Protein Studies

PubMed Central

Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

2014-01-01

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation. PMID:24797261

The physical boundaries of public goods cooperation between surface-attached bacterial cells

PubMed Central

Weigert, Michael; Kümmerli, Rolf

2017-01-01

Bacteria secrete a variety of compounds important for nutrient scavenging, competition mediation and infection establishment. While there is a general consensus that secreted compounds can be shared and therefore have social consequences for the bacterial collective, we know little about the physical limits of such bacterial social interactions. Here, we address this issue by studying the sharing of iron-scavenging siderophores between surface-attached microcolonies of the bacterium Pseudomonas aeruginosa. Using single-cell fluorescent microscopy, we show that siderophores, secreted by producers, quickly reach non-producers within a range of 100 µm, and significantly boost their fitness. Producers in turn respond to variation in sharing efficiency by adjusting their pyoverdine investment levels. These social effects wane with larger cell-to-cell distances and on hard surfaces. Thus, our findings reveal the boundaries of compound sharing, and show that sharing is particularly relevant between nearby yet physically separated bacteria on soft surfaces, matching realistic natural conditions such as those encountered in soft tissue infections. PMID:28701557

UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

2014-09-01

The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

PubMed

Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

2013-09-01

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

NASA Astrophysics Data System (ADS)

Martin, Daniel R.; Matyushov, Dmitry V.

2015-04-01

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc1 bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ˜0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

PubMed Central

Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

2015-01-01

The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts 1-3. Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both R- and S- diastereoisomers of Met-O, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting Met residues from oxidation should prompt search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum (ER). PMID:26641313

Learning and evolution in bacterial taxis: an operational amplifier circuit modeling the computational dynamics of the prokaryotic 'two component system' protein network.

PubMed

Di Paola, Vieri; Marijuán, Pedro C; Lahoz-Beltra, Rafael

2004-01-01

Adaptive behavior in unicellular organisms (i.e., bacteria) depends on highly organized networks of proteins governing purposefully the myriad of molecular processes occurring within the cellular system. For instance, bacteria are able to explore the environment within which they develop by utilizing the motility of their flagellar system as well as a sophisticated biochemical navigation system that samples the environmental conditions surrounding the cell, searching for nutrients or moving away from toxic substances or dangerous physical conditions. In this paper we discuss how proteins of the intervening signal transduction network could be modeled as artificial neurons, simulating the dynamical aspects of the bacterial taxis. The model is based on the assumption that, in some important aspects, proteins can be considered as processing elements or McCulloch-Pitts artificial neurons that transfer and process information from the bacterium's membrane surface to the flagellar motor. This simulation of bacterial taxis has been carried out on a hardware realization of a McCulloch-Pitts artificial neuron using an operational amplifier. Based on the behavior of the operational amplifier we produce a model of the interaction between CheY and FliM, elements of the prokaryotic two component system controlling chemotaxis, as well as a simulation of learning and evolution processes in bacterial taxis. On the one side, our simulation results indicate that, computationally, these protein 'switches' are similar to McCulloch-Pitts artificial neurons, suggesting a bridge between evolution and learning in dynamical systems at cellular and molecular levels and the evolutive hardware approach. On the other side, important protein 'tactilizing' properties are not tapped by the model, and this suggests further complexity steps to explore in the approach to biological molecular computing.

Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface.

PubMed

Swasthi, Hema M; Mukhopadhyay, Samrat

2017-12-01

Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Fuel of the Bacterial Flagellar Type III Protein Export Apparatus.

PubMed

Minamino, Tohru; Kinoshita, Miki; Namba, Keiichi

2017-01-01

The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.

Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

PubMed

Bi, Xiaobao; Yin, Juan; Chen Guanbang, Ashley; Liu, Chuan-Fa

2018-06-07

The cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of a Bacterial Dynamin-like Protein Lipid Tube Provides a Mechanism For Assembly and Membrane Curving

PubMed Central

Low, Harry H.; Sachse, Carsten; Amos, Linda A.; Löwe, Jan

2009-01-01

Summary Proteins of the dynamin superfamily mediate membrane fission, fusion, and restructuring events by polymerizing upon lipid bilayers and forcing regions of high curvature. In this work, we show the electron cryomicroscopy reconstruction of a bacterial dynamin-like protein (BDLP) helical filament decorating a lipid tube at ∼11 Å resolution. We fitted the BDLP crystal structure and produced a molecular model for the entire filament. The BDLP GTPase domain dimerizes and forms the tube surface, the GTPase effector domain (GED) mediates self-assembly, and the paddle region contacts the lipids and promotes curvature. Association of BDLP with GMPPNP and lipid induces radical, large-scale conformational changes affecting polymerization. Nucleotide hydrolysis seems therefore to be coupled to polymer disassembly and dissociation from lipid, rather than membrane restructuring. Observed structural similarities with rat dynamin 1 suggest that our results have broad implication for other dynamin family members. PMID:20064379

Druggable protein interaction sites are more predisposed to surface pocket formation than the rest of the protein surface.

PubMed

Johnson, David K; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically "druggable" by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that "druggability" is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

PubMed

Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R

2005-09-01

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.

Surface chemical studies on selective separation of pyrite and galena in the presence of bacterial cells and metabolic products of Paenibacillus polymyxa.

PubMed

Patra, Partha; Natarajan, K A

2006-06-15

Selective separation of pyrite and galena from mixture of the two minerals was achieved through interaction with cells and metabolic products from a culture of Paenibacillus polymyxa. Adsorption of cells and metabolic products onto minerals and electrokinetic studies of minerals after interaction with cells and metabolic products were carried out to examine the resulting surface modification on the mineral surfaces. Flocculation and flotation techniques were successfully applied in the selective separation of minerals after bacterial interaction. The effect of varying conditions for production of extracellular polysaccharides and protein provided an insight into the possible mechanism involved in microbially induced flocculation and flotation of pyrite and galena.

