Epitopes of Microbial and Human Heat Shock Protein 60 and Their Recognition in Myalgic Encephalomyelitis

PubMed Central

Elfaitouri, Amal; Herrmann, Björn; Bölin-Wiener, Agnes; Wang, Yilin; Gottfries, Carl-Gerhard; Zachrisson, Olof; Pipkorn, Rϋdiger; Rönnblom, Lars; Blomberg, Jonas

2013-01-01

Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity. PMID:24312270

Separation and determination of peptide metabolite of Bacillus licheniformis in a microbial fuel cell by high-speed capillary micellar electrokinetic chromatography.

PubMed

Wang, Wei; Bai, Ruiguang; Cai, Xiaoyu; Lin, Ping; Ma, Lihong

2017-11-01

A method using high-speed capillary micellar electrokinetic chromatography and a microbial fuel cell was applied to determine the metabolite of the peptides released by Bacillus licheniformis. Two peptides, l-carnosine and l-alanyl-l-glutamine were used as the substrate to feed Bacillus licheniformis in a microbial fuel cell. The metabolism process of the bacterium was monitored by analyzing the voltage outputs of the microbial fuel cell. A home-made spontaneous injection device was applied to perform high-speed capillary micellar electrokinetic chromatography. Under the optimized conditions, tryptophan, glycine, valine, tyrosine and the two peptides could be rapidly separated within 2.5 min with micellar electrokinetic chromatography mode. Then the method was applied to analyze the solutions sampled from the microbial fuel cell. After 92 h running, valine, as the metabolite, was successfully detected with concentration 3.90 × 10 -5 M. The results demonstrated that Bacillus licheniformis could convert l-carnosine and l-alanyl-l-glutamine into valine. The method employed in this work was proved to have great potential in analysis of metabolites, such as amino acids, for microorganisms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements.

PubMed

Qian, Chen; Hettich, Robert L

2017-07-07

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling. Liquid chromatography coupled to high-performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to proteome extraction and subsequent MS measurement. To this end, we have designed an experimental method to improve microbial proteome measurement by removing the soil-borne humic substances coextraction from soils. Our approach employs an in situ detergent-based microbial lysis/TCA precipitation coupled to an additional cleanup step involving acidified precipitation and filtering at the peptide level to remove most of the humic acid interferences prior to proteolytic peptide measurement. The novelty of this approach is an integration to exploit two different characteristics of humic acids: (1) Humic acids are insoluble in acidic solution but should not be removed at the protein level, as undesirable protein removal may also occur. Rather it is better to leave the humics acids in the samples until the peptide level, at which point the significant differential solubility of humic acids versus peptides at low pH can be exploited very efficiently. (2) Most of the humic acids have larger molecule weights than the peptides. Therefore, filtering a pH 2 to 3 peptide solution with a 10 kDa filter will remove most of the humic acids. This method is easily interfaced with normal proteolytic processing approaches and provides a reliable and straightforward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or biasing protein identification in mass spectrometry. In general, this humic acid removal step is universal and can be adopted by any workflow to effectively remove humic acids to avoid them negatively competing with peptides for binding with reversed-phase resin or ionization in the electrospray.

Plant peptides in defense and signaling.

PubMed

Marmiroli, Nelson; Maestri, Elena

2014-06-01

This review focuses on plant peptides involved in defense against pathogen infection and those involved in the regulation of growth and development. Defense peptides, defensins, cyclotides and anti-microbial peptides are compared and contrasted. Signaling peptides are classified according to their major sites of activity. Finally, a network approach to creating an interactomic peptide map is described. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Pathogenesis of Rickettsioses and Development of Novel Anti-Rickettsia Treatment by Comginatorial Peptide-Based Libraries

DTIC Science & Technology

2005-02-01

in many parts of the world and include Rocky Mountain spotted fever (the most common rickettsiosis in the US), epidemic and endemic typhus (1-4). The... Mountain spotted fever and other rickettsioses. In: Schaecter M, Engleberg NC, Eisenstein BI, MedoffG, editors. Mechanisms of microbial disease. Baltimore...Brouqui P, editors. Rickettsiae and rickettsial diseases at the turn of the millenium. Paris: Elsevier; 1999. p. 274-277. 3. Walker DH. Rocky

A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone

PubMed Central

Pruitt, Rory N.; Joe, Anna; Zhang, Weiguo; Feng, Wei; Stewart, Valley; Schwessinger, Benjamin; Dinneny, José R.; Ronald, Pamela C.

2018-01-01

Summary The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides.Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides.Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence.These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide. PMID:28556915

Recent Research in Antihypertensive Activity of Food Protein-derived Hydrolyzates and Peptides.

PubMed

Saleh, Ahmed S M; Zhang, Qing; Shen, Qun

2016-01-01

Year to year obesity prevalence, reduced physical activities, bad habits/or stressful lifestyle, and other environmental and physiological impacts lead to increase in diseases such as coronary heart disease, stroke, cancer, diabetes, and hypertension worldwide. Hypertension is considered as one of the most common serious chronic diseases; however, discovery of medications with high efficacy and without side effects for treatment of patients remains a challenge for scientists. Recent trends in functional foods have evidenced that food bioactive proteins play a major role in the concepts of illness and curing; therefore, nutritionists, biomedical scientists, and food scientists are working together to develop improved systems for the discovery of peptides with increased potency and therapeutic benefits. This review presents a recent research carried out to date for the purpose of isolation and identification of bioactive hydrolyzates and peptides with angiotensin I converting enzyme inhibitory activity and antihypertensive effect from animal, marine, microbial, and plant food proteins. Effects of food processing and hydrolyzation conditions as well as some other impacts on formation, activity, and stability of these hydrolyzates and peptides are also presented.

A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone.

PubMed

Pruitt, Rory N; Joe, Anna; Zhang, Weiguo; Feng, Wei; Stewart, Valley; Schwessinger, Benjamin; Dinneny, José R; Ronald, Pamela C

2017-07-01

The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides. Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides. Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide. © 2017 UT-Battelle LLC. New Phytologist © 2017 New Phytologist Trust.

Mapping membrane activity in undiscovered peptide sequence space using machine learning

PubMed Central

Fulan, Benjamin M.; Wong, Gerard C. L.

2016-01-01

There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its “antimicrobialness”) and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide’s minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences. PMID:27849600

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration.

PubMed

Werner, Jeffrey J; Ptak, A Celeste; Rahm, Brian G; Zhang, Sheng; Richardson, Ruth E

2009-10-01

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Systemic Lupus Erythematosus: Molecular Mimicry between Anti-dsDNA CDR3 Idiotype, Microbial and Self Peptides-As Antigens for Th Cells.

PubMed

Aas-Hanssen, Kristin; Thompson, Keith M; Bogen, Bjarne; Munthe, Ludvig A

2015-01-01

Systemic lupus erythematosus (SLE) is marked by a T helper (Th) cell-dependent B cell hyperresponsiveness, with frequent germinal center reactions, and gammaglobulinemia. A feature of SLE is the finding of IgG autoantibodies specific for dsDNA. The specificity of the Th cells that drive the expansion of anti-dsDNA B cells is unresolved. However, anti-microbial, anti-histone, and anti-idiotype Th cell responses have been hypothesized to play a role. It has been entirely unclear if these seemingly disparate Th cell responses and hypotheses could be related or unified. Here, we describe that H chain CDR3 idiotypes from IgG(+) B cells of lupus mice have sequence similarities with both microbial and self peptides. Matched sequences were more frequent within the mutated CDR3 repertoire and when sequences were derived from lupus mice with expanded anti-dsDNA B cells. Analyses of histone sequences showed that particular histone peptides were similar to VDJ junctions. Moreover, lupus mice had Th cell responses toward histone peptides similar to anti-dsDNA CDR3 sequences. The results suggest that Th cells in lupus may have multiple cross-reactive specificities linked to the IgVH CDR3 Id-peptide sequences as well as similar DNA-associated protein motifs.

Natural History of Innate Host Defense Peptides.

PubMed

Linde, A; Wachter, B; Höner, O P; Dib, L; Ross, C; Tamayo, A R; Blecha, F; Melgarejo, T

2009-12-01

Host defense peptides act on the forefront of innate immunity, thus playing a central role in the survival of animals and plants. Despite vast morphological changes in species through evolutionary history, all animals examined to date share common features in their innate immune defense strategies, hereunder expression of host defense peptides (HDPs). Most studies on HDPs have focused on humans, domestic and laboratory animals. More than a thousand different sequences have been identified, yet data on HDPs in wild-living animals are sparse. The biological functions of HDPs include broad-spectrum antimicrobial activity and immunomodulation. Natural selection and coevolutionary host-pathogen arms race theory suggest that the extent and specificity of the microbial load influences the spectrum and potency of HDPs in different species. Individuals of extant species-that have lived for an extended period in evolutionary history amid populations with intact processes of natural selection-likely possess the most powerful and well-adapted "natural antibiotics". Research on the evolutionary history of the innate defense system and the host in context of the consequences of challenges as well as the efficacy of the innate immune system under natural conditions is therefore of immediate interest. This review focuses on evolutionary aspects of immunophysiology, with emphasis on innate effector molecules. Studies on host defense in wild-living animals may significantly enhance our understanding of inborn immune mechanisms, and help identify molecules that may assist us to cope better with the increasing microbial challenges that likely follow from the continuous amplification of biodiversity levels on Earth.

Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.

PubMed

Pöppel, Anne-Kathrin; Vogel, Heiko; Wiesner, Jochen; Vilcinskas, Andreas

2015-05-01

The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin.

PubMed

Xiang, Ning; Lyu, Yuan; Zhu, Xiao; Bhunia, Arun K; Narsimhan, Ganesan

2016-11-01

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization and Function of the First Antibiotic Isolated from a Vent Organism: The Extremophile Metazoan Alvinella pompejana

PubMed Central

Tasiemski, Aurélie; Jung, Sascha; Boidin-Wichlacz, Céline; Jollivet, Didier; Cuvillier-Hot, Virginie; Pradillon, Florence; Vetriani, Costantino; Hecht, Oliver; Sönnichsen, Frank D.; Gelhaus, Christoph; Hung, Chien-Wen; Tholey, Andreas; Leippe, Matthias; Grötzinger, Joachim; Gaill, Françoise

2014-01-01

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side. PMID:24776651

Health benefits of fermented foods.

PubMed

Şanlier, Nevin; Gökcen, Büşra Başar; Sezgin, Aybüke Ceyhun

2017-09-25

In the past, the beneficial effects of fermented foods on health were unknown, and so people primarily used fermentation to preserve foods, enhance shelf life, and improve flavour. Fermented foods became an important part of the diet in many cultures, and over time fermentation has been associated with many health benefits. Because of this, the fermentation process and the resulting fermented products have recently attracted scientific interest. In addition, microorganisms contributing to the fermentation process have recently been associated with many health benefits, and so these microorganisms have become another focus of attention. Lactic acid bacteria (LAB) have been some of the most studied microorganisms. During fermentation, these bacteria synthesize vitamins and minerals, produce biologically active peptides with enzymes such as proteinase and peptidase, and remove some non-nutrients. Compounds known as biologically active peptides, which are produced by the bacteria responsible for fermentation, are also well known for their health benefits. Among these peptides, conjugated linoleic acids (CLA) have a blood pressure lowering effect, exopolysaccharides exhibit prebiotic properties, bacteriocins show anti-microbial effects, sphingolipids have anti-carcinogenic and anti-microbial properties, and bioactive peptides exhibit anti-oxidant, anti-microbial, opioid antagonist, anti-allergenic, and blood pressure lowering effects. As a result, fermented foods provide many health benefits such as anti-oxidant, anti-microbial, anti-fungal, anti-inflammatory, anti-diabetic and anti-atherosclerotic activity. However, some studies have shown no relationship between fermented foods and health benefits. Therefore, this paper aims to investigate the health effects of fermented foods.

Identification and screening of potent antimicrobial peptides in arthropod genomes.

PubMed

Duwadi, Deepesh; Shrestha, Anishma; Yilma, Binyam; Kozlovski, Itamar; Sa-Eed, Munaya; Dahal, Nikesh; Jukosky, James

2018-05-01

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2-16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13-25 μg/ml against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5 μg/ml against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection. Copyright © 2018 Elsevier Inc. All rights reserved.

A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs.

PubMed

Gordon, Y Jerold; Romanowski, Eric G; McDermott, Alison M

2005-07-01

Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Literature review. Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

PubMed

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of the antimicrobial peptide protegrine 1 on sperm viability and bacterial load of boar seminal doses.

PubMed

Sancho, S; Briz, M; Yeste, M; Bonet, S; Bussalleu, E

2017-10-01

The presence of bacteria adversely affects boar sperm quality of seminal doses intended for artificial insemination. Currently, the most common measure to prevent bacteriospermia is the addition of antibiotics in semen extenders; however, mounting evidence shows that microbial resistance exists. A promising alternative to replace antibiotics are antimicrobial peptides. In this study, the effects of the antimicrobial peptide protegrine 1 (PG1) on the sperm viability and bacterial load of boar seminal doses were evaluated. Three different concentrations of PG1 (2.5, 25 and 100 μg/ml) were tested over a storing period of 10 days at 17°C. Sperm viability was analysed by fluorescence microscopy (SYBR14/propidium iodide), and bacterial load was assessed by plating 100 μl of each sample in Luria-Bertani medium and incubated at 37°C for 72 hr under aerobic conditions. Protegrine 1 was effective in controlling the bacterial load in all the assessed concentrations (p < .05), reaching the lowest values at the highest concentrations of the antimicrobial peptide. Nevertheless, sperm viability was significantly (p < .05) reduced by all tested concentrations of this peptide, the most cytotoxic effects being observed at the highest PG1 concentrations. Despite these results, the use of PG1 as an alternative to antibiotics cannot be totally discarded, as further studies using the truncated form of this peptide are needed. © 2017 Blackwell Verlag GmbH.

Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and Secreted Aspartyl Proteinase (SAPS) 2, 4, 5 and 6 Expressions

DTIC Science & Technology

2013-04-01

1 AWARD NUMBER: W81XWH-12-2-0025 TITLE: Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition...drugs using various synthetic and naturally occurring antimicrobial molecules. Natural antimicrobial peptides , such as defensins produced by...These antimicrobial peptides generally exhibit selective toxicity for microorganisms and show fewer propensities to induce microbial resistance

Short AntiMicrobial Peptides (SAMPs) as a class of extraordinary promising therapeutic agents.

PubMed

Ramesh, Suhas; Govender, Thavendran; Kruger, Hendrik G; de la Torre, Beatriz G; Albericio, Fernando

2016-07-01

The emergence of multidrug resistant bacteria has a direct impact on global public health because of the reduced potency of existing antibiotics against pathogens. Hence, there is a pressing need for new drugs with different modes of action that can kill microorganisms. Antimicrobial peptides (AMPs) can be regarded as an alternative tool for this purpose because they are proven to have therapeutic effects with broad-spectrum activities. There are some hurdles in using AMPs as clinical candidates such as toxicity, lack of stability and high budgets required for manufacturing. This can be overcome by developing shorter and more easily accessible AMPs, the so-called Short AntiMicrobial Peptides (SAMPs) that contain between two and ten amino acid residues. These are emerging as an attractive class of therapeutic agents with high potential for clinical use and possessing multifunctional activities. In this review we attempted to compile those SAMPs that have exhibited biological properties which are believed to hold promise for the future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Oral pathogens change proliferation properties of oral tumor cells by affecting gene expression of human defensins.

PubMed

Hoppe, T; Kraus, D; Novak, N; Probstmeier, R; Frentzen, M; Wenghoefer, M; Jepsen, S; Winter, J

2016-10-01

The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.

A Review of Antimicrobial Peptides and Their Therapeutic Potential as Anti-Infective Drugs

PubMed Central

Gordon, Y. Jerold; Romanowski, Eric G.; McDermott, Alison M.

2006-01-01

Purpose. Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Methods. Literature review. Results. Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. Conclusions. While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven. PMID:16020284

The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens.

PubMed

Chakraborty, Sandeep; Phu, My; de Morais, Tâmara Prado; Nascimento, Rafael; Goulart, Luiz Ricardo; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya M

2014-01-01

The therapeutic potential of α-helical anti-microbial peptides (AH-AMP) to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizing α-helical peptides (PAGAL), we elucidate a search methodology (SCALPEL) that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We provide in vitro validation of SCALPEL on plant pathogens ( Xylella fastidiosa, Xanthomonas arboricola and Liberibacter crescens) by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP. The identified peptides include a linear AH-AMP present within the existing structure of phosphoenolpyruvate carboxylase (PPC20), and an AH-AMP mimicing the properties of the two α-helices of cecropin B from chitinase (CHITI25). The minimum inhibitory concentration of these peptides are comparable to that of cecropin B, while anionic peptides used as control failed to show any inhibitory effect on these pathogens. Substitute therapies in place of conventional chemotherapies using membrane permeabilizing peptides like these might also prove effective to target cancer cells. The use of native structures from the same organism could possibly ensure that administration of such peptides will be better tolerated and not elicit an adverse immune response. We suggest a similar approach to target Ebola epitopes, enumerated using PAGAL recently, by selecting suitable peptides from the human proteome, especially in wake of recent reports of cationic amphiphiles inhibiting virus entry and infection.

A pocket guide to explorations of the defensin field.

PubMed

Selsted, Michael E

2007-01-01

Antimicrobial peptides are among the most ancient effectors of host defense. Intersecting lines of research demonstrate that life forms as diverse as plants, insects, and vertebrates employ antimicrobial peptides to kill or neutralize a wide variety of microbial species. Defensins, of which there are three structural sub-families, constitute a major category of host defense peptides in vertebrates. Presented here is a brief history of the emergence of the defensin field with an emphasis on the role of these peptides in mammalian innate immunity.

Metaproteogenomics reveals the soil microbial communities active in nutrient cycling processes under different tree species

NASA Astrophysics Data System (ADS)

Keiblinger, Katharina Maria; Masse, Jacynthe; Zühlke, Daniela; Riedel, Katharina; Zechmeister-Boltenstern, Sophie; Prescott, Cindy E.; Grayston, Sue

2016-04-01

Tree species exert strong effects on microbial communities in litter and soil and may alter rates of soil processes fundamental to nutrient cycling and carbon fluxes (Prescott and Grayston 2013). However, the influence of tree species on decomposition processes are still contradictory and poorly understood. An understanding of the mechanisms underlying plant influences on soil processes is important for our ability to predict ecosystem response to altered global/environmental conditions. In order to link microbial community structure and function to forest-floor nutrient cycling processes, we sampled forest floors under western redcedar (Thuja plicata), Douglas-fir (Pseudotsuga menziesii) and Sitka spruce (Picea sitchensis) grown in nutrient-poor sites in common garden experiments on Vancouver island (Canada). We measured forest-floor total N, total C, initial NH4+ and NO3- concentrations, DOC, Cmic and Nmic. Gross rates of ammonification and NH4+ consumption were measured using the 15N pool-dilution method. Organic carbon quality was assessed through FTIR analyses. Microbial community structure was analysed by a metaproteogenomic approach using 16S and ITS amplification and sequencing with MiSeq platform. Proteins were extracted and peptides characterized via LC-MS/MS on a Velos Orbitrap to assess the active microbial community. Different microbial communities were active under the three tree species and variation in process rates were observed and will be discussed. This research provides new insights on microbial processes during organic matter decomposition. The metaproteogenomic approach enables us to investigate these changes with respect to possible effects on soil C-storage at even finer taxonomic resolution.

A Review of the Latest Advances in Encrypted Bioactive Peptides from Protein-Rich Waste

PubMed Central

Lemes, Ailton Cesar; Sala, Luisa; Ores, Joana da Costa; Braga, Anna Rafaela Cavalcante; Egea, Mariana Buranelo; Fernandes, Kátia Flávia

2016-01-01

Bioactive peptides are considered the new generation of biologically active regulators that not only prevent the mechanism of oxidation and microbial degradation in foods but also enhanced the treatment of various diseases and disorders, thus increasing quality of life. This review article emphasizes recent advances in bioactive peptide technology, such as: (i) new strategies for transforming bioactive peptides from residual waste into added-value products; (ii) nanotechnology for the encapsulation, protection and release of controlled peptides; and (iii) use of techniques of large-scale recovery and purification of peptides aiming at future applications to pharmaceutical and food industries. PMID:27322241

Mass Spectrometry Imaging of Complex Microbial Communities

PubMed Central

2016-01-01

Conspectus In the two decades since mass spectrometry imaging (MSI) was first applied to visualize the distribution of peptides across biological tissues and cells, the technique has become increasingly effective and reliable. MSI excels at providing complementary information to existing methods for molecular analysis—such as genomics, transcriptomics, and metabolomics—and stands apart from other chemical imaging modalities through its capability to generate information that is simultaneously multiplexed and chemically specific. Today a diverse family of MSI approaches are applied throughout the scientific community to study the distribution of proteins, peptides, and small-molecule metabolites across many biological models. The inherent strengths of MSI make the technique valuable for studying microbial systems. Many microbes reside in surface-attached multicellular and multispecies communities, such as biofilms and motile colonies, where they work together to harness surrounding nutrients, fend off hostile organisms, and shield one another from adverse environmental conditions. These processes, as well as many others essential for microbial survival, are mediated through the production and utilization of a diverse assortment of chemicals. Although bacterial cells are generally only a few microns in diameter, the ecologies they influence can encompass entire ecosystems, and the chemical changes that they bring about can occur over time scales ranging from milliseconds to decades. Because of their incredible complexity, our understanding of and influence over microbial systems requires detailed scientific evaluations that yield both chemical and spatial information. MSI is well-positioned to fulfill these requirements. With small adaptations to existing methods, the technique can be applied to study a wide variety of chemical interactions, including those that occur inside single-species microbial communities, between cohabitating microbes, and between microbes and their hosts. In recognition of this potential for scientific advancement, researchers have adapted MSI methodologies for the specific needs of the microbiology research community. As a result, workflows exist for imaging microbial systems with many of the common MSI ionization methods. Despite this progress, there is substantial room for improvements in instrumentation, sample preparation, and data interpretation. This Account provides a brief overview of the state of technology in microbial MSI, illuminates selected applications that demonstrate the potential of the technique, and highlights a series of development challenges that are needed to move the field forward. In the coming years, as microbial MSI becomes easier to use and more universally applicable, the technique will evolve into a fundamental tool widely applied throughout many divisions of science, medicine, and industry. PMID:28001363

Mass Spectrometry Imaging of Complex Microbial Communities.

PubMed

Dunham, Sage J B; Ellis, Joseph F; Li, Bin; Sweedler, Jonathan V

2017-01-17

In the two decades since mass spectrometry imaging (MSI) was first applied to visualize the distribution of peptides across biological tissues and cells, the technique has become increasingly effective and reliable. MSI excels at providing complementary information to existing methods for molecular analysis-such as genomics, transcriptomics, and metabolomics-and stands apart from other chemical imaging modalities through its capability to generate information that is simultaneously multiplexed and chemically specific. Today a diverse family of MSI approaches are applied throughout the scientific community to study the distribution of proteins, peptides, and small-molecule metabolites across many biological models. The inherent strengths of MSI make the technique valuable for studying microbial systems. Many microbes reside in surface-attached multicellular and multispecies communities, such as biofilms and motile colonies, where they work together to harness surrounding nutrients, fend off hostile organisms, and shield one another from adverse environmental conditions. These processes, as well as many others essential for microbial survival, are mediated through the production and utilization of a diverse assortment of chemicals. Although bacterial cells are generally only a few microns in diameter, the ecologies they influence can encompass entire ecosystems, and the chemical changes that they bring about can occur over time scales ranging from milliseconds to decades. Because of their incredible complexity, our understanding of and influence over microbial systems requires detailed scientific evaluations that yield both chemical and spatial information. MSI is well-positioned to fulfill these requirements. With small adaptations to existing methods, the technique can be applied to study a wide variety of chemical interactions, including those that occur inside single-species microbial communities, between cohabitating microbes, and between microbes and their hosts. In recognition of this potential for scientific advancement, researchers have adapted MSI methodologies for the specific needs of the microbiology research community. As a result, workflows exist for imaging microbial systems with many of the common MSI ionization methods. Despite this progress, there is substantial room for improvements in instrumentation, sample preparation, and data interpretation. This Account provides a brief overview of the state of technology in microbial MSI, illuminates selected applications that demonstrate the potential of the technique, and highlights a series of development challenges that are needed to move the field forward. In the coming years, as microbial MSI becomes easier to use and more universally applicable, the technique will evolve into a fundamental tool widely applied throughout many divisions of science, medicine, and industry.

ClusterMine360: a database of microbial PKS/NRPS biosynthesis

PubMed Central

Conway, Kyle R.; Boddy, Christopher N.

2013-01-01

ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and non-ribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has >200 gene clusters from >185 compound families. It also features a unique sequence repository containing >10 000 polyketide synthase/non-ribosomal peptide synthetase domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. We are confident that this database will be a useful resource for members of the polyketide synthases/non-ribosomal peptide synthetases research community, enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information. PMID:23104377

Labeled Antimicrobial Peptides for Detection of Microorganisms

DTIC Science & Technology

2008-12-01

1. INTRODUCTION Antimicrobial peptides (AMPs) are part of the innate defense system found in all organisms to protect them from microbial infection...2005) with antimicrobial activity against predominantly gram-negative bacteria. SMAP29 is from the cathelicidin family of peptides found in sheep ...in buffer, milk and apple juice. Cells were grown and prepared in PBST as described above. 20 III anti-£. coli 0157 paramagnetic Dyna-beads (Dynal

Transport Functions Dominate the SAR11 Metaproteome at Low-Nutrient Extremes in the Sargasso Sea

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

Sowell, Sarah M.; Wilhelm, Larry; Norbeck, Angela D.

2009-01-01

The northwestern Sargasso Sea is part of the North Atlantic subtropical oceanic gyre that is characterized as seasonally oligotrophic with pronounced stratification in the summer and autumn. Essentially a marine desert, the biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorous and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary LC-tandem mass spectrometry to study the composition of microbial proteomes in surface samples collected in September 2005. A total of 2279 peptides that mapped to 236 SAR11 proteins, andmore » 3208 peptides that mapped to 404 Synechococcus proteins, were detected. Mass spectra from SAR11 periplasmic binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars, and spermidine. Although the data showed that a large fraction of microbial protein synthesis in the Sargasso Sea is devoted to inorganic and organic nutrient acquisition, the proteomes of both SAR11 and Synechococcus also indicated that these populations were actively growing. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme but sufficient to sustain microbial community activity.« less

Metagenomic Taxonomy-Guided Database-Searching Strategy for Improving Metaproteomic Analysis.

PubMed

Xiao, Jinqiu; Tanca, Alessandro; Jia, Ben; Yang, Runqing; Wang, Bo; Zhang, Yu; Li, Jing

2018-04-06

Metaproteomics provides a direct measure of the functional information by investigating all proteins expressed by a microbiota. However, due to the complexity and heterogeneity of microbial communities, it is very hard to construct a sequence database suitable for a metaproteomic study. Using a public database, researchers might not be able to identify proteins from poorly characterized microbial species, while a sequencing-based metagenomic database may not provide adequate coverage for all potentially expressed protein sequences. To address this challenge, we propose a metagenomic taxonomy-guided database-search strategy (MT), in which a merged database is employed, consisting of both taxonomy-guided reference protein sequences from public databases and proteins from metagenome assembly. By applying our MT strategy to a mock microbial mixture, about two times as many peptides were detected as with the metagenomic database only. According to the evaluation of the reliability of taxonomic attribution, the rate of misassignments was comparable to that obtained using an a priori matched database. We also evaluated the MT strategy with a human gut microbial sample, and we found 1.7 times as many peptides as using a standard metagenomic database. In conclusion, our MT strategy allows the construction of databases able to provide high sensitivity and precision in peptide identification in metaproteomic studies, enabling the detection of proteins from poorly characterized species within the microbiota.