Pole-to-pole biogeography of surface and deep marine bacterial communities

PubMed Central

Ghiglione, Jean-François; Galand, Pierre E.; Pommier, Thomas; Pedrós-Alió, Carlos; Maas, Elizabeth W.; Bakker, Kevin; Bertilson, Stefan; Kirchman, David L.; Lovejoy, Connie; Yager, Patricia L.; Murray, Alison E.

2012-01-01

The Antarctic and Arctic regions offer a unique opportunity to test factors shaping biogeography of marine microbial communities because these regions are geographically far apart, yet share similar selection pressures. Here, we report a comprehensive comparison of bacterioplankton diversity between polar oceans, using standardized methods for pyrosequencing the V6 region of the small subunit ribosomal (SSU) rRNA gene. Bacterial communities from lower latitude oceans were included, providing a global perspective. A clear difference between Southern and Arctic Ocean surface communities was evident, with 78% of operational taxonomic units (OTUs) unique to the Southern Ocean and 70% unique to the Arctic Ocean. Although polar ocean bacterial communities were more similar to each other than to lower latitude pelagic communities, analyses of depths, seasons, and coastal vs. open waters, the Southern and Arctic Ocean bacterioplankton communities consistently clustered separately from each other. Coastal surface Southern and Arctic Ocean communities were more dissimilar from their respective open ocean communities. In contrast, deep ocean communities differed less between poles and lower latitude deep waters and displayed different diversity patterns compared with the surface. In addition, estimated diversity (Chao1) for surface and deep communities did not correlate significantly with latitude or temperature. Our results suggest differences in environmental conditions at the poles and different selection mechanisms controlling surface and deep ocean community structure and diversity. Surface bacterioplankton may be subjected to more short-term, variable conditions, whereas deep communities appear to be structured by longer water-mass residence time and connectivity through ocean circulation. PMID:23045668

Role of Streptococcus sanguinis sortase A in bacterial colonization.

PubMed

Yamaguchi, Masaya; Terao, Yutaka; Ogawa, Taiji; Takahashi, Toshihito; Hamada, Shigeyuki; Kawabata, Shigetada

2006-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.

An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

PubMed

Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

2013-04-15

NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Resistance of bacterial biofilms formed on stainless steel surface to disinfecting agent.

PubMed

Królasik, Joanna; Zakowska, Zofia; Krepska, Milena; Klimek, Leszek

2010-01-01

The natural ability of microorganisms for adhesion and biofilm formation on various surfaces is one of the factors causing the inefficiency of a disinfection agent, despite its proven activity in vitro. The aim of the study was to determine the effectiveness of disinfecting substances on bacterial biofilms formed on stainless steel surface. A universally applied disinfecting agent was used in the tests. Bacterial strains: Listeria innocua, Pseudomonas putida, Micrococcus luteus, Staphylococcus hominis strains, were isolated from food contact surfaces, after a cleaning and disinfection process. The disinfecting agent was a commercially available acid specimen based on hydrogen peroxide and peroxyacetic acid, the substance that was designed for food industry usage. Model tests were carried out on biofilm formed on stainless steel (type 304, no 4 finish). Biofilms were recorded by electron scanning microscope. The disinfecting agent in usable concentration, 0.5% and during 10 minutes was ineffective for biofilms. The reduction of cells in biofilms was only 1-2 logarithmic cycles. The use of the agent in higher concentration--1% for 30 minutes caused reduction of cell number by around 5 logarithmic cycles only in the case of one microorganism, M. luteus. For other types: L. innocua, P. putida, S. hominis, the requirements placed on disinfecting agents were not fulfilled. The results of experiments proved that bacterial biofilms are resistant to the disinfectant applied in its operational parameters. Disinfecting effectiveness was achieved after twofold increase of the agent's concentration.

Impact of fluorescent protein fusions on the bacterial flagellar motor.

PubMed

Heo, M; Nord, A L; Chamousset, D; van Rijn, E; Beaumont, H J E; Pedaci, F

2017-10-03

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation- new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Cyclic di-GMP differentially tunes a bacterial flagellar motor through a novel class of CheY-like regulators.

PubMed

Nesper, Jutta; Hug, Isabelle; Kato, Setsu; Hee, Chee-Seng; Habazettl, Judith Maria; Manfredi, Pablo; Grzesiek, Stephan; Schirmer, Tilman; Emonet, Thierry; Jenal, Urs

2017-11-01

The flagellar motor is a sophisticated rotary machine facilitating locomotion and signal transduction. Owing to its important role in bacterial behavior, its assembly and activity are tightly regulated. For example, chemotaxis relies on a sensory pathway coupling chemical information to rotational bias of the motor through phosphorylation of the motor switch protein CheY. Using a chemical proteomics approach, we identified a novel family of CheY-like (Cle) proteins in Caulobacter crescentus , which tune flagellar activity in response to binding of the second messenger c-di-GMP to a C-terminal extension. In their c-di-GMP bound conformation Cle proteins interact with the flagellar switch to control motor activity. We show that individual Cle proteins have adopted discrete cellular functions by interfering with chemotaxis and by promoting rapid surface attachment of motile cells. This study broadens the regulatory versatility of bacterial motors and unfolds mechanisms that tie motor activity to mechanical cues and bacterial surface adaptation.

Physicochemical factors influencing bacterial transfer from contact lenses to surfaces with different roughness and wettability.

PubMed

Vermeltfoort, Pit B J; van der Mei, Henny C; Busscher, Henk J; Hooymans, Johanna M M; Bruinsma, Gerda M

2004-11-15

The aim of this study was to determine the transfer of Pseudomonas aeruginosa No. 3 and Staphylococcus aureus 835 from contact lenses to surfaces with different hydrophobicity and roughness. Bacteria were allowed to adhere to contact lenses (Surevue, PureVision, or Focus Night & Day) by incubating the lenses in a bacterial suspension for 30 min. The contaminated lenses were put on a glass, poly(methylmethacrylate), or silicone rubber substratum, shaped to mimic the eye. After 2 and 16 h, lenses were separated from the substrata and bacteria were swabbed off from the respective surfaces and resuspended in saline. Appropriate serial dilutions of these suspensions were made, from which aliquots were plated on agar for enumeration. Bacterial transfer varied between 4 and 60%, depending on the combination of strain, contact time, contact lens, and substratum surface. For P. aeruginosa No. 3, transfer was significantly higher after 16 h than after 2 h, whereas less increase with time was seen for S. aureus 835. Bacterial transfer from all tested contact lenses was least to silicone rubber, the most hydrophobic and roughest substratum surface included. (c) 2004 Wiley Periodicals, Inc.

Druggable Protein Interaction Sites Are More Predisposed to Surface Pocket Formation than the Rest of the Protein Surface

PubMed Central

Johnson, David K.; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically “druggable” by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that “druggability” is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention. PMID:23505360

Water at protein surfaces studied with femtosecond nonlinear spectroscopy

NASA Astrophysics Data System (ADS)

Bakker, Huib J.

We report on an investigation of the structure and dynamics of water molecules near protein surfaces with femtosecond nonlinear spectroscopic techniques. We measured the reorientation dynamics of water molecules near the surface of several globular protein surfaces, using polarization-resolved femtosecond infrared spectroscopy. We found that water molecules near the protein surface have a much slower reorientation than water molecules in bulk liquid water. The number of slow water molecules scales scales with the size of the hydrophobic surface of the protein. When we denature the proteins by adding an increasing amount of urea to the protein solution, we observe that the water-exposed surface increases by 50% before the secondary structure of the proteins changes. This finding indicates that protein unfolding starts with the protein structure becoming less tight, thereby allowing water to enter. With surface vibrational sum frequency generation (VSFG) spectroscopy, we studied the structure of water at the surface of antifreeze protein III. The measured VSFG spectra showed the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution, at temperatures well above the freezing point. This ordered ice-like hydration layers at the protein surface likely plays an important role in the specific recognition and binding of anti-freeze protein III to nascent ice crystallites, and thus in its anti-freeze mechanism. This research is supported by the ''Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO).

Anticancer Activity of Bacterial Proteins and Peptides.

PubMed

Karpiński, Tomasz M; Adamczak, Artur

2018-04-30

Despite much progress in the diagnosis and treatment of cancer, tumour diseases constitute one of the main reasons of deaths worldwide. The side effects of chemotherapy and drug resistance of some cancer types belong to the significant current therapeutic problems. Hence, searching for new anticancer substances and medicines are very important. Among them, bacterial proteins and peptides are a promising group of bioactive compounds and potential anticancer drugs. Some of them, including anticancer antibiotics (actinomycin D, bleomycin, doxorubicin, mitomycin C) and diphtheria toxin, are already used in the cancer treatment, while other substances are in clinical trials (e.g., p28, arginine deiminase ADI) or tested in in vitro research. This review shows the current literature data regarding the anticancer activity of proteins and peptides originated from bacteria: antibiotics, bacteriocins, enzymes, nonribosomal peptides (NRPs), toxins and others such as azurin, p28, Entap and Pep27anal2. The special attention was paid to the still poorly understood active substances obtained from the marine sediment bacteria. In total, 37 chemical compounds or groups of compounds with antitumor properties have been described in the present article.

Surface energetics and protein-protein interactions: analysis and mechanistic implications

PubMed Central

Peri, Claudio; Morra, Giulia; Colombo, Giorgio

2016-01-01

Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

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.

A coarse grain model for protein-surface interactions

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2013-09-01

The interaction of proteins with surfaces is important in numerous applications in many fields—such as biotechnology, proteomics, sensors, and medicine—but fundamental understanding of how protein stability and structure are affected by surfaces remains incomplete. Over the last several years, molecular simulation using coarse grain models has yielded significant insights, but the formalisms used to represent the surface interactions have been rudimentary. We present a new model for protein surface interactions that incorporates the chemical specificity of both the surface and the residues comprising the protein in the context of a one-bead-per-residue, coarse grain approach that maintains computational efficiency. The model is parameterized against experimental adsorption energies for multiple model peptides on different types of surfaces. The validity of the model is established by its ability to quantitatively and qualitatively predict the free energy of adsorption and structural changes for multiple biologically-relevant proteins on different surfaces. The validation, done with proteins not used in parameterization, shows that the model produces remarkable agreement between simulation and experiment.

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

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

PubMed Central

Ohnishi, S; Murata, M; Hato, M

1998-01-01

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves. PMID:9449346

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.

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

PubMed

Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

2018-06-01

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

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

Shrestha, Ruben; Chen, Xuejie; Ramyar, Kasra X.

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H 2O 2 activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Bluemore » 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.« less

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.

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.

Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

PubMed Central

Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

A mobile precursor determines protein resistance on nanostructured surfaces.

PubMed

Wang, Kang; Chen, Ye; Gong, Xiangjun; Xia, Jianlong; Zhao, Junpeng; Shen, Lei

2018-05-09

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

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

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis.

PubMed

Mueller, R F; Characklis, W G; Jones, W L; Sears, J T

1992-05-01

The processes leading to bacterial colonization on solid-water interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 microm (for silicon) to 0.015 microm (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varied by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.

PubMed

Upadhyay, Vaibhav; Singh, Anupam; Jha, Divya; Singh, Akansha; Panda, Amulya K

2016-06-08

Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form. Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize

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.