Transmembrane Pores Formed by Human Antimicrobial Peptide LL-37

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

Qian, Shuo

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the {alpha}-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observedmore » an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 2333 {angstrom}. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.« less

Engineered chimeric peptides with antimicrobial and titanium-binding functions to inhibit biofilm formation on Ti implants.

PubMed

Geng, Hongjuan; Yuan, Yang; Adayi, Aidina; Zhang, Xu; Song, Xin; Gong, Lei; Zhang, Xi; Gao, Ping

2018-01-01

Titanium (Ti) implants have been commonly used in oral medicine. However, despite their widespread clinical application, these implants are susceptible to failure induced by microbial infection due to bacterial biofilm formation. Immobilization of chimeric peptides with antibacterial properties on the Ti surface may be a promising antimicrobial approach to inhibit biofilm formation. Here, chimeric peptides were designed by connecting three sequences (hBD-3-1/2/3) derived from human β-defensin-3 (hBD-3) with Ti-binding peptide-l (TBP-l: RKLPDAGPMHTW) via a triple glycine (G) linker to modify Ti surfaces. Using X-ray photoelectron spectroscopy (XPS), the properties of individual domains of the chimeric peptides were evaluated for their binding activity toward the Ti surface. The antimicrobial and anti-biofilm efficacy of the peptides against initial settlers, Streptococcus oralis (S. oralis), Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis), was evaluated with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Transmission electron microscopy (TEM) and real-time quantitative PCR (qRT-PCR) were used to study cell membrane changes and the underlying antimicrobial mechanism. Compared with the other two peptides, TBP-1-GGG-hBD3-3 presented stronger antibacterial activity and remained stable in saliva and serum. Therefore, it was chosen as the best candidate to modify Ti surfaces in this study. This peptide inhibited the growth of initial streptococci and biofilm formation on Ti surfaces with no cytotoxicity to MC3T3-E1 cells. Disruption of the integrity of bacterial membranes and decreased expression of adhesion protein genes from S. gordonii revealed aspects of the antibacterial mechanism of TBP-1-GGG-hBD3-3. We conclude that engineered chimeric peptides with antimicrobial activity provide a potential solution for inhibiting biofilm formation on Ti surfaces to reduce or prevent the occurrence of peri-implant diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics.

PubMed

Nikolouli, Katerina; Mossialos, Dimitris

2012-08-01

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.

Molecular mimicry modulates plant host responses to pathogens.

PubMed

Ronald, Pamela; Joe, Anna

2018-01-25

Pathogens often secrete molecules that mimic those present in the plant host. Recent studies indicate that some of these molecules mimic plant hormones required for development and immunity. This Viewpoint reviews the literature on microbial molecules produced by plant pathogens that functionally mimic molecules present in the plant host. This article includes examples from nematodes, bacteria and fungi with emphasis on RaxX, a microbial protein produced by the bacterial pathogen Xanthomonas oryzae pv. oryzae. RaxX mimics a plant peptide hormone, PSY (plant peptide containing sulphated tyrosine). The rice immune receptor XA21 detects sulphated RaxX but not the endogenous peptide PSY. Studies of the RaxX/XA21 system have provided insight into both host and pathogen biology and offered a framework for future work directed at understanding how XA21 and the PSY receptor(s) can be differentially activated by RaxX and endogenous PSY peptides. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Directed evolution of FLS2 towards novel flagellin peptide recognition

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

Helft, Laura; Thompson, Mikayla; Bent, Andrew F.

Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wildtype Arabidopsis FLS2 confers little or no response. A targeted approachmore » generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. Furthermore, these studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.« less

Directed evolution of FLS2 towards novel flagellin peptide recognition

DOE PAGES

Helft, Laura; Thompson, Mikayla; Bent, Andrew F.

2016-06-06

Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wildtype Arabidopsis FLS2 confers little or no response. A targeted approachmore » generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. Furthermore, these studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.« less

Targeted Modification of a Novel Amphibian Antimicrobial Peptide from Phyllomedusa tarsius to Enhance Its Activity against MRSA and Microbial Biofilm

PubMed Central

Gao, Yitian; Wu, Di; Wang, Lei; Lin, Chen; Ma, Chengbang; Xi, Xinping; Zhou, Mei; Duan, Jinao; Bininda-Emonds, Olaf R. P.; Chen, Tianbao; Shaw, Chris

2017-01-01

Antimicrobial peptides (AMPs) in the skin secretions of amphibians are fundamental components of a unique defense system that has evolved to protect these hosts from microbial invasion. Medusins constitute a recently-discovered AMP family from phyllomedusine leaf frog skin and exhibit highly-conserved structural characteristics. Here, we report a novel medusin, medusin-PT, from the skin secretion of the Tarsier Leaf Frog, Phyllomedusa tarsius. The mature peptide was initially identified from its cloned biosynthetic precursor-encoding cDNA as obtained by the rapid amplification of cDNA ends (RACE) method. Reverse-phase HPLC and tandem mass spectrometry confirmed both the presence of medusin-PT in the skin secretion and its primary structure. In a range of bioassays, medusin-PT exhibited antimicrobial activity against only the Gram-positive bacterium Staphylococcus aureus at 64 μg/ml. However, after directed changes to enhance the cationicity and amphipathicity of the peptide structure, three analog showed more potent antimicrobial activity against several additional bacteria including the antibiotic-resistant bacterium MRSA. In addition, these analog exhibited activity against microbial biofilm (minimum biofilm inhibitory and eradication concentrations of 32 μg/ml and over 64 μg/ml, respectively). These data provide evidence that medusins might be promising candidates as novel antibiotic leads and that the targeted modification of a natural AMP can both improve its efficacy so as to provide new insights into antibiotic design and development. PMID:28469603

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

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

Qian, Chen; Hettich, Robert L.

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

DOE PAGES

Qian, Chen; Hettich, Robert L.

2017-05-24

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Accumulation of deaminated peptides in anoxic sediments of Santa Barbara Basin

NASA Astrophysics Data System (ADS)

Abdulla, Hussain A.; Burdige, David J.; Komada, Tomoko

2018-02-01

Proteins represent the most abundant class of biomolecules in marine sinking particles and microbial biomass, yet their cycling in marine sediments is not fully understood. To investigate whether some portion of hydrolyzed proteins escapes complete remineralization and accumulate in the pore waters, we analyzed dissolved organic matter from the anoxic sediments of Santa Barbara Basin, California, by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS). The results showed an increase in the molecular diversity and abundance of dissolved organic nitrogen (DON) formulas with depth. A comparison of the detected DON formulas to a database of small peptides (2-4 amino acid sequences) returned 119 matches, and these formulas were most abundant near the sediment surface. When we compared our detected formulas to all possible structures that would result from deamination of peptides in the database, we found 680 formula matches. However, these molecular formulas can represent hundreds of different structural isomers (in the present case as many as 3257 different deaminated peptide structures), which cannot be distinguished by the FTICR-MS settings that were used. Analysis of amino acid sequences suggests that these deaminated peptides may be the products of selective degradation of source proteins in marine sediments. We hypothesize that these deaminated peptides accumulate in the pore waters due to extracellular proteinases being inhibited from completely hydrolyzing specific peptides to free amino acids. We suggest that anaerobic microbes deaminate peptides largely to produce H2, which is ultimately used as a reducing agent by other sediment microbes (e.g. CO2 reduction by methanogens). Simple calculations suggest that deaminated peptides may represent ∼25-45% of DOC accumulating in these sediment pore waters. Unlike rapid remineralization of free amino acids, peptide deamination leaves behind the peptide carbon skeleton. Molecular structures of these remnant carbon skeletons may hold important clues about specific microbial processes influencing organic matter remineralization and accumulation.

Anti-Mycobacterium activity of microbial peptides in a silkworm infection model with Mycobacterium smegmatis.

PubMed

Yagi, Akiho; Uchida, Ryuji; Hamamoto, Hiroshi; Sekimizu, Kazuhisa; Kimura, Ken-Ichi; Tomoda, Hiroshi

2017-05-01

An in vivo-mimic silkworm infection model with Mycobacterium smegmatis was established. When silkworms were raised at 37 °C following an injection of M. smegmatis cells (1.25 × 10 7 CFU larva -1  g -1 ) into the silkworm hemolymph, they died within 48 h. Under these conditions, four microbial peptides with anti-M. smegmatis activity, lariatin A, calpinactam, lysocin E and propeptin, exerted therapeutic effects in a dose-dependent manner, and these are also clinically used agents that are active against Mycobacterium tuberculosis. These results indicate that the silkworm infection model with M. smegmatis is practically useful for the screening of therapeutically effective anti-M. tuberculosis antibiotics.

Cloning of cDNAs encoding new peptides of the dermaseptin-family.

PubMed

Wechselberger, C

1998-10-14

Dermaseptins are a group of basic (lysine-rich) peptides, 27-34 amino acids in length and involved in the defense of frog skin against microbial invasion. By using a degenerated oligonucleotide primer binding to the 5'-untranslated region of previously characterized cDNAs of these peptides, it was possible to identify new members of the dermaseptin family in the South American frogs Agalychnis annae and Pachymedusa dacnicolor. Amino acid alignment and secondary structure prediction reveals, that only five of the deduced peptides can be supposed to be also functional homologs to the known dermaseptins from Phyllomedusa bicolor and Phyllomedusa sauvagei. The remaining six peptides described in this paper have not been isolated and characterized yet.

Role of Host-Defence Peptides in Eye Diseases

PubMed Central

Kolar, Satya S.; McDermott, Alison M.

2013-01-01

The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarized. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed. PMID:21584809

Lab-on-fiber optofluidic platform for in-situ study of therapeutic peptides and bacterial response (Rising Researcher Presentation) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tian, Fei; Yang, Fan; Liang, Junfeng

2017-05-01

Hospital acquired infections in indwelling device have become a life-threatening issue accompanied by the wide use of medical devices and implants. The infection process typically involves the attachment, growth and eventual assemblage of microbial cells into biofilms, with the latter exhibiting extremely higher antibiotic tolerance than planktonic bacteria. Surface constructed antimicrobial coatings offer a viable solution for bacteria responsive antibiotic strategy in medical devices such as catheter and stents. Therapeutic peptide has pioneered the field for their attractive pharmacological profile with broad antibacterial spectrum, great efficacy and long life-span. It has been a common practice to separately assess bacteria responses through commercially available activity assay kits after their exposure to antibiotic coatings, limiting the assessment of their activity in vitro with a discontinuous fashion. We developed and demonstrated an innovative all-optical lab-on-fiber optofluidic platform (LOFOP) to fill in this technical gap by allowing in situ measurement of the bacteria attachment in a continuous manner. This LOFOP allows for evaluation of drug release and resultant bacterial response by integrating glass capillary with lytic peptide-containing LbL-coated long period graing (LPG) as its core. S. aureus suspension is introduced through the assembled optofluidic platform with the capillary and the peptide-coated LPG. The efficacy of the peptide-containing coating is evaluated in situ by monitoring the attachment of bacteria and the ensuing development of biofilms using the LPG. LPG without antimicrobial coatings will be explored and compared as control.

Rumen degradable protein supply affects microbial efficiency in continuous culture and growth in steers.

PubMed

Brooks, M A; Harvey, R M; Johnson, N F; Kerley, M S

2012-12-01

We hypothesized that microbial efficiency and output from fermentation in the rumen would be optimized when peptide supply was balanced with peptide requirement of ruminal microflora. This study was conducted to measure response of varying rumen degradable peptide (RDPep) supply on ruminal fermentation characteristics and steer growth. A continuous culture experiment was conducted with diets formulated to achieve a predicted RDPep balance (RDPep supplied above RDPep required) of -0.30 to 1.45% CP with rumen degradable N (RDN) balance (RDN supplied above RDN required) above dietary ammonia-N requirement of microbes. Two additional treatments had RDPep balances of -0.30 and 0.78% CP with insufficient ammonia-N supply to meet microbial requirements. Single-flow fermenters (N = 24; n = 6) were inoculated with rumen fluid and maintained anaerobically at 39°C with a 0.06 h(-1) dilution rate. Inadequate RDN decreased OM digestion and microbial N flow, and increased rumen undegradable N (P < 0.01). Microbial efficiency decreased in RDN-deficient diets and was greatest when RDPep balance did not excessively exceed microbial requirement of RDPep predicted (P < 0.01). A growth study was conducted with 49 yearling, crossbred, Angus steers (initial BW 370 ± 34 kg). Animals were assigned to 1 of 4 treatment groups by BW and further divided into 3 pens with 4 steers per pen to achieve similar initial pen weights. Treatments consisted of 4 isonitrogenous diets balanced for RDN but varying in predicted RDPep balance (0.55%, -0.02%, -0.25%, and -0.65% CP). Animals were maintained on treatment for 70 d with individual BW taken on d 0, 1, 21, 42, 70, and 71. Final BW decreased linearly with decreasing RDPep (P = 0.05). Average daily gain and G:F displayed a quadratic effect with greater ADG and G:F at greater and lesser RDPep levels (P = 0.02). We concluded that balancing RDPep supply to predicted requirement improved fermentation efficiency and microbial output, which in turn improved animal performance.

Human host defense peptides - role in maintaining human homeostasis and pathological processes.

PubMed

Dawgul, Malgorzata Anna; Greber, Katarzyna Ewa; Sawicki, Wieslaw; Kamysz, Wojciech

2016-12-12

The human body expresses over 100 host defense peptides and proteins (antimicrobial peptides, AMPs). The compounds are produced by tissues and mucosal surfaces, e.g. skin, the digestive and urinary tract, the ocular surface and neutrophils, and are believed to play a crucial role in defense from microbial infection. They are considered to protect the human body against microbial infections due to their antimicrobial and immunomodulatory activities. As well as having strong antimicrobial activity towards a broad spectrum of microorganisms, AMPs have been found to interact with neutrophils, monocytes and T-cells and promote the production of cytokines. They also neutralize the action of lipopolysaccharide (LPS) and play a crucial role in wound healing processes. In response to the microbial stimuli the AMPs are released in order to fight the infection, however there are several microorganisms evading the human immune system by downregulation of AMPs. Decreased or elevated expression of AMPs is associated also with several non-infectious diseases. Despite numerous studies conducted in the field of AMPs over the last few decades, their exact role in physiological and pathological processes remains to be explained. In this paper, we review the most significant human AMPs and their potential roles in maintaining human homeostasis as well as in pathological processes.

Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

PubMed

da Silva, Ronivaldo Rodrigues

2017-09-01

Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

Functional significance of bioactive peptides derived from soybean.

PubMed

Singh, Brij Pal; Vij, Shilpa; Hati, Subrota

2014-04-01

Biologically active peptides play an important role in metabolic regulation and modulation. Several studies have shown that during gastrointestinal digestion, food processing and microbial proteolysis of various animals and plant proteins, small peptides can be released which possess biofunctional properties. These peptides are to prove potential health-enhancing nutraceutical for food and pharmaceutical applications. The beneficial health effects of bioactive peptides may be several like antihypertensive, antioxidative, antiobesity, immunomodulatory, antidiabetic, hypocholesterolemic and anticancer. Soybeans, one of the most abundant plant sources of dietary protein, contain 36-56% of protein. Recent studies showed that soy milk, an aqueous extract of soybean, and its fermented product have great biological properties and are a good source of bioactive peptides. This review focuses on bioactive peptides derived from soybean; we illustrate their production and biofunctional attributes. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineering microbial factories for synthesis of value-added products

PubMed Central

Du, Jing; Shao, Zengyi; Zhao, Huimin

2011-01-01

Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed. PMID:21526386

T cell receptor cross-reactivity between similar foreign and self peptides influences naïve cell population size and autoimmunity

PubMed Central

Nelson, Ryan W.; Beisang, Daniel; Tubo, Noah J.; Dileepan, Thamotharampillai; Wiesner, Darin L.; Nielsen, Kirsten; Wüthrich, Marcel; Klein, Bruce S.; Kotov, Dmitri I.; Spanier, Justin A.; Fife, Brian T.; Moon, James J.; Jenkins, Marc K.

2014-01-01

SUMMARY T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naïve CD4+ T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant since systemic expression of a self peptide reduced the size of a naïve cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naïve T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations, and may allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection. PMID:25601203

Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity.

PubMed

Shandala, Tetyana; Woodcock, Joanna M; Ng, Yeap; Biggs, Lisa; Skoulakis, Efthimios M C; Brooks, Doug A; Lopez, Angel F

2011-07-01

The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

Ribonuclease 7, an antimicrobial peptide upregulated during infection, contributes to microbial defense of the human urinary tract.

PubMed

Spencer, John David; Schwaderer, Andrew L; Wang, Huanyu; Bartz, Julianne; Kline, Jennifer; Eichler, Tad; DeSouza, Kristin R; Sims-Lucas, Sunder; Baker, Peter; Hains, David S

2013-04-01

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and enzyme-linked immunosorbant assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection and has antibacterial activity against uropathogens at micromolar concentrations.

Ribonuclease 7, an antimicrobial peptide up-regulated during infection, contributes to microbial defense of the human urinary tract

PubMed Central

Spencer, John David; Schwaderer, Andrew L.; Wang, Huanyu; Bartz, Julianne; Kline, Jennifer; Eichler, Tad; DeSouza, Kristin R.; Sims-Lucas, Sunder; Baker, Peter; Hains, David S.

2012-01-01

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and ELISA assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection, and has antibacterial activity against uropathogens at micromolar concentrations. PMID:23302724

Harnessing the power of microbial nanowires.

PubMed

Reguera, Gemma

2018-05-27

The reduction of iron oxide minerals and uranium in model metal reducers in the genus Geobacter is mediated by conductive pili composed primarily of a structurally divergent pilin peptide that is otherwise recognized, processed and assembled in the inner membrane by a conserved Type IVa pilus apparatus. Electronic coupling among the peptides is promoted upon assembly, allowing the discharge of respiratory electrons at rates that greatly exceed the rates of cellular respiration. Harnessing the unique properties of these conductive appendages and their peptide building blocks in metal bioremediation will require understanding of how the pilins assemble to form a protein nanowire with specialized sites for metal immobilization. Also important are insights into how cells assemble the pili to make an electroactive matrix and grow on electrodes as biofilms that harvest electrical currents from the oxidation of waste organic substrates. Genetic engineering shows promise to modulate the properties of the peptide building blocks, protein nanowires and current-harvesting biofilms for various applications. This minireview discusses what is known about the pilus material properties and reactions they catalyse and how this information can be harnessed in nanotechnology, bioremediation and bioenergy applications. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Recent updates of marine antimicrobial peptides.

PubMed

Semreen, Mohammad H; El-Gamal, Mohammed I; Abdin, Shifaa; Alkhazraji, Hajar; Kamal, Leena; Hammad, Saba; El-Awady, Faten; Waleed, Dima; Kourbaj, Layal

2018-03-01

Antimicrobial peptides are group of proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens. This class of compounds contributed to solving the microbial resistance dilemma that limited the use of many potent antimicrobial agents. The marine environment is known to be one of the richest sources for antimicrobial peptides, yet this environment is not fully explored. Hence, the scientific research attention should be directed toward the marine ecosystem as enormous amount of useful discoveries could be brought to the forefront. In the current article, the marine antimicrobial peptides reported from mid 2012 to 2017 have been reviewed.

Are highly morphed peptide frameworks lurking silently in microbial genomes valuable as next generation antibiotic scaffolds?

PubMed

Walsh, Christopher T

2017-07-01

Antibiotics are a therapeutic class that, once deployed, select for resistant bacterial pathogens and so shorten their useful life cycles. As a consequence new versions of antibiotics are constantly needed. Among the antibiotic natural products, morphed peptide scaffolds, converting conformationally mobile, short-lived linear peptides into compact, rigidified small molecule frameworks, act on a wide range of bacterial targets. Advances in bacterial genome mining, biosynthetic gene cluster prediction and expression, and mass spectroscopic structure analysis suggests many more peptides, modified both in side chains and peptide backbones, await discovery. Such molecules may turn up new bacterial targets and be starting points for combinatorial or semisynthetic manipulations to optimize activity and pharmacology parameters.

Polymer Multilayers Loaded with Antifungal β-Peptides Kill Planktonic Candida albicans and Reduce Formation of Fungal Biofilms on the Surfaces of Flexible Catheter Tubes

PubMed Central

Raman, Namrata; Lee, Myung-Ryul

2014-01-01

Candida albicans is the most common fungal pathogen responsible for hospital-acquired infections. Most C albicans infections are associated with the implantation of medical devices that act as points of entry for the pathogen and as substrates for the growth of fungal biofilms that are notoriously difficult to eliminate by systemic administration of conventional antifungal agents. In this study, we report a fill-and-purge approach to the layer-by-layer fabrication of biocompatible, nanoscale ‘polyelectrolyte multilayers’ (PEMs) on the luminal surfaces of flexible catheters, and an investigation of this platform for the localized, intraluminal release of a cationic β-peptide-based antifungal agent. We demonstrate that polyethylene catheter tubes with luminal surfaces coated with multilayers ~700 nm thick fabricated from poly-L-glutamic acid (PGA) and poly-L-lysine (PLL) can be loaded, post-fabrication, by infusion with β-peptide, and that this approach promotes extended intraluminal release of this agent (over ~4 months) when incubated in physiological media. The β-peptide remained potent against intraluminal inoculation of the catheters with C albicans and substantially reduced the formation of C albicans biofilms on the inner surfaces of film-coated catheters. Finally, we report that these β-peptide-loaded coatings exhibit antifungal activity under conditions that simulate intermittent catheter use and microbial challenge for at least three weeks. We conclude that β-peptide-loaded PEMs offer a novel and promising approach to kill C albicans and prevent fungal biofilm formation on surfaces, with the potential to substantially reduce the incidence of device-associated infections in indwelling catheters. β-Peptides comprise a promising new class of antifungal agents that could help address problems associated with the use of conventional antifungal agents. The versatility of the layer-by-layer approach used here thus suggests additional opportunities to exploit these new agents in other biomedical and personal care applications in which fungal infections are endemic. PMID:24862322

Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

PubMed Central

Haynie, S L; Crum, G A; Doele, B A

1995-01-01

A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

PubMed Central

Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2017-01-01

In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

Fatty acid conjugation enhances the activities of antimicrobial peptides.

PubMed

Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

2013-04-01

Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications

PubMed Central

Scorciapino, Mariano A.; Serra, Ilaria; Manzo, Giorgia; Rinaldi, Andrea C.

2017-01-01

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells. PMID:28273806

Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications.

PubMed

Scorciapino, Mariano A; Serra, Ilaria; Manzo, Giorgia; Rinaldi, Andrea C

2017-03-03

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells.

Identification of Rothia Bacteria as Gluten-Degrading Natural Colonizers of the Upper Gastro-Intestinal Tract

PubMed Central

Zamakhchari, Maram; Wei, Guoxian; Dewhirst, Floyd; Lee, Jaeseop; Schuppan, Detlef; Oppenheim, Frank G.; Helmerhorst, Eva J.

2011-01-01

Background Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes. Methodology/Principal Findings Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD620 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10). Conclusion/Significance While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides. PMID:21957450

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract.

PubMed

Zamakhchari, Maram; Wei, Guoxian; Dewhirst, Floyd; Lee, Jaeseop; Schuppan, Detlef; Oppenheim, Frank G; Helmerhorst, Eva J

2011-01-01

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes. Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD(620) 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70-75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3-10). While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.

Effects of cinnamaldehyde and garlic oil on rumen microbial fermentation in a dual flow continuous culture.

PubMed

Busquet, M; Calsamiglia, S; Ferret, A; Cardozo, P W; Kamel, C

2005-07-01

Eight continuous culture fermentors inoculated with ruminal liquor from heifers fed a 50:50 alfalfa hay:concentrate diet (17.6% crude protein, 28.0% neutral detergent fiber) were used in 3 replicated periods to study the effects of cinnamaldehyde (CIN) and garlic oil (GAR) on rumen microbial fermentation. Treatments were no additive (negative control); 1.25 mg/L (MON) and 12.5 mg/L (MON10) of the ionophore antibiotic monensin (positive control); 31.2 mg/L CIN (CIN) and 312 mg/L (CIN10) of CIN; and 31.2 mg/L GAR (GAR) and 312 mg/L (GAR10) of GAR (Allium sativa). The MON10 caused expected changes in microbial fermentation patterns (a decrease in fiber digestion, ammonia N concentration, and proportions of acetate and butyrate; an increase in the proportion of propionate; and a trend to increase small peptide plus AA N concentration). The CIN decreased the proportion of acetate and branch-chained volatile fatty acids (VFA) and increased the proportion of propionate; CIN10 decreased the proportion of acetate and increased the proportion of butyrate compared with the control. The GAR10 increased the proportion of propionate and butyrate and decreased the proportion of acetate and branch-chained VFA compared with the control. The GAR10 also increased the small peptide plus amino acid N concentration, although no effects were observed on large peptides or ammonia N concentrations. The CIN and GAR10 resulted in similar effects as monensin, with the exception of the effects on the molar proportion of butyrate, which suggests that they might have a different mode of action in affecting in vitro microbial fermentation.

Enteric defensins are essential regulators of intestinal microbial ecology.

PubMed

Salzman, Nita H; Hung, Kuiechun; Haribhai, Dipica; Chu, Hiutung; Karlsson-Sjöberg, Jenny; Amir, Elad; Teggatz, Paul; Barman, Melissa; Hayward, Michael; Eastwood, Daniel; Stoel, Maaike; Zhou, Yanjiao; Sodergren, Erica; Weinstock, George M; Bevins, Charles L; Williams, Calvin B; Bos, Nicolaas A

2010-01-01

Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell alpha-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if alpha-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human alpha-defensin gene (DEFA5) and in mice lacking an enzyme required for the processing of mouse alpha-defensins. In these complementary models, we detected significant alpha-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5-expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to alpha-defensins in regulating the makeup of the commensal microbiota.

New milk protein-derived peptides with potential antimicrobial activity: an approach based on bioinformatic studies.

PubMed

Dziuba, Bartłomiej; Dziuba, Marta

2014-08-20

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

PubMed Central

Dziuba, Bartłomiej; Dziuba, Marta

2014-01-01

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins. PMID:25141106

Fighting microbial infections: A lesson from amphibian skin-derived esculentin-1 peptides.

PubMed

Mangoni, Maria Luisa; Luca, Vincenzo; McDermott, Alison M

2015-09-01

Due to the growing emergence of resistance to commercially available antibiotics/antimycotics in virtually all clinical microbial pathogens, the discovery of alternative anti-infective agents, is greatly needed. Gene-encoded antimicrobial peptides (AMPs) hold promise as novel therapeutics. In particular, amphibian skin is one of the richest storehouses of AMPs, especially that of the genus Rana, with esculentins-1 being among the longest (46 amino acids) AMPs found in nature to date. Here, we report on the recently discovered in vitro and in vivo activities and mechanism of action of two derivatives of the N-terminal part of esculentin-1a and -1b peptides, primarily against two relevant opportunistic microorganisms causing a large number of life-threatening infections worldwide; i.e. the Gram-negative bacterium Pseudomonas aeruginosa and the yeast Candida albicans. Because of distinct advantages compared to several mammalian AMPs, the two selected frog skin AMP-derivatives represent attractive candidates for the development of new antimicrobial compounds with expanded properties, for both human and veterinary medicine. Copyright © 2015 Elsevier Inc. All rights reserved.

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase, and bovine and porcine fibrinogen/thrombin in restructured meat.

PubMed

Jira, Wolfgang; Schwägele, Fredi

2017-12-15

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase (TG) from Streptomyces mobaraensis, and bovine and porcine fibrinogen/thrombin in restructured meat was developed using tryptic marker peptides of TG (five markers), and bovine and porcine fibrinogen (six markers each). Meat binding experiments with beef and pork were performed using a technical TG mixture (Activa, Ajinomoto), and bovine and porcine plasmapowder FG (PPFG; Sonac B.V.). The method developed allows the simultaneous detection of the use of these cold-set binders in raw and heated samples. The peak areas of the fibrinogen marker peptides were increased by a factor of about 100, compared to blank values originating from the occurrence of residual blood in meat, using a concentration of 0.6% bovine and porcine PPFG. A differentiation between the use of blood plasma powder and PPFG using the ratios of fibrinogen to serotransferrin peptide peak areas seems to be possible. Copyright © 2017 Elsevier Ltd. All rights reserved.