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

PubMed

Spahn, Christoph; Glaesmann, Mathilda; Gao, Yunfeng; Foo, Yong Hwee; Lampe, Marko; Kenney, Linda J; Heilemann, Mike

2017-01-01

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

PubMed Central

2010-01-01

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

Unique surface adsorption behaviors of serum proteins on chemically uniform and alternating surfaces

NASA Astrophysics Data System (ADS)

Song, Sheng

With increasing interests of studying proteins adsorption on the surfaces with nanoscale features in biomedical field, it is crucial to have fundamental understandings on how the proteins are adsorbed on such a surface and what factors contribute to the driving forces of adsorption. Besides, exploring more available nanoscale templates would greatly offer more possibilities one could design surface bio-detection methods with favorable protein-surface interactions. Thus, to fulfill the purpose, the work in this dissertation has been made into three major sections. First, to probe the intermediate states which possibly exist between stable and unstable phases described in mean-field theory diagram, a solvent vapor annealing method is chosen to slowly induce the copolymer polystyrene-block-polyvinylpyridine (PS-b-PVP)'s both blocks undergoing micro-phase separations from initial spherical nanodomains into terminal cylindrical nanodomains. During this process, real time atomic force microscopy (AFM) has been conducted to capture other six intermediate states with different morphologies on the polymeric film surfaces. Secondly, upon recognizing each intermediate state, the solution of immunoglobulin gamma (IgG) proteins has been deposited on the surface and been rinsed off with buffer solution before the protein-bounded surface is imaged by AFM. It has been found IgG showing a strong adsorption preference on PS over P4VP block. Among all the six intermediate states, the proteins are almost exclusively adsorbed on PS nanodomains regardless the concentration and deposition time. Thirdly, a trinodular shape protein fibrinogen (Fg) is selected for investigating how geometry and surface charge of proteins would interplay with cylindrical nanodomains on a surface developed from Polystyrene -block-Poly-(methyl methacrylate) PS-b-PMMA. Also, Fg adsorptions on chemically homogeneous surfaces are included here to have a better contrast of showing how much difference it can make

Can the Bacterial Community of a High Arctic Glacier Surface Escape Viral Control?

PubMed Central

Rassner, Sara M. E.; Anesio, Alexandre M.; Girdwood, Susan E.; Hell, Katherina; Gokul, Jarishma K.; Whitworth, David E.; Edwards, Arwyn

2016-01-01

Glacial ice surfaces represent a seasonally evolving three-dimensional photic zone which accumulates microbial biomass and potentiates positive feedbacks in ice melt. Since viruses are abundant in glacial systems and may exert controls on supraglacial bacterial production, we examined whether changes in resource availability would promote changes in the bacterial community and the dynamics between viruses and bacteria of meltwater from the photic zone of a Svalbard glacier. Our results indicated that, under ambient nutrient conditions, low estimated viral decay rates account for a strong viral control of bacterial productivity, incurring a potent viral shunt of a third of bacterial carbon in the supraglacial microbial loop. Moreover, it appears that virus particles are very stable in supraglacial meltwater, raising the prospect that viruses liberated in melt are viable downstream. However, manipulating resource availability as dissolved organic carbon, nitrogen, and phosphorous in experimental microcosms demonstrates that the photic zone bacterial communities can escape viral control. This is evidenced by a marked decline in virus-to-bacterium ratio (VBR) concomitant with increased bacterial productivity and number. Pyrosequencing shows a few bacterial taxa, principally Janthinobacterium sp., dominate both the source meltwater and microcosm communities. Combined, our results suggest that viruses maintain high VBR to promote contact with low-density hosts, by the manufacture of robust particles, but that this necessitates a trade-off which limits viral production. Consequently, dominant bacterial taxa appear to access resources to evade viral control. We propose that a delicate interplay of bacterial and viral strategies affects biogeochemical cycling upon glaciers and, ultimately, downstream ecosystems. PMID:27446002

Binding host proteins to the M protein contributes to the mortality associated with influenza-Streptococcus pyogenes superinfections.

PubMed

Herrera, Andrea L; Suso, Kuta; Allison, Stephanie; Simon, Abby; Schlenker, Evelyn; Huber, Victor C; Chaussee, Michael S

2017-09-25

The mortality associated with influenza A virus (IAV) is often due to the development of secondary bacterial infections known as superinfections. The group A streptococcus (GAS) is a relatively uncommon cause of IAV superinfections, but the mortality of these infections is high. We used a murine model to determine whether the surface-localized GAS M protein contributes to the outcome of IAV-GAS superinfections. A comparison between wild-type GAS and an M protein mutant strain (emm3) showed that the M3 protein was essential to virulence. To determine whether the binding, or recruitment, of host proteins to the bacterial surface contributed to virulence, GAS was suspended with BALF collected from mice that had recovered from a sub-lethal infection with IAV. Following intranasal inoculation of naïve mice, the mortality associated with the wild-type strain, but not the emm3 mutant strain, was greater compared to mice inoculated with GAS suspended with either BALF from uninfected mice or PBS. Further analyses showed that both albumin and fibrinogen (Fg) were more abundant in the respiratory tract 8 days after IAV infection, that M3 bound both proteins to the bacterial surface, and that suspension of GAS with either protein increased GAS virulence in the absence of antecedent IAV infection. Overall, the results showed that M3 is essential to the virulence of GAS in an IAV superinfection and suggested that increased abundance of albumin and Fg in the respiratory tract following IAV infection enhanced host susceptibility to secondary GAS infection.

Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model.

PubMed

Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

2017-02-10

In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease.

Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model

PubMed Central

Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

2017-01-01

In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease. PMID:28186208

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

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

The oral bacterial microbiome of occlusal surfaces in children and its association with diet and caries.

PubMed

Ribeiro, Apoena Aguiar; Azcarate-Peril, Maria Andrea; Cadenas, Maria Belen; Butz, Natasha; Paster, Bruce J; Chen, Tsute; Bair, Eric; Arnold, Roland R

2017-01-01

Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index [DMFSm Index, including active white spot lesions (AWSL)] value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

PubMed Central

Kesel, S.; Mader, A.; Seeberger, P. H.; Lieleg, O.

2014-01-01

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future. PMID:25038098

Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation.

PubMed

Kilsgård, Ola; Karlsson, Christofer; Malmström, Erik; Malmström, Johan

2016-11-01

Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could

Attenuation of Streptococcus suis virulence by the alteration of bacterial surface architecture

PubMed Central

Feng, Youjun; Cao, Min; Shi, Jie; Zhang, Huimin; Hu, Dan; Zhu, Jing; Zhang, Xianyun; Geng, Meiling; Zheng, Feng; Pan, Xiuzhen; Li, Xianfu; Hu, Fuquan; Tang, Jiaqi; Wang, Changjun

2012-01-01

NeuB, a sialic acid synthase catalyzes the last committed step of the de novo biosynthetic pathway of sialic acid, a major element of bacterial surface structure. Here we report a functional NeuB homologue of Streptococcus suis, a zoonotic agent, and systematically address its molecular and immunological role in bacterial virulence. Disruption of neuB led to thinner capsules and more susceptibility to pH, and cps2B inactivation resulted in complete absence of capsular polysaccharides. These two mutants both exhibited increased adhesion and invasion to Hep-2 cells and improved sensibility to phagocytosis. Not only do they retain the capability of inducing the release of host pro-inflammatory cytokines, but also result in the faster secretion of IL-8. Easier cleaning up of the mutant strains in whole blood is consistent with virulence attenuation seen with experimental infections of both mice and SPF-piglets. Therefore we concluded that altered architecture of S. suis surface attenuates its virulence. PMID:23050094

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.

Electro-induced protein deposition on low-fouling surfaces

NASA Astrophysics Data System (ADS)

Cole, M. A.; Voelcker, N. H.; Thissen, H.

2007-12-01

Control over protein adsorption is a key issue for numerous biomedical applications ranging from diagnostic microarrays to tissue-engineered medical devices. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which upon application of an external trigger can be transformed into surfaces showing high protein adsorption on demand. Silicon wafers were used as substrate materials upon which thin functional coatings were constructed by the deposition of an allylamine plasma polymer followed by high-density grafting of poly(ethylene oxide) aldehyde, resulting in a low-fouling surface. When the underlying highly doped silicon substrate was used as an electrode, the resulting electrostatic attraction between the electrode and charged proteins in solution induced protein deposition at the low-fouling interface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to characterize the surface modifications. Controlled protein adsorption experiments were carried out using horseradish peroxidase. The amount of protein deposited at the surface was then investigated by means of a colorimetric assay. It is expected that the concept described here will find use in a variety of biotechnological and biomedical applications, particularly in the area of biochips.

[Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique].

PubMed

Zhang, Yu; Yao, Youlin; Jiang, Siyuan; Lu, Yilu; Liu, Yunqiang; Tao, Dachang; Zhang, Sizhong; Ma, Yongxin

2015-04-01

To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique. Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed. Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins. Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

Denaturation of proteins near polar surfaces

NASA Astrophysics Data System (ADS)

Starzyk, Anna; Cieplak, Marek

2011-12-01

All-atom molecular dynamics simulations for proteins placed near a model mica surface indicate existence of two types of evolution. One type leads to the surface-induced unfolding and the other just to a deformation. The two behaviors are characterized by distinct properties of the radius of gyration and of a novel distortion parameter that distinguishes between elongated, globular, and planar shapes. They also differ in the nature of their single site diffusion and two-site distance fluctuations. The four proteins chosen for the studies, the tryptophan cage, protein G, hydrophobin and lyzozyme, are small to allow for a fair determination of the forces generated by the surface as the effects of finite cutoffs in the Coulombic interactions are thus minimized. When the net charge on the surface is set to zero artificially, infliction of deformation is seen to persists but no unfolding takes place. Unfolding may also be prevented by a cluster of disulfide bonds, as we observe in simulations of hydrophobin.

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.

ABI domain containing proteins contribute to surface protein display and cell division in Staphylococcus aureus

PubMed Central

Frankel, Matthew B.; Wojcik, Brandon; DeDent, Andrea C.; Missiakas, Dominique M.; Schneewind, Olaf

2012-01-01

Summary The human pathogen Staphyloccocus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harbored transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross walls and in the relative abundance of staphylococci with cross walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. PMID:20923422

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

PubMed

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Comprehensive assessment and performance improvement of effector protein predictors for bacterial secretion systems III, IV and VI.

PubMed

An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor

2018-01-01

Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The oral bacterial microbiome of occlusal surfaces in children and its association with diet and caries

PubMed Central

Azcarate-Peril, Maria Andrea; Cadenas, Maria Belen; Butz, Natasha; Paster, Bruce J.; Chen, Tsute; Bair, Eric

2017-01-01

Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index [DMFSm Index, including active white spot lesions (AWSL)] value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and

Surface modification of protein enhances encapsulation in chitosan nanoparticles

NASA Astrophysics Data System (ADS)

Koyani, Rina D.; Andrade, Mariana; Quester, Katrin; Gaytán, Paul; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2018-04-01

Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

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.

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

Legionella pneumophila effector WipA, a bacterial PPP protein phosphatase with PTP activity.

PubMed

Jia, Qian; Lin, Yun; Gou, Xuejing; He, Lei; Shen, Dong; Chen, Dongni; Xie, Wei; Lu, Yongjun

2018-04-26

The gram-negative bacterium Legionella pneumophila invades human's lung and causes Legionnaires' disease. To benefit its survival and replication in cellular milieu, L. pneumophila secrets at least 330 effector proteins into host cells. We found that the effector WipA has the protein tyrosine phosphatase (PTP) activity but does not depend on the classical CX5R motif for activity, suggesting that WipA is an unconventional PTP. Meanwhile, the presence of three other highly conserved motifs typically seen in protein serine/threonine phosphatases and the poor inhibition of WipA activity by okadaic acid led us to propose that WipA is a bacterial protein phosphatase. In addition, the determination of the 2.55-Å crystal structure of WipA revealed that WipA resembles cold-active protein tyrosine phosphatase (CAPTPase), and therefore very likely shares the same catalytic mechanism.

Surface Relaxation in Protein Crystals

NASA Technical Reports Server (NTRS)

Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

2002-01-01

Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

Modulating surface rheology by electrostatic protein/polysaccharide interactions.

PubMed

Ganzevles, Renate A; Zinoviadou, Kyriaki; van Vliet, Ton; Cohen, Martien A; de Jongh, Harmen H

2006-11-21

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.

Applications of yeast surface display for protein engineering

PubMed Central

Cherf, Gerald M.; Cochran, Jennifer R.