Milk derived bioactive peptides and their impact on human health - A review.

PubMed

Mohanty, D P; Mohapatra, S; Misra, S; Sahu, P S

2016-09-01

Milk-derived bioactive peptides have been identified as potential ingredients of health-promoting functional foods. These bioactive peptides are targeted at diet-related chronic diseases especially the non-communicable diseases viz., obesity, cardiovascular diseases and diabetes. Peptides derived from the milk of cow, goat, sheep, buffalo and camel exert multifunctional properties, including anti-microbial, immune modulatory, anti-oxidant, inhibitory effect on enzymes, anti-thrombotic, and antagonistic activities against various toxic agents. Majority of those regulate immunological, gastrointestinal, hormonal and neurological responses, thereby playing a vital role in the prevention of cancer, osteoporosis, hypertension and other disorders as discussed in this review. For the commercial production of such novel bioactive peptides large scale technologies based on membrane separation and ion exchange chromatography methods have been developed. Separation and identification of those peptides and their pharmacodynamic parameters are necessary to transfer their potent functional properties into food applications. The present review summarizes the preliminary classes of bioactive milk-derived peptides along with their physiological functions, general characteristics and potential applications in health-care.

Biodegradable nanostructures with selective lysis of microbial membranes

NASA Astrophysics Data System (ADS)

Nederberg, Fredrik; Zhang, Ying; Tan, Jeremy P. K.; Xu, Kaijin; Wang, Huaying; Yang, Chuan; Gao, Shujun; Guo, Xin Dong; Fukushima, Kazuki; Li, Lanjuan; Hedrick, James L.; Yang, Yi-Yan

2011-05-01

Macromolecular antimicrobial agents such as cationic polymers and peptides have recently been under an increased level of scrutiny because they can combat multi-drug-resistant microbes. Most of these polymers are non-biodegradable and are designed to mimic the facially amphiphilic structure of peptides so that they may form a secondary structure on interaction with negatively charged microbial membranes. The resulting secondary structure can insert into and disintegrate the cell membrane after recruiting additional polymer molecules. Here, we report the first biodegradable and in vivo applicable antimicrobial polymer nanoparticles synthesized by metal-free organocatalytic ring-opening polymerization of functional cyclic carbonate. We demonstrate that the nanoparticles disrupt microbial walls/membranes selectively and efficiently, thus inhibiting the growth of Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and fungi, without inducing significant haemolysis over a wide range of concentrations. These biodegradable nanoparticles, which can be synthesized in large quantities and at low cost, are promising as antimicrobial drugs, and can be used to treat various infectious diseases such as MRSA-associated infections, which are often linked with high mortality.

Antimicrobial Peptides in 2014

PubMed Central

Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

2015-01-01

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

Versatile microbial surface-display for environmental remediation and biofuels production

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

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

PubMed

Chuan, Yap P; Wibowo, Nani; Connors, Natalie K; Wu, Yang; Hughes, Fiona K; Batzloff, Michael R; Lua, Linda H L; Middelberg, Anton P J

2014-06-01

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 μg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries. © 2013 Wiley Periodicals, Inc.

Hidden weapons of microbial destruction in plant genomes

PubMed Central

Manners, John M

2007-01-01

Recent bioinformatic analyses of sequenced plant genomes reveal a previously unrecognized abundance of genes encoding antimicrobial cysteine-rich peptides, representing a formidable and dynamic defense arsenal against plant pests and pathogens. PMID:17903311

Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp.

PubMed

Federico, Baruzzi; Pinto, Loris; Quintieri, Laura; Carito, Antonia; Calabrese, Nicola; Caputo, Leonardo

2015-12-23

The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfected fresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptides in controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolates were collected from ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonas media, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas putida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was found when Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarole chicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papain and rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability. As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using the peptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced (P ≤ 0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTE lettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable cell density in the inoculated wounds. This is the first paper providing direct evidence regarding the application of an antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage.

Synergy of SOCS-1 Inhibition and Microbial-Based Cancer Vaccines

DTIC Science & Technology

2014-11-01

response without causing additional risk to the patient. The goal of our proposal is to modify a live- attenuated vaccine vector based on the food -borne...response after vaccination with a live-­‐‑attenuated L. monocytogenes. Aim 3: Test the hypothesis that secretion of a SOCS-­‐‑1 small peptide ...efficient internalization of pathogens and dying cells, processing of this material into peptide antigens that are presented in the context of major

In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU.

PubMed

Cole, Nerida; Hume, Emma B H; Vijay, Ajay K; Sankaridurg, Padmaja; Kumar, Naresh; Willcox, Mark D P

2010-01-01

One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens-induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses. A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P < or = 0.05). Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

Avian Antimicrobial Host Defense Peptides: From Biology to Therapeutic Applications

PubMed Central

Zhang, Guolong; Sunkara, Lakshmi T.

2014-01-01

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomoduatory properties. Because they act on the microbial membranes or host immune cells, HDPs pose a low risk of triggering microbial resistance and therefore, are being actively investigated as a novel class of antimicrobials and vaccine adjuvants. Cathelicidins and β-defensins are two major families of HDPs in avian species. More than a dozen HDPs exist in birds, with the genes in each HDP family clustered in a single chromosomal segment, apparently as a result of gene duplication and diversification. In contrast to their mammalian counterparts that adopt various spatial conformations, mature avian cathelicidins are mostly α-helical. Avian β-defensins, on the other hand, adopt triple-stranded β-sheet structures similar to their mammalian relatives. Besides classical β-defensins, a group of avian-specific β-defensin-related peptides, namely ovodefensins, exist with a different six-cysteine motif. Like their mammalian counterparts, avian cathelicidins and defensins are derived from either myeloid or epithelial origin expressed in a majority of tissues with broad-spectrum antibacterial and immune regulatory activities. Structure-function relationship studies with several avian HDPs have led to identification of the peptide analogs with potential for use as antimicrobials and vaccine adjuvants. Dietary modulation of endogenous HDP synthesis has also emerged as a promising alternative approach to disease control and prevention in chickens. PMID:24583933

Polymer multilayers loaded with antifungal β-peptides kill planktonic Candida albicans and reduce formation of fungal biofilms on the surfaces of flexible catheter tubes.

PubMed

Raman, Namrata; Lee, Myung-Ryul; Palecek, Sean P; Lynn, David M

2014-10-10

Candida albicans is the most common fungal pathogen responsible for hospital-acquired infections. Most C. albicans infections are associated with the implantation of medical devices that act as points of entry for the pathogen and as substrates for the growth of fungal biofilms that are notoriously difficult to eliminate by systemic administration of conventional antifungal agents. In this study, we report a fill-and-purge approach to the layer-by-layer fabrication of biocompatible, nanoscale 'polyelectrolyte multilayers' (PEMs) on the luminal surfaces of flexible catheters, and an investigation of this platform for the localized, intraluminal release of a cationic β-peptide-based antifungal agent. We demonstrate that polyethylene catheter tubes with luminal surfaces coated with multilayers ~700nm thick fabricated from poly-l-glutamic acid (PGA) and poly-l-lysine (PLL) can be loaded, post-fabrication, by infusion with β-peptide, and that this approach promotes extended intraluminal release of this agent (over ~4months) when incubated in physiological media. The β-peptide remained potent against intraluminal inoculation of the catheters with C. albicans and substantially reduced the formation of C. albicans biofilms on the inner surfaces of film-coated catheters. Finally, we report that these β-peptide-loaded coatings exhibit antifungal activity under conditions that simulate intermittent catheter use and microbial challenge for at least three weeks. We conclude that β-peptide-loaded PEMs offer a novel and promising approach to kill C. albicans and prevent fungal biofilm formation on surfaces, with the potential to substantially reduce the incidence of device-associated infections in indwelling catheters. β-Peptides comprise a promising new class of antifungal agents that could help address problems associated with the use of conventional antifungal agents. The versatility of the layer-by-layer approach used here thus suggests additional opportunities to exploit these new agents in other biomedical and personal care applications in which fungal infections are endemic. Copyright © 2014 Elsevier B.V. All rights reserved.

Antimicrobial Peptides: An Emerging Category of Therapeutic Agents.

PubMed

Mahlapuu, Margit; Håkansson, Joakim; Ringstad, Lovisa; Björn, Camilla

2016-01-01

Antimicrobial peptides (AMPs), also known as host defense peptides, are short and generally positively charged peptides found in a wide variety of life forms from microorganisms to humans. Most AMPs have the ability to kill microbial pathogens directly, whereas others act indirectly by modulating the host defense systems. Against a background of rapidly increasing resistance development to conventional antibiotics all over the world, efforts to bring AMPs into clinical use are accelerating. Several AMPs are currently being evaluated in clinical trials as novel anti-infectives, but also as new pharmacological agents to modulate the immune response, promote wound healing, and prevent post-surgical adhesions. In this review, we provide an overview of the biological role, classification, and mode of action of AMPs, discuss the opportunities and challenges to develop these peptides for clinical applications, and review the innovative formulation strategies for application of AMPs.

Identification of peptides in functional Scamorza ovine milk cheese.

PubMed

Albenzio, M; Santillo, A; Marino, R; Della Malva, A; Caroprese, M; Sevi, A

2015-12-01

Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations - A review.

PubMed

Zhao, Cindy J; Schieber, Andreas; Gänzle, Michael G

2016-11-01

Fermented foods are valued for their rich and complex odour and taste. The metabolic activity of food-fermenting microorganisms determines food quality and generates odour and taste compounds. This communication reviews the formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations. Pathways of the generation of taste compounds are presented for soy sauce, cheese, fermented meats, and bread. Proteolysis or autolysis during food fermentations generates taste-active amino acids and peptides; peptides derived from proteolysis particularly impart umami taste (e.g. α-glutamyl peptides) or bitter taste (e.g. hydrophobic peptides containing proline). Taste active peptide derivatives include pyroglutamyl peptides, γ-glutamyl peptides, and succinyl- or lactoyl amino acids. The influence of fermentation microbiota on proteolysis, and peptide hydrolysis, and the metabolism of glutamate and arginine is well understood, however, the understanding of microbial metabolic activities related to the formation of taste-active peptide derivatives is incomplete. Improved knowledge of the interactions between taste-active compounds will enable the development of novel fermentation strategies to develop tastier, less bitter, and low-salt food products, and may provide novel and "clean label" ingredients to improve the taste of other food products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bioactive peptides released from in vitro digestion of human milk with or without pasteurization.

PubMed

Wada, Yasuaki; Lönnerdal, Bo

2015-04-01

Pasteurized donor human milk (HM) serves as the best alternative for breast-feeding when availability of mother's milk is limited. Pasteurization is also applied to mother's own milk for very low birth weight infants, who are vulnerable to microbial infection. Whether pasteurization affects protein digestibility and therefore modulates the profile of bioactive peptides released from HM proteins by gastrointestinal digestion, has not been examined to date. HM with and without pasteurization (62.5 °C for 30 min) were subjected to in vitro gastrointestinal digestion, followed by peptidomic analysis to compare the formation of bioactive peptides. Some of the bioactive peptides, such as caseinophosphopeptide homologues, a possible opioid peptide (or propeptide), and an antibacterial peptide, were present in undigested HM and showed resistance to in vitro digestion, suggesting that these peptides are likely to exert their bioactivities in the gastrointestinal lumen, or be stably transported to target organs. In vitro digestion of HM released a large variety of bioactive peptides such as angiotensin I-converting enzyme-inhibitory, antioxidative, and immunomodulatory peptides. Bioactive peptides were released largely in the same manner with and without pasteurization. Provision of pasteurized HM may be as beneficial as breast-feeding in terms of milk protein-derived bioactive peptides.

Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis

NASA Astrophysics Data System (ADS)

Kirkpatrick, Christine L.; Parsley, Nicole C.; Bartges, Tessa E.; Cooke, Madeline E.; Evans, Wilaysha S.; Heil, Lilian R.; Smith, Thomas J.; Hicks, Leslie M.

2018-05-01

Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety. [Figure not available: see fulltext.

Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis

NASA Astrophysics Data System (ADS)

Kirkpatrick, Christine L.; Parsley, Nicole C.; Bartges, Tessa E.; Cooke, Madeline E.; Evans, Wilaysha S.; Heil, Lilian R.; Smith, Thomas J.; Hicks, Leslie M.

2018-02-01

Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety. [Figure not available: see fulltext.

Giardia co-infection promotes the secretion of antimicrobial peptides beta-defensin 2 and trefoil factor 3 and attenuates attaching and effacing bacteria-induced intestinal disease.

PubMed

Manko, Anna; Motta, Jean-Paul; Cotton, James A; Feener, Troy; Oyeyemi, Ayodele; Vallance, Bruce A; Wallace, John L; Buret, Andre G

2017-01-01

Our understanding of polymicrobial gastrointestinal infections and their effects on host biology remains incompletely understood. Giardia duodenalis is an ubiquitous intestinal protozoan parasite infecting animals and humans. Concomitant infections with Giardia and other gastrointestinal pathogens commonly occur. In countries with poor sanitation, Giardia infection has been associated with decreased incidence of diarrheal disease and fever, and reduced serum inflammatory markers release, via mechanisms that remain obscure. This study analyzed Giardia spp. co-infections with attaching and effacing (A/E) pathogens, and assessed whether and how the presence of Giardia modulates host responses to A/E enteropathogens, and alters intestinal disease outcome. In mice infected with the A/E pathogen Citrobacter rodentium, co-infection with Giardia muris significantly attenuated weight loss, macro- and microscopic signs of colitis, bacterial colonization and translocation, while concurrently enhancing the production and secretion of antimicrobial peptides (AMPs) mouse β-defensin 3 and trefoil factor 3 (TFF3). Co-infection of human intestinal epithelial cells (Caco-2) monolayers with G. duodenalis trophozoites and enteropathogenic Escherichia coli (EPEC) enhanced the production of the AMPs human β-defensin 2 (HBD-2) and TFF3; this effect was inhibited with treatment of G. duodenalis with cysteine protease inhibitors. Collectively, these results suggest that Giardia infections are capable of reducing enteropathogen-induced colitis while increasing production of host AMPs. Additional studies also demonstrated that Giardia was able to directly inhibit the growth of pathogenic bacteria. These results reveal novel mechanisms whereby Giardia may protect against gastrointestinal disease induced by a co-infecting A/E enteropathogen. Our findings shed new light on how microbial-microbial interactions in the gut may protect a host during concomitant infections.