2015-01-01

The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine. PMID:26060074

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

PubMed

Eldridge, Matthew J G; Shenoy, Avinash R

2015-02-01

Inflammasomes - molecular platforms for caspase-1 activation - have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1β and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to 'canonical' and 'non-canonical' activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

PubMed

Duraj-Thatte, Anna M; Praveschotinunt, Pichet; Nash, Trevor R; Ward, Frederick R; Joshi, Neel S

2018-02-22

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

NASA Astrophysics Data System (ADS)

Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

2006-03-01

Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

PubMed Central

Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

2010-01-01

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family*

PubMed Central

Broussard, Tyler C.; Miller, Darcie J.; Jackson, Pamela; Nourse, Amanda; White, Stephen W.; Rock, Charles O.

2016-01-01

Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

PubMed

Tomalty, Heather E; Walker, Virginia K

2014-09-26

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Surface contact stimulates the just-in-time deployment of bacterial adhesins.

PubMed

Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

2012-01-01

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive. © 2011 Blackwell Publishing Ltd.

Human Common Salivary Protein 1 (CSP-1) Promotes Binding of Streptococcus mutans to Experimental Salivary Pellicle and Glucans Formed on Hydroxyapatite Surface

PubMed Central

Ambatipudi, Kiran S.; Hagen, Fred K.; Delahunty, Claire M.; Han, Xuemei; Shafi, Rubina; Hryhorenko, Jennifer; Gregoire, Stacy; Marquis, Robert E.; Melvin, James E.; Koo, Hyun; Yates, John R.

2010-01-01

Summary The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins which interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/ml). However, S. mutans cells exposed to rCSP-1 (10 μg/ml) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto tooth surface. PMID:20858015

Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis.

PubMed

Wang, Jiang; Li, Chang; Jiang, Yingjian; Zheng, Hongmei; Li, Dehui; Liang, Yibo; Deng, Wensheng; Zhang, Dianliang

2017-02-01

The aim of the study was to investigate the effects of ceramide-1-phosphate transfer protein (CPTP) on the intestinal epithelial tight junction proteins in patients with severe acute pancreatitis (SAP). Fifty patients with SAP were classified into two groups according to the presence of bacterial translocation (BT) in the blood. Thirty healthy individuals were included in the control group. The presence of BT was analyzed by polymerase chain reaction. The expression of tight junction proteins and CPTP was determined using immunohistochemistry and western blotting. Bacterial DNA was detected in the peripheral blood of 62.0% of the patients with SAP. The expression of CPTP and tight junction proteins in SAP patients was lower than that in healthy controls. Among the patients with SAP, those positive for BT(+) showed a lower level of CPTP and occluding (OC) and zonula occludens-1 (ZO-1) expression and a higher level of IVA cPLA2 expression than BT(-) patients. Moreover, the expression of CPTP was significantly associated with ZO-1 and showed a negative correlation with expression of IVA cPLA2 in SAP-BT(+) patients. CPTP affects the expression of tight junction proteins and may protects the intestinal epithelial barrier by downregulating the expression of IVA cPLA2. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

PubMed Central

Polansky, Ondrej; Babak, Vladimir; Kulich, Pavel

2016-01-01

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors. PMID:27868063

Mapping hydration dynamics and coupled water-protein fluctuations around a protein surface

NASA Astrophysics Data System (ADS)

Zhang, Luyuan; Wang, Lijuan; Kao, Ya-Ting; Qiu, Weihong; Yang, Yi; Okobiah, Oghaghare; Zhong, Dongping

2009-03-01

Elucidation of the molecular mechanism of water-protein interactions is critical to understanding many fundamental aspects of protein science, such as protein folding and misfolding and enzyme catalysis. We recently carried out a global mapping of protein-surface hydration dynamics around a globular α-helical protein apomyoglobin. The intrinsic optical probe tryptophan was employed to scan the protein surface one at a time by site-specific mutagenesis. With femtosecond resolution, we mapped out the dynamics of water-protein interactions with more than 20 mutants and for two states, native and molten globular. A robust bimodal distribution of time scales was observed, representing two types of water motions: local relaxation and protein-coupled fluctuations. The time scales show a strong correlation with the local protein structural rigidity and chemical identity. We also resolved two distinct contributions to the overall Stokes-shifts from the two time scales. These results are significant to understanding the role of hydration water on protein structural stability, dynamics and function.

Terahertz vibrational signature of bacterial spores arising from nanostructure decorated endospore surface.

PubMed

Datta, Debopam; Stroscio, Michael A; Dutta, Mitra; Zhang, Weidong; Brown, Elliott R

2018-05-03

This theoretical effort is the first to explore the possible hypothesis that terahertz optical activity of Bacillus spores arises from normal vibrational modes of spore coat subcomponents in the terahertz frequency range. Bacterial strains like Bacillus and Clostridium form spores with a hardened coating made of peptidoglycan to protect its genetic material in harsh conditions. In recent years, electron microscopy and atomic force microscopy has revealed that bacterial spore surfaces are decorated with nanocylinders and honeycomb nanostructures. In this article, a simple elastic continuum model is used to describe the vibration of these nanocylinders mainly in Bacillus subtilis, which also leads to the conclusion that the terahertz signature of these spores arises from the vibration of these nanostructures. Three vibrating modes: radial/longitudinal, torsional and flexural, have been identified and discussed for the nanocylinders. The effect of bound water, which shifts the vibration frequency, is also discussed. The peptidoglycan molecule consists of polar and charged amino acids; hence, the sporal surface local vibrations interact strongly with the terahertz radiation. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photocatalytic bacterial inactivation by TiO2-coated surfaces

PubMed Central

2013-01-01

The aim of this study was the evaluation of the photoactivated antibacterial activity of titanium dioxide (TiO2)-coated surfaces. Bacterial inactivation was evaluated using TiO2-coated Petri dishes. The experimental conditions optimized with Petri dishes were used to test the antibacterial effect of TiO2-coated ceramic tiles. The best antibacterial effect with Petri dishes was observed at 180, 60, 30 and 20 min of exposure for Escherichia coli, Staphylococcus aureus, Pseudomonas putida and Listeria innocua, respectively. The ceramic tiles demonstrated a photoactivated bactericidal effect at the same exposure time. In general, no differences were observed between the antibacterial effect obtained with Petri dishes and tiles. However, the photochemical activity of Petri dishes was greater than the activity of the tiles. Results obtained indicates that the TiO2-coated surfaces showed a photoactivated bactericidal effect with all bacteria tested highlighting that the titania could be used in the ceramic and building industry for the production of coated surfaces to be placed in microbiologically sensitive environments, such as the hospital and food industry. PMID:24090112

Three-dimensional visualization of nanostructured surfaces and bacterial attachment using Autodesk® Maya®.

PubMed

Boshkovikj, Veselin; Fluke, Christopher J; Crawford, Russell J; Ivanova, Elena P

2014-02-28

There has been a growing interest in understanding the ways in which bacteria interact with nano-structured surfaces. As a result, there is a need for innovative approaches to enable researchers to visualize the biological processes taking place, despite the fact that it is not possible to directly observe these processes. We present a novel approach for the three-dimensional visualization of bacterial interactions with nano-structured surfaces using the software package Autodesk Maya. Our approach comprises a semi-automated stage, where actual surface topographic parameters, obtained using an atomic force microscope, are imported into Maya via a custom Python script, followed by a 'creative stage', where the bacterial cells and their interactions with the surfaces are visualized using available experimental data. The 'Dynamics' and 'nDynamics' capabilities of the Maya software allowed the construction and visualization of plausible interaction scenarios. This capability provides a practical aid to knowledge discovery, assists in the dissemination of research results, and provides an opportunity for an improved public understanding. We validated our approach by graphically depicting the interactions between the two bacteria being used for modeling purposes, Staphylococcus aureus and Pseudomonas aeruginosa, with different titanium substrate surfaces that are routinely used in the production of biomedical devices.

Three-dimensional visualization of nanostructured surfaces and bacterial attachment using Autodesk® Maya®

NASA Astrophysics Data System (ADS)

Boshkovikj, Veselin; Fluke, Christopher J.; Crawford, Russell J.; Ivanova, Elena P.

2014-02-01

There has been a growing interest in understanding the ways in which bacteria interact with nano-structured surfaces. As a result, there is a need for innovative approaches to enable researchers to visualize the biological processes taking place, despite the fact that it is not possible to directly observe these processes. We present a novel approach for the three-dimensional visualization of bacterial interactions with nano-structured surfaces using the software package Autodesk Maya. Our approach comprises a semi-automated stage, where actual surface topographic parameters, obtained using an atomic force microscope, are imported into Maya via a custom Python script, followed by a `creative stage', where the bacterial cells and their interactions with the surfaces are visualized using available experimental data. The `Dynamics' and `nDynamics' capabilities of the Maya software allowed the construction and visualization of plausible interaction scenarios. This capability provides a practical aid to knowledge discovery, assists in the dissemination of research results, and provides an opportunity for an improved public understanding. We validated our approach by graphically depicting the interactions between the two bacteria being used for modeling purposes, Staphylococcus aureus and Pseudomonas aeruginosa, with different titanium substrate surfaces that are routinely used in the production of biomedical devices.

Three-dimensional visualization of nanostructured surfaces and bacterial attachment using Autodesk® Maya®

PubMed Central

Boshkovikj, Veselin; Fluke, Christopher J.; Crawford, Russell J.; Ivanova, Elena P.

2014-01-01

There has been a growing interest in understanding the ways in which bacteria interact with nano-structured surfaces. As a result, there is a need for innovative approaches to enable researchers to visualize the biological processes taking place, despite the fact that it is not possible to directly observe these processes. We present a novel approach for the three-dimensional visualization of bacterial interactions with nano-structured surfaces using the software package Autodesk Maya. Our approach comprises a semi-automated stage, where actual surface topographic parameters, obtained using an atomic force microscope, are imported into Maya via a custom Python script, followed by a ‘creative stage', where the bacterial cells and their interactions with the surfaces are visualized using available experimental data. The ‘Dynamics' and ‘nDynamics' capabilities of the Maya software allowed the construction and visualization of plausible interaction scenarios. This capability provides a practical aid to knowledge discovery, assists in the dissemination of research results, and provides an opportunity for an improved public understanding. We validated our approach by graphically depicting the interactions between the two bacteria being used for modeling purposes, Staphylococcus aureus and Pseudomonas aeruginosa, with different titanium substrate surfaces that are routinely used in the production of biomedical devices. PMID:24577105

Comparative Genomic Analyses of the Bacterial Phosphotransferase System

PubMed Central

Barabote, Ravi D.; Saier, Milton H.

2005-01-01

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. PMID:16339738

Physicochemical code for quinary protein interactions in Escherichia coli

PubMed Central

Mu, Xin; Choi, Seongil; Lang, Lisa; Mowray, David; Danielsson, Jens; Oliveberg, Mikael

2017-01-01

How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical−chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein’s behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics. PMID:28536196

Selective molecular transport through the protein shell of a bacterial microcompartment organelle

DOE PAGES

Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; ...

2015-02-23

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. In this paper, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux ofmore » propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Finally and overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.« less

Protein Oxidation: Key to Bacterial Desiccation Resistance?

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

Fredrickson, Jim K.; Li, Shu-Mei W.; Gaidamakova, E.