Giardia co-infection promotes the secretion of antimicrobial peptides beta-defensin 2 and trefoil factor 3 and attenuates attaching and effacing bacteria-induced intestinal disease

PubMed Central

Manko, Anna; Motta, Jean-Paul; Cotton, James A.; Feener, Troy; Oyeyemi, Ayodele; Vallance, Bruce A.; Wallace, John L.

2017-01-01

Our understanding of polymicrobial gastrointestinal infections and their effects on host biology remains incompletely understood. Giardia duodenalis is an ubiquitous intestinal protozoan parasite infecting animals and humans. Concomitant infections with Giardia and other gastrointestinal pathogens commonly occur. In countries with poor sanitation, Giardia infection has been associated with decreased incidence of diarrheal disease and fever, and reduced serum inflammatory markers release, via mechanisms that remain obscure. This study analyzed Giardia spp. co-infections with attaching and effacing (A/E) pathogens, and assessed whether and how the presence of Giardia modulates host responses to A/E enteropathogens, and alters intestinal disease outcome. In mice infected with the A/E pathogen Citrobacter rodentium, co-infection with Giardia muris significantly attenuated weight loss, macro- and microscopic signs of colitis, bacterial colonization and translocation, while concurrently enhancing the production and secretion of antimicrobial peptides (AMPs) mouse β-defensin 3 and trefoil factor 3 (TFF3). Co-infection of human intestinal epithelial cells (Caco-2) monolayers with G. duodenalis trophozoites and enteropathogenic Escherichia coli (EPEC) enhanced the production of the AMPs human β-defensin 2 (HBD-2) and TFF3; this effect was inhibited with treatment of G. duodenalis with cysteine protease inhibitors. Collectively, these results suggest that Giardia infections are capable of reducing enteropathogen-induced colitis while increasing production of host AMPs. Additional studies also demonstrated that Giardia was able to directly inhibit the growth of pathogenic bacteria. These results reveal novel mechanisms whereby Giardia may protect against gastrointestinal disease induced by a co-infecting A/E enteropathogen. Our findings shed new light on how microbial-microbial interactions in the gut may protect a host during concomitant infections. PMID:28622393

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.

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans.

PubMed

Mowlds, Peter; Barron, Aoife; Kavanagh, Kevin

2008-05-01

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

PubMed

Kim, Young-Min; Kim, Nam-Hong; Lee, Jong-Wan; Jang, Jin-Sun; Park, Yung-Hoon; Park, Seong-Cheol; Jang, Mi-Kyeong

2015-07-31

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 μM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications.

PubMed

Salazar-Marroquín, Elma Laura; Galán-Wong, Luis J; Moreno-Medina, Víctor Ricardo; Reyes-López, Miguel Ángel; Pereyra-Alférez, Benito

2016-07-01

The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area.

Metabolic profiling of sourdough fermented wheat and rye bread.

PubMed

Koistinen, Ville M; Mattila, Outi; Katina, Kati; Poutanen, Kaisa; Aura, Anna-Marja; Hanhineva, Kati

2018-04-09

Sourdough fermentation by lactic acid bacteria is commonly used in bread baking, affecting several attributes of the final product. We analyzed whole-grain wheat and rye breads and doughs prepared with baker's yeast or a sourdough starter including Candida milleri, Lactobacillus brevis and Lactobacillus plantarum using non-targeted metabolic profiling utilizing LC-QTOF-MS. The aim was to determine the fermentation-induced changes in metabolites potentially contributing to the health-promoting properties of whole-grain wheat and rye. Overall, we identified 118 compounds with significantly increased levels in sourdough, including branched-chain amino acids (BCAAs) and their metabolites, small peptides with high proportion of BCAAs, microbial metabolites of phenolic acids and several other potentially bioactive compounds. We also identified 69 compounds with significantly decreased levels, including phenolic acid precursors, nucleosides, and nucleobases. Intensive sourdough fermentation had a higher impact on the metabolite profile of whole-grain rye compared to milder whole-grain wheat sourdough fermentation. We hypothesize that the increased amount of BCAAs and potentially bioactive small peptides may contribute to the insulin response of rye bread, and in more general, the overall protective effect against T2DM and CVD.

Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability

PubMed Central

Redmile-Gordon, M.A.; Evershed, R.P.; Hirsch, P.R.; White, R.P.; Goulding, K.W.T.

2015-01-01

An emerging paradigm in soil science suggests microbes can perform ‘N mining’ from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs. PMID:26339106

NF-κB/Rel Proteins and the Humoral Immune Responses of Drosophila melanogaster

PubMed Central

Ganesan, Sandhya; Aggarwal, Kamna; Paquette, Nicholas; Silverman, Neal

2011-01-01

Nuclear Factor-κB (NF-κB)/Rel transcription factors form an integral part of innate immune defenses and are conserved throughout the animal kingdom. Studying the function, mechanism of activation and regulation of these factors is crucial for understanding host responses to microbial infections. The fruit fly Drosophila melanogaster has proved to be a valuable model system to study these evolutionarily conserved NF-κB mediated immune responses. Drosophila combats pathogens through humoral and cellular immune responses. These humoral responses are well characterized and are marked by the robust production of a battery of anti-microbial peptides. Two NF-κB signaling pathways, the Toll and the IMD pathways, are responsible for the induction of these antimicrobial peptides. Signal transduction in these pathways is strikingly similar to that in mammalian TLR pathways. In this chapter, we discuss in detail the molecular mechanisms of microbial recognition, signal transduction and NF-κB regulation, in both the Toll and the IMD pathways. Similarities and differences relative to their mammalian counterparts are discussed, and recent advances in our understanding of the intricate regulatory networks in these NF-κB signaling pathways are also highlighted. PMID:20852987

Identification of Quorum-Sensing Inhibitors Disrupting Signaling between Rgg and Short Hydrophobic Peptides in Streptococci

PubMed Central

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Lee, Hyun; Chlipala, George E.; Ratia, Kiira

2015-01-01

ABSTRACT Bacteria coordinate a variety of social behaviors, important for both environmental and pathogenic bacteria, through a process of intercellular chemical signaling known as quorum sensing (QS). As microbial resistance to antibiotics grows more common, a critical need has emerged to develop novel anti-infective therapies, such as an ability to attenuate bacterial pathogens by means of QS interference. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development in response to the short hydrophobic peptides SHP2 and SHP3. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that could specifically inhibit Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt complexes of Rgg and FITC (fluorescein isothiocyanate)-conjugated SHP using a fluorescence polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with submicromolar affinities, to specifically abolish transcription of Rgg2/3-controlled genes, and to prevent biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit, cyclosporine A, as well as its nonimmunosuppressive analog, valspodar, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The Rgg-FITC-peptide-based screen provides a platform to identify inhibitors specific for each Rgg type. Discovery of Rgg inhibitors constitutes a step toward the goal of manipulating bacterial behavior for purposes of improving health. PMID:25968646

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D–dependent mechanism

PubMed Central

Schauber, Jürgen; Dorschner, Robert A.; Coda, Alvin B.; Büchau, Amanda S.; Liu, Philip T.; Kiken, David; Helfrich, Yolanda R.; Kang, Sewon; Elalieh, Hashem Z.; Steinmeyer, Andreas; Zügel, Ulrich; Bikle, Daniel D.; Modlin, Robert L.; Gallo, Richard L.

2007-01-01

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-β1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-β1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection. PMID:17290304

A lesson from Bombinins H, mildly cationic diastereomeric antimicrobial peptides from Bombina skin.

PubMed

Mangoni, Maria Luisa

2013-12-01

Gene-encoded peptide antibiotics represent fascinating molecules for the development of new antimicrobials with a new mode of action: and one of the richest sources is amphibian skin. In particular, the skin of the fire-bellied toad Bombina genus contains mildly cationic antimicrobial peptides (AMPs), named bombinins H, with attractive properties. Indeed, some members of this peptide family coexist in skin secretions as isomers in which a single D-amino acid (alloisoleucine or leucine) is incorporated as a result of a post-translational modification of the respective gene-encoded Lamino acid. Here, a brief overview of the genes coding for these peptides, their spectrum of antimicrobial activities, mechanism of action and interactions with biological or model membranes is reported. Remarkably, a single D-amino acid substitution represents a unique approach developed by Nature not only to modulate the peptide stability in vivo, but also to confer the all-L peptide and its diastereomer distinctive biological features. Overall, such findings should assist in the generation of new peptide-based anti-infective agents, which are urgently needed because of the growing emergence of microbial strains resistant to conventional antimicrobials.

Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor

NASA Astrophysics Data System (ADS)

Kumar, Amit; Delogu, Francesco

2017-02-01

The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR’s cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

PubMed Central

Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

2016-01-01

ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity. PMID:27440904

Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and Secreted Aspartyl Proteinase (SAPS) 2, 4, 5 and 6 Expressions

DTIC Science & Technology

2016-07-01

broad range of antibacterial activity and could play a role in preventing microbial infections(Decanis et al., 2009), (Zaslof, 2002). These antimicrobial...range of antibacterial activity and could play a role in preventing microbial infections(Decanis et al., 2009),(Zaslof, 2002). These antimicrobial...KSL- W (KKVVFWVKFK)(Na et al., 2007), which possess a broad range of antibacterial activity . It killed selected strains of non-oral and oral

Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity

NASA Astrophysics Data System (ADS)

Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.; Dong, He

2015-11-01

Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05233e

Involvement of granulysin-producing T cells in the development of superficial microbial folliculitis.

PubMed

Oono, T; Morizane, S; Yamasaki, O; Shirafuji, Y; Huh, W-K; Akiyama, H; Iwatsuki, K

2004-05-01

Granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defence mechanisms against mycobacterial infection. Superficial microbial folliculitis is a common skin disease. In a previous report, we showed that, as a first line of defence, alpha-defensin, a human neutrophil peptide, and beta-defensin (human beta-defensin-2) were expressed in infiltrating neutrophils and in lesional epidermal keratinocytes, respectively, in superficial folliculitis. As we also observed many infiltrating lymphocytes in lesional dermis, we hypothesized that infiltrating lymphocytes may possess antimicrobial substances, such as granulysin, and play a role in the defence mechanism as a second line of defence. Seven specimens of superficial microbial folliculitis diagnosed clinically and histologically were examined by means of immunohistochemistry. To identify the phenotype of cells expressing granulysin, confocal laser microscopic examination was performed. A dense lymphoid cell infiltrate was observed in pustules, in the perivascular regions. A large number of these lymphoid cells were positive for granulysin. Phenotypically, cells consisted of CD3+ T cells, CD8+ T cells and UCHL-1+ T cells. CD20+ cells and CD56+ cells were not observed. Microscopic examination with a confocal laser showed that the lymphocytes producing granulysin were CD3+ and CD4+ T cells but not CD8+ T cells. We showed that many granulysin-bearing T cells infiltrated affected follicles and perilesional dermis in superficial microbial folliculitis. However, few granulysin-positive lymphoid cells were observed in sterile pustular lesions. Our observations indicated that adaptive immunity such as granulysin, a lymphocyte-produced antimicrobial protein, may play an important role in the cutaneous defence mechanism.

Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

PubMed

López, Constanza M; Bru, Elena; Vignolo, Graciela M; Fadda, Silvina G

2015-06-01

In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers. Copyright © 2015 Elsevier Ltd. All rights reserved.

The novel antimicrobial peptides from skin of Chinese broad-folded frog, Hylarana latouchii (Anura:Ranidae).

PubMed

Wang, Hui; Lu, Yi; Zhang, Xiuqing; Hu, Yuhong; Yu, Haining; Liu, Jingze; Sun, Junshe

2009-02-01

Broad-folded frogs (Hylarana latouchii), one member of 12 species of the genus Hylarana in the Chinese frog fauna, are widely distributed in the South of China. In this study, we purified and characterized three antimicrobial peptides from the skin secretion of H. latouchii. Five different cDNA fragments encoding the precursors of these antimicrobial peptides were cloned, and five mature antimicrobial peptides belonging to two different families were deduced from the five cDNAs. Structural characterization of the mature peptides had identified them as members of the brevinin-1 and temporin families. They were named brevinin-1LTa (FFGTALKIAANVLPTAICKILKKC), brevinin-1LTb (FFGTALKIAANILPTAICKILKKC), temporin-LTa (FFPLVLGALGSILPKIF-NH(2)), temporin-LTb (FIITGLVRGLTKLF-NH(2)) and temorin-LTc (SLSRFLSFLKIVYPPAF-NH(2)). Brevinin-1LTa, temporin-LTa, temporin-LTb and temporin-LTc with different antimicrobial activities induced significant morphological alterations of the tested microbial surfaces as shown by scanning electron microscopy, which indicated strong membrane disruption.

The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate.

PubMed

O'Connor, Stephen; Szwej, Emilia; Nikodinovic-Runic, Jasmina; O'Connor, Aisling; Byrne, Annette T; Devocelle, Marc; O'Donovan, Norma; Gallagher, William M; Babu, Ramesh; Kenny, Shane T; Zinn, Manfred; Zulian, Qun Ren; O'Connor, Kevin E

2013-04-01

The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthetic peptides that cause F-actin bundling and block actin depolymerization

DOEpatents

Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

2011-10-18

Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

Peptides conjugated to silver nanoparticles in biomedicine - a "value-added" phenomenon.