For extremely ionizing radiation resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the U.S. Department of Energy’s Hanford Site in Washington state. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of themore » genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms which limit protein oxidation during dehydration.« less

Copper effects on bacterial activity of estuarine silty sediments

NASA Astrophysics Data System (ADS)

Almeida, Adelaide; Cunha, Ângela; Fernandes, Sandra; Sobral, Paula; Alcântara, Fernanda

2007-07-01

Bacteria of silty estuarine sediments were spiked with copper to 200 μg Cu g -1 dry weight sediment in order to assess the impact of copper on bacterial degradation of organic matter and on bacterial biomass production. Bacterial density was determined by direct counting under epifluorescence microscopy and bacterial production by the incorporation of 3H-Leucine. Leucine turnover rate was evaluated by 14C-leucine incorporation and ectoenzymatic activities were estimated as the hydrolysis rate of model substrates for β-glucosidase and leucine-aminopeptidase. The presence of added copper in the microcosms elicited, after 21 days of incubation, generalised anoxia and a decrease in organic matter content. The non-eroded surface of the copper-spiked sediment showed, when compared to the control, a decrease in bacterial abundance and significant lower levels of bacterial production and of leucine turnover rate. Bacterial production and leucine turnover rate decreased to 1.4% and 13% of the control values, respectively. Ectoenzymatic activities were also negatively affected but by smaller factors. After erosion by the water current in laboratory flume conditions, the eroded surface of the control sediment showed a generalised decline in all bacterial activities. The erosion of the copper-spiked sediment showed, however, two types of responses with respect to bacterial activities at the exposed surface: positive responses of bacterial production and leucine turnover rate contrasting with slight negative responses of ectoenzymatic activities. The effects of experimental erosion in the suspended cells were also different in the control and in the copper-spiked sediment. Bacterial cells in the control microcosm exhibited, when compared to the non-eroded sediment cells, decreases in all activities after the 6-h suspension. The response of the average suspended copper-spiked sediment cell differed from the control by a less sharp decrease in ectoenzymatic activities and

Improved protein surface comparison and application to low-resolution protein structure data.

PubMed

Sael, Lee; Kihara, Daisuke

2010-12-14

Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

Improved protein surface comparison and application to low-resolution protein structure data

PubMed Central

2010-01-01

Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy. PMID:21172052

Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria.

PubMed

Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Li, Chunbao; Lu, Chunbao; Zhou, Guanghong

2015-10-14

Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host.

Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria

PubMed Central

Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Lu, Chunbao; Zhou, Guanghong

2015-01-01

Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host. PMID:26463271

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.

Bacterial Iron–Sulfur Regulatory Proteins As Biological Sensor-Switches

PubMed Central

Crack, Jason C.; Green, Jeffrey; Hutchings, Matthew I.; Thomson, Andrew J.

2012-01-01

Abstract Significance: In recent years, bacterial iron–sulfur cluster proteins that function as regulators of gene transcription have emerged as a major new group. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions. This can range from mounting a response to oxidative or nitrosative stress to switching between anaerobic and aerobic respiratory pathways. The sensitivity of these ancient cofactors to small molecule reactive oxygen and nitrogen species, in particular, makes them ideally suited to function as sensors. Recent Advances: An important challenge is to obtain mechanistic and structural information about how these regulators function and, in particular, how the chemistry occurring at the cluster drives the subsequent regulatory response. For several regulators, including FNR, SoxR, NsrR, IscR, and Wbl proteins, major advances in understanding have been gained recently and these are reviewed here. Critical Issues: A common theme emerging from these studies is that the sensitivity and specificity of the cluster of each regulatory protein must be exquisitely controlled by the protein environment of the cluster. Future Directions: A major future challenge is to determine, for a range of regulators, the key factors for achieving control of sensitivity/specificity. Such information will lead, eventually, to a system understanding of stress response, which often involves more than one regulator. Antioxid. Redox Signal. 17, 1215–1231. PMID:22239203

Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

PubMed

Merchant, Mark; Kinney, Clint; Sanders, Paige

2009-12-01

Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins.

PubMed

Glenting, Jacob; Beck, Hans Christian; Vrang, Astrid; Riemann, Holger; Ravn, Peter; Hansen, Anne Maria; Antonsson, Martin; Ahrné, Siv; Israelsen, Hans; Madsen, Søren

2013-06-12

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated. Copyright © 2013 Elsevier GmbH. All rights reserved.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

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

Slowdown of surface diffusion during early stages of bacterial colonization

NASA Astrophysics Data System (ADS)

Vourc'h, T.; Peerhossaini, H.; Léopoldès, J.; Méjean, A.; Chauvat, F.; Cassier-Chauvat, C.

2018-03-01

We study the surface diffusion of the model cyanobacterium Synechocystis sp. PCC6803 during the incipient stages of cell contact with a glass surface in the dilute regime. We observe a twitching motility with alternating immobile tumble and mobile run periods, resulting in a normal diffusion described by a continuous-time random walk with a coefficient of diffusion D . Surprisingly, D is found to decrease with time down to a plateau. This is observed only when the cyanobacterial cells are able to produce released extracellular polysaccharides, as shown by a comparative study between the wild-type strain and various polysaccharides-depleted mutants. The analysis of the trajectories taken by the bacterial cells shows that the temporal characteristics of their intermittent motion depend on the instantaneous fraction of visited sites during diffusion. This describes quantitatively the time dependence of D , related to the progressive surface coverage by the polysaccharides. The observed slowdown of the surface diffusion may constitute a basic precursor mechanism for microcolony formation and provides clues for controlling biofilm formation.

The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

PubMed

Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

2016-06-13

The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

PubMed

Cowles, Kimberly N; Gitai, Zemer

2010-06-01

Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

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...

Predicting stability of alpha-helical, orthogonal-bundle proteins on surfaces

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2010-09-01

The interaction of proteins with surfaces is a key phenomenon in many applications, but current understanding of the biophysics involved is lacking. At present, rational design of such emerging technologies is difficult as no methods or theories exist that correctly predict how surfaces influence protein behavior. Using molecular simulation and a coarse-grain model, this study illustrates for the first time that stability of proteins on surfaces can be correlated with tertiary structural elements for alpha-helical, orthogonal-bundle proteins. Results show that several factors contribute to stability on surfaces including the nature of the loop region where the tether is placed and the ability of the protein to freely rotate on the surface. A thermodynamic analysis demonstrates that surfaces stabilize proteins entropically and that any destabilization is an enthalpic effect. Moreover, the entropic effects are concentrated on the unfolded state of the protein while the ethalpic effects are focused on the folded state.

Deciphering fine molecular details of proteins' structure and function with a Protein Surface Topography (PST) method.

PubMed

Koromyslova, Anna D; Chugunov, Anton O; Efremov, Roman G