PubMed

Ramesh, Suhas; Grijalva, Marcelo; Debut, Alexis; de la Torre, Beatriz G; Albericio, Fernando; Cumbal, Luis H

2016-11-15

Nanotechnology is gaining impetus in the present century and particularly the use of nanoparticles (NPs), whose properties are significantly different from the larger matter. These have found wider and potential applications in the fields of medicine, energy, cosmetics, environment and biomedicine. Among the NPs, silver nanoparticles (AgNPs) are of particular interest for scientists and technologists due to their unique physico-chemical and biological properties. Besides, AgNPs by themselves also possess broad-spectrum microbial activity, which has further expanded their application in both academia and industries. On the other hand, research and drug discovery in the field of peptides is surging. Chemistry and biology of peptides have seen a renaissance in this century as many of the peptide-based therapeutics have entered the market and many more are in the different phases of clinical trials. To fuel this, peptides have also found numerous applications in nanotechnology. Taking advantage of these two scenarios, namely, AgNPs and peptides, conjugation of these entities have emerged as a powerful technique and have opened the doors for a new revolution. Keeping this motivation in mind, we here present a mini-review on the combined concept of AgNPs and peptides.

A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions*

PubMed Central

Weston, Andrew J.; Dunlap, Walter C.; Shick, J. Malcolm; Klueter, Anke; Iglic, Katrina; Vukelic, Ana; Starcevic, Antonio; Ward, Malcolm; Wells, Mark L.; Trick, Charles G.; Long, Paul F.

2012-01-01

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal “bleaching” stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure. PMID:22351649

Sequence diversity and evolution of antimicrobial peptides in invertebrates.

PubMed

Tassanakajon, Anchalee; Somboonwiwat, Kunlaya; Amparyup, Piti

2015-02-01

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that act as the key components in the invertebrate innate immunity against invading pathogens. Several AMPs have been identified and characterized in invertebrates, and found to display considerable diversity in their amino acid sequence, structure and biological activity. AMP genes appear to have rapidly evolved, which might have arisen from the co-evolutionary arms race between host and pathogens, and enabled organisms to survive in different microbial environments. Here, the sequence diversity of invertebrate AMPs (defensins, cecropins, crustins and anti-lipopolysaccharide factors) are presented to provide a better understanding of the evolution pattern of these peptides that play a major role in host defense mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Antimicrobial peptides: a review of how peptide structure impacts antimicrobial activity

NASA Astrophysics Data System (ADS)

Soares, Jason W.; Mello, Charlene M.

2004-03-01

Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins.

Precision-guided antimicrobial peptide as a targeted modulator of human microbial ecology.

PubMed

Guo, Lihong; McLean, Jeffrey S; Yang, Youngik; Eckert, Randal; Kaplan, Christopher W; Kyme, Pierre; Sheikh, Omid; Varnum, Brian; Lux, Renate; Shi, Wenyuan; He, Xuesong

2015-06-16

One major challenge to studying human microbiome and its associated diseases is the lack of effective tools to achieve targeted modulation of individual species and study its ecological function within multispecies communities. Here, we show that C16G2, a specifically targeted antimicrobial peptide, was able to selectively kill cariogenic pathogen Streptococcus mutans with high efficacy within a human saliva-derived in vitro oral multispecies community. Importantly, a significant shift in the overall microbial structure of the C16G2-treated community was revealed after a 24-h recovery period: several bacterial species with metabolic dependency or physical interactions with S. mutans suffered drastic reduction in their abundance, whereas S. mutans' natural competitors, including health-associated Streptococci, became dominant. This study demonstrates the use of targeted antimicrobials to modulate the microbiome structure allowing insights into the key community role of specific bacterial species and also indicates the therapeutic potential of C16G2 to achieve a healthy oral microbiome.

Diagnostic value of anti-microbial peptide, cathelicidin in congenital pneumonia.

PubMed

Gad, Ghada I; Abushady, Nancy M; Fathi, Marwa S; Elsaadany, Wafaa

2015-01-01

To evaluate the diagnostic value of anti-microbial peptide (cathelicidin), LL-37, in congenital pneumonia and its relation to 25 hydroxycholecalciferol [(25 OH)D] status. The study included 30 neonates with congenital pneumonia and culture proven sepsis admitted to neonatal intensive care unit of Ain Shams University and 30 healthy neonates as control group. All neonates were subjected to history taking, clinical examination and measurement of serum 25(OH)D and cathelicidin. Neonates with congenital pneumonia had significantly higher serum cathelicidin and lower serum 25(OH)D compared to controls. Serum cathelicidin was negatively correlated with Apgar score at 1 and 5 min and positively correlated with length of stay among patient group. Cut-off value of cathelicidin to diagnose congenital pneumonia was 17 pg/mmol with 93% sensitivity and 86% specificity. Neonates with congenital pneumonia had significantly high cathelicidin and low 25(OH)D suggesting a possible role of fetal 25(OH)D deficiency as predisposing factor for congenital pneumonia.

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

PubMed Central

2013-01-01

Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. PMID:23586331

Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications

PubMed Central

Salazar-Marroquín, Elma Laura; Galán-Wong, Luis J.; Moreno-Medina, Víctor Ricardo; Reyes-López, Miguel Ángel; Pereyra-Alférez, Benito

2016-01-01

The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area. PMID:27340340

Increased gut permeability in cancer cachexia: mechanisms and clinical relevance.

PubMed

Bindels, Laure B; Neyrinck, Audrey M; Loumaye, Audrey; Catry, Emilie; Walgrave, Hannah; Cherbuy, Claire; Leclercq, Sophie; Van Hul, Matthias; Plovier, Hubert; Pachikian, Barbara; Bermúdez-Humarán, Luis G; Langella, Philippe; Cani, Patrice D; Thissen, Jean-Paul; Delzenne, Nathalie M

2018-04-06

Intestinal disorders often occur in cancer patients, in association with body weight loss, and this alteration is commonly attributed to the chemotherapy. Here, using a mouse model of cancer cachexia induced by ectopic transplantation of C26 cancer cells, we discovered a profound alteration in the gut functions (gut permeability, epithelial turnover, gut immunity, microbial dysbiosis) independently of any chemotherapy. These alterations occurred independently of anorexia and were driven by interleukin 6. Gut dysfunction was found to be resistant to treatments with an anti-inflammatory bacterium ( Faecalibacterium prausnitzii ) or with gut peptides involved in intestinal cell renewal (teduglutide, a glucagon-like peptide 2 analogue). The translational value of our findings was evaluated in 152 colorectal and lung cancer patients with or without cachexia. The serum level of the lipopolysaccharide-binding protein, often presented as a reflection of the bacterial antigen load, was not only increased in cachectic mice and cancer patients, but also strongly correlated with the serum IL-6 level and predictive of death and cachexia occurrence in these patients. Altogether, our data highlight profound alterations of the intestinal homeostasis in cancer cachexia occurring independently of any chemotherapy and food intake reduction, with potential relevance in humans. In addition, we point out the lipopolysaccharide-binding protein as a new biomarker of cancer cachexia related to gut dysbiosis.

Increased gut permeability in cancer cachexia: mechanisms and clinical relevance

PubMed Central

Bindels, Laure B.; Neyrinck, Audrey M.; Loumaye, Audrey; Catry, Emilie; Walgrave, Hannah; Cherbuy, Claire; Leclercq, Sophie; Van Hul, Matthias; Plovier, Hubert; Pachikian, Barbara; Bermúdez-Humarán, Luis G.; Langella, Philippe; Cani, Patrice D.; Thissen, Jean-Paul; Delzenne, Nathalie M.

2018-01-01

Intestinal disorders often occur in cancer patients, in association with body weight loss, and this alteration is commonly attributed to the chemotherapy. Here, using a mouse model of cancer cachexia induced by ectopic transplantation of C26 cancer cells, we discovered a profound alteration in the gut functions (gut permeability, epithelial turnover, gut immunity, microbial dysbiosis) independently of any chemotherapy. These alterations occurred independently of anorexia and were driven by interleukin 6. Gut dysfunction was found to be resistant to treatments with an anti-inflammatory bacterium (Faecalibacterium prausnitzii) or with gut peptides involved in intestinal cell renewal (teduglutide, a glucagon-like peptide 2 analogue). The translational value of our findings was evaluated in 152 colorectal and lung cancer patients with or without cachexia. The serum level of the lipopolysaccharide-binding protein, often presented as a reflection of the bacterial antigen load, was not only increased in cachectic mice and cancer patients, but also strongly correlated with the serum IL-6 level and predictive of death and cachexia occurrence in these patients. Altogether, our data highlight profound alterations of the intestinal homeostasis in cancer cachexia occurring independently of any chemotherapy and food intake reduction, with potential relevance in humans. In addition, we point out the lipopolysaccharide-binding protein as a new biomarker of cancer cachexia related to gut dysbiosis. PMID:29719601

Blufensin1 Negatively Impacts Basal Defense in Response to Barley Powdery Mildew

USDA-ARS?s Scientific Manuscript database

Plants have evolved complex regulatory mechanisms to control the defense response against microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSIN1 (BLN1), a small peptide ...

Immunity-dependent reduction of segmented filamentous bacteria in mice infected with the helminthic parasite Nippostrongylus brasiliensis

USDA-ARS?s Scientific Manuscript database

Immune modulation by helminth (worm) parasites could protect the host against autoimmune diseases. We report that the parasitic nematode Nippostrongylus brasiliensis induces changes in the expression of antimicrobial peptides that are associated with marked microbial composition shifts, including re...

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

PubMed

Neela, Venkata Sanjeev Kumar; Devalraju, Kamakshi Prudhula; Pydi, Satya Sudheer; Sunder, Sharada Ramaseri; Adiraju, Kameswara Rao; Singh, Surya Satyanarayana; Anandaraj, M P J S; Valluri, Vijaya Lakshmi

2016-09-01

Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients. © The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Therapeutic Potential of Animal β-hairpin Antimicrobial Peptides.

PubMed

Panteleev, Pavel V; Balandin, Sergey V; Ivanov, Vadim T; Ovchinnikova, Tatiana V

2017-01-01

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play the key role in host defense. Because of the low resistance rate, AMPs have caught extensive attention as possible alternatives to conventional antibiotics. Over the last years, it has become evident that biological functions of AMPs are beyond direct killing of microbial cells. This review focuses on a relatively small family of animal host defense peptides with the β-hairpin structure stabilized by disulfide bridges. Their small size, rigid structure, stability to proteases, and plethora of biological functions, including antibacterial, antifungal, antiviral, anticancer, endotoxin-binding, metabolism- and immune- modulating activities, make natural β-hairpin AMPs an attractive molecular basis for drug design. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Bacteriocins from Gram-Negative Bacteria: A Classification?

NASA Astrophysics Data System (ADS)

Rebuffat, Sylvie

Bacteria produce an arsenal of toxic peptides and proteins, which are termed bacteriocins and play a role in mediating the dynamics of microbial populations and communities. Bacteriocins from Gram-negative bacteria arise mainly from Enterobacteriaceae. They assemble into two main families: high molecular mass modular proteins (30-80 kDa) termed colicins and low molecular mass peptides (between 1 and 10 kDa) termed microcins. The production of colicins is mediated by the SOS response regulon, which plays a role in the response of many bacteria to DNA damages. Microcins are highly stable hydrophobic peptides that are produced under stress conditions, particularly nutrient depletion. Colicins and microcins are found essentially in Escherichia coli, but several other Gram-negative species also produce bacteriocin-like substances. This chapter presents the basis of a classification of colicins and microcins.

Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.

PubMed

Tang, Xiang-Shan; Tang, Zhi-Ru; Wang, Sheng-Ping; Feng, Ze-Meng; Zhou, Dong; Li, Tie-Jun; Yin, Yu-Long

2012-02-01

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat as preservative.

PubMed

Przybylski, Rémi; Firdaous, Loubna; Châtaigné, Gabrielle; Dhulster, Pascal; Nedjar, Naïma

2016-11-15

Bovine cruor, a slaughterhouse by-product, contains mainly hemoglobin, broadly described as a rich source of antimicrobial peptides. In the current context of food safety, bioactive peptides could be of interest as preservatives in the distribution of food products. The aim of this work was to study the α137-141 fragment of hemoglobin (Thr-Ser-Lys-Tyr-Arg), a small (653Da) and hydrophilic antimicrobial peptide. Its production was fast, with more 65% finally produced at 24h already produced after 30min of hydrolysis with pepsin. Moreover, increasing substrate concentration (from 1 to 8% (w/v)) resulted in a proportional augmentation of α137-141 production (to 807.95±41.03mgL(-1)). The α137-141 application on meat as preservative (0.5%, w/w) reduced the lipid oxidation about 60% to delay meat rancidity. The α137-141 peptide also inhibited the microbial growths under refrigeration during 14days. These antimicrobial effects were close to those of the butylated hydroxytoluene (BHT). Copyright © 2016 Elsevier Ltd. All rights reserved.

Theoretical Sum Frequency Generation Spectroscopy of Peptides

PubMed Central

2015-01-01

Vibrational sum frequency generation (SFG) has become a very promising technique for the study of proteins at interfaces, and it has been applied to important systems such as anti-microbial peptides, ion channel proteins, and human islet amyloid polypeptide. Moreover, so-called “chiral” SFG techniques, which rely on polarization combinations that generate strong signals primarily for chiral molecules, have proven to be particularly discriminatory of protein secondary structure. In this work, we present a theoretical strategy for calculating protein amide I SFG spectra by combining line-shape theory with molecular dynamics simulations. We then apply this method to three model peptides, demonstrating the existence of a significant chiral SFG signal for peptides with chiral centers, and providing a framework for interpreting the results on the basis of the dependence of the SFG signal on the peptide orientation. We also examine the importance of dynamical and coupling effects. Finally, we suggest a simple method for determining a chromophore’s orientation relative to the surface using ratios of experimental heterodyne-detected signals with different polarizations, and test this method using theoretical spectra. PMID:25203677

Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide against Pseudomonas spp.

PubMed Central

Eckert, Randal; Qi, Fengxia; Yarbrough, Daniel K.; He, Jian; Anderson, Maxwell H.; Shi, Wenyuan

2006-01-01

Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designed Pseudomonas-specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics against Pseudomonas spp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminate Pseudomonas spp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics. PMID:16569868

Bioactivity and the First Transmission Electron Microscopy Immunogold Studies of Short De Novo-Designed Antimicrobial Peptides▿

PubMed Central

Azad, Marisa Ann; Huttunen-Hennelly, Heidi Esther Katrina; Ross Friedman, Cynthia

2011-01-01

In light of the era of microbial drug resistance, the current study aimed to better understand the relationships between sequence, higher-order structure, and mechanism of action for five designed peptides against multidrug-resistant (MDR) pathogens. All peptides studied were 15 residues long, were polycationic, adopted alpha-helical structures within hydrophobic environments (excluding the d-amino acid-substituted peptide MA-d), and contained N-terminal glycine residues, a novel antimicrobial peptide (AMP) design principle. Increasing hydrophobicity enhanced MICs (≤500 μg/ml to ≤7.4 μg/ml) without significantly increasing hemolytic activity (18% maximum hemolysis at 3,400 μg/ml). To the best of our knowledge, this is the first study to have successfully adapted and used a transmission electron microscopy (TEM) immunogold method to investigate the mechanism of action of short (∼15 residues long) AMPs within bacteria. We propose a “floodgate” mechanism to possibly explain membrane deformation and the relative absence of membrane-associated peptides 10 h into incubation. PMID:21300831

Antimicrobial Peptides and Wound Healing: Biological and Therapeutic Considerations

PubMed Central

Mangoni, Maria Luisa; McDermott, Alison M.; Zasloff, Michael

2016-01-01

Repair of tissue wounds is a fundamental process to re-establish tissue integrity and regular function. Importantly, infection is a major factor that hinders wound healing. Multicellular organisms have evolved an arsenal of host-defence molecules, including antimicrobial peptides (AMPs), aimed at controlling microbial proliferation and at modulating the host's immune response to a variety of biological or physical insults. In this brief review we provide the evidence for a role of AMPs as endogenous mediators of wound healing and their promising therapeutic potential for treatment of non-life threatening skin and other epithelial injuries. PMID:26738772

Machine learning in the rational design of antimicrobial peptides.

PubMed

Rondón-Villarreal, Paola; Sierra, Daniel A; Torres, Rodrigo

2014-01-01

One of the most important public health issues is the microbial and bacterial resistance to conventional antibiotics by pathogen microorganisms. In recent years, many researches have been focused on the development of new antibiotics. Among these, antimicrobial peptides (AMPs) have raised as a promising alternative to combat antibioticresistant microorganisms. For this reason, many theoretical efforts have been done in the development of new computational tools for the rational design of both better and effective AMPs. In this review, we present an overview of the rational design of AMPs using machine learning techniques and new research fields.

Characterization of peptides from common bean protein isolates and their potential to inhibit markers of type-2 diabetes, hypertension and oxidative stress.

PubMed

Mojica, Luis; Luna-Vital, Diego A; González de Mejía, Elvira

2017-06-01

Diabetes and hypertension are diseases affecting a high proportion of the world population; the use of food-based products such as common bean peptides may contribute to reduce the risk of complications associated to chronic diseases. The aim was to produce and characterize peptides from common bean protein isolates and evaluate their potential to inhibit markers of type-2 diabetes, hypertension and oxidative stress. Mexican black and Brazilian Carioca bean isolated proteins were characterized after pepsin/pancreatin digestion. Also, four synthesized pure peptides, originally found in these beans, were evaluated. Bean protein digests and pure peptides exerted dipeptidyl peptidase-IV (DPP-IV) inhibition (IC 50 = 0.03-0.87 mg dry weight (DW) mL -1 ). Lineweaver-Burk plots and computational modeling showed competitive inhibition of DPP-IV. Angiotensin-converting enzyme (ACE) inhibition ranged from IC 50 = 0.09 to 0.99 mg DW mL -1 , and α-glucosidase inhibition ranged from 36.3 to 50.1% mg -1 DW. Carioca Perola bean digested proteins presented the highest antioxidant capacity (269.3 mmol L -1 Trolox equivalent g -1 DW) as the peptide KTYGL (P > 0.05) with the most potent DPP-IV and ACE inhibition. Peptides from common bean have antidiabetic and antihypertensive potential regardless of their antioxidant capacity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Antimicrobial peptides from skin secretions of Hypsiboas pulchellus (Anura: Hylidae).

PubMed

Siano, Alvaro; Húmpola, María Verónica; de Oliveira, Eliandre; Albericio, Fernando; Simonetta, Arturo C; Lajmanovich, Rafael; Tonarelli, Georgina G

2014-04-25

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 μM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 μM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides.

Designing Antibacterial Peptides with Enhanced Killing Kinetics

PubMed Central

Waghu, Faiza H.; Joseph, Shaini; Ghawali, Sanket; Martis, Elvis A.; Madan, Taruna; Venkatesh, Kareenhalli V.; Idicula-Thomas, Susan

2018-01-01

Antimicrobial peptides (AMPs) are gaining attention as substitutes for antibiotics in order to combat the risk posed by multi-drug resistant pathogens. Several research groups are engaged in design of potent anti-infective agents using natural AMPs as templates. In this study, a library of peptides with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family were screened using popular online prediction algorithms. These peptide variants were designed in a manner to retain the conserved residues within the MAP family. The prediction algorithms were found to effectively classify peptides based on their antimicrobial nature. In order to improve the activity of the identified peptides, molecular dynamics (MD) simulations, using bilayer and micellar systems could be used to design and predict effect of residue substitution on membranes of microbial and mammalian cells. The inference from MD simulation studies well corroborated with the wet-lab observations indicating that MD-guided rational design could lead to discovery of potent AMPs. The effect of the residue substitution on membrane activity was studied in greater detail using killing kinetic analysis. Killing kinetics studies on Gram-positive, negative and human erythrocytes indicated that a single residue change has a drastic effect on the potency of AMPs. An interesting outcome was a switch from monophasic to biphasic death rate constant of Staphylococcus aureus due to a single residue mutation in the peptide. PMID:29527201

Inducible factors with antimicrobial activity after immune challenge in the haemolymph of Red Palm Weevil (Insecta).

PubMed

Mastore, Maristella; Binda Rossetti, Simona; Giovannardi, Stefano; Scarì, Giorgio; Brivio, Maurizio F

2015-05-01

Insects are capable of innate immune responses elicited after microbial infection. In this process, the receptor-mediated recognition of foreign bodies and the subsequent activation of immunocompetent cells lead to the synthesis ex novo of a peptide pool with antimicrobial activity. We investigated the inducible immune response of a coleopteran, Rhynchophorus ferrugineus, challenged with both Gram-negative and Gram-positive bacteria. After immunization, we evaluated the presence of antimicrobial peptides using either biochemical analyses or microbiological techniques. The antimicrobial properties of the newly synthesized protein pool, detectable in haemolymph fractions of low molecular mass, showed strong antibacterial activity against various bacterial strains (Escherichia coli, Pseudomonas sp. OX1, Bacillus subtilis and Micrococcus luteus). In addition to the preliminary study of the mechanism of action of the pool of antimicrobial peptides, we also investigated its effects on bacterial cell walls by means of fluorescence microscopy and scanning electron microscopy. The data suggest that the main effects seem to be directed at destabilizing and damaging the bacterial wall. This study provides data that help us to understand some aspects of the inducible innate immunity in a system model that lacks anticipatory responses. However, the weevil has finely tuned its defensive strategies to counteract effectively microbial infection. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Characterization of the Antimicrobial Peptide Penisin, a Class Ia Novel Lantibiotic from Paenibacillus sp. Strain A3

PubMed Central

Baindara, Piyush; Chaudhry, Vasvi; Mittal, Garima; Liao, Luciano M.; Matos, Carolina O.; Khatri, Neeraj; Franco, Octavio L.; Patil, Prabhu B.

2015-01-01

Attempts to isolate novel antimicrobial peptides from microbial sources have been on the rise recently, despite their low efficacy in therapeutic applications. Here, we report identification and characterization of a new efficient antimicrobial peptide from a bacterial strain designated A3 that exhibited highest identity with Paenibacillus ehimensis. Upon purification and subsequent molecular characterization of the antimicrobial peptide, referred to as penisin, we found the peptide to be a bacteriocin-like peptide. Consistent with these results, RAST analysis of the entire genome sequence revealed the presence of a lantibiotic gene cluster containing genes necessary for synthesis and maturation of a lantibiotic. While circular dichroism and one-dimension nuclear magnetic resonance experiments confirmed a random coil structure of the peptide, similar to other known lantibiotics, additional biochemical evidence suggests posttranslational modifications of the core peptide yield six thioether cross-links. The deduced amino acid sequence of the putative biosynthetic gene penA showed approximately 74% similarity with elgicin A and 50% similarity with the lantibiotic paenicidin A. Penisin effectively killed methicillin-resistant Staphylococcus aureus (MRSA) and did not exhibit hemolysis activity. Unlike other lantibiotics, it effectively inhibited the growth of Gram-negative bacteria. Furthermore, 80 mg/kg of body weight of penisin significantly reduced bacterial burden in a mouse thigh infection model and protected BALB/c mice in a bacteremia model entailing infection with Staphylococcus aureus MTCC 96, suggesting that it could be a promising new antimicrobial peptide. PMID:26574006

Fermented functional foods based on probiotics and their biogenic metabolites.

PubMed

Stanton, Catherine; Ross, R Paul; Fitzgerald, Gerald F; Van Sinderen, Douwe

2005-04-01

The claimed health benefits of fermented functional foods are expressed either directly through the interaction of ingested live microorganisms, bacteria or yeast with the host (probiotic effect) or indirectly as a result of ingestion of microbial metabolites produced during the fermentation process (biogenic effect). Although still far from fully understood, several probiotic mechanisms of action have been proposed, including competitive exclusion, competition for nutrients and/or stimulation of an immune response. The biogenic properties of fermented functional foods result from the microbial production of bioactive metabolites such as certain vitamins, bioactive peptides, organic acids or fatty acids during fermentation.

LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

PubMed Central

Lai, Yvonne; Adhikarakunnathu, Sreedevi; Bhardwaj, Kanchan; Ranjith-Kumar, C. T.; Wen, Yahong; Jordan, Jarrat L.; Wu, Linda H.; Dragnea, Bogdan; Mateo, Lani San; Kao, C. Cheng

2011-01-01

Background Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. Methodology/Principal Findings Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. Conclusions/Significance LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA. PMID:22039520

Antibacterial function of the human cathelicidin-18 peptide (LL-37) between theory and practice.

PubMed

Iacob, Simona A; Iacob, Diana G

2014-01-01

The human cathelicidin-18 is an antimicrobial, immunomodulatory and tissue repair peptide. The LL-37 fragment of this peptide which is in fact the active domain of the cathelicidin-18 is critical for the human antibacterial defense and epithelial integrity. It's activity against resistant pathogens, the potential of epithelial healing after microbial injury and the neutralization of bacterial endotoxin underlie the most important benefits of this peptide. However, there are still a number of questions that remain to be answered regarding the precise interactions of cathelicidin-18 within the immune system, the exact tissue concentrations or its possible pro-tumoral activity. In this respect, the therapeutic potential of cathelicidin-18 in various infections has been proved by in vitro experiments, but additional detailed clinical studies are still required to ascertain its antimicrobial role in vivo. We present a short review on the antibacterial activity of human cathelicidin-18 (LL-37) according to in vitro experiments while discussing its potential use in the clinical practice.

IL-10 inhibits while calcitriol reestablishes placental antimicrobial peptides gene expression.

PubMed

Olmos-Ortiz, Andrea; Noyola-Martínez, Nancy; Barrera, David; Zaga-Clavellina, Verónica; Avila, Euclides; Halhali, Ali; Biruete, Benjamín; Larrea, Fernando; Díaz, Lorenza

2015-04-01

IL-10 and calcitriol help to achieve a successful pregnancy by suppressing active maternal immunity; however, these factors exert opposite effects upon microbial infections. In the skin and immune cells, IL-10 downregulates β-defensins while calcitriol induces cathelicidin gene expression in various tissues including placenta. Though, the regulation of human placental β-defensins by IL-10 and calcitriol has not been studied. Therefore, we explored the regulation of these antimicrobial peptides expression in cultured placental cells by calcitriol and IL-10 alone and combined. Real time PCR showed that calcitriol stimulated, while IL-10 inhibited, β-defensins and cathelicidin gene expression (P<0.05). In coincubations studies, calcitriol was able to maintain antimicrobial peptides gene expression above control values, overriding IL-10 inhibitory effects. Calcitriol downregulated endogenous IL-10 secretion. Interestingly, calcitriol and TNF-α cooperatively enhanced β-defensins, while TNF-α reduced basal and calcitriol-stimulated cathelicidin gene expression. In summary, calcitriol and IL-10 exerted opposite effects on antimicrobial peptides expression in the human placenta, suggesting that unbalanced production of IL-10 and calcitriol could be deleterious to innate immune responses during gestation. Our results suggest that calcitriol enhancement of placental defenses involves two mechanisms: (1) downregulation of IL-10 secretion and (2) direct upregulation of β-defensins and cathelicidin gene expression. Considering that IL-10 and calcitriol differentially regulate the innate immune response in the placenta, in the case of an infection, calcitriol might restrict IL-10 permissive actions towards microbial invasion while restrains inflammation, allowing for pregnancy to continue in quiescence. These results strongly advice maternal vitamin D sufficiency during pregnancy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Trametes versicolor extract modifies human fecal microbiota composition in vitro.

PubMed

Yu, Zhuo-Teng; Liu, Bo; Mukherjee, Purna; Newburg, David S

2013-06-01

Trametes versicolor is a mushroom used as a traditional Chinese medicine (Yun-zhi) for a wide array of seemingly disparate conditions. We hypothesized that many of its multiple purported activities could be mediated through stimulation of beneficial mutualist components of the microbiota. Human fecal microbiota was cultured anaerobically to determine its ability to ferment a common extract of T. versicolor, designated polysaccharide peptide (PSP), and the ability of PSP to alter the composition of the microbial community. The presence of PSP and fructooligosaccharides (FOS, a common prebiotic) in the medium, but not cellulose, significantly increased levels of Bifidobacterium spp. PSP also elevated Lactobacillus spp., while reducing Clostridium spp., Staphylococcus spp. and Enterococcus spp. Levels of Streptococcus spp., Bacteroides spp. and Escherichia did not significantly change. Fermentation of PSP increased the concentration of organic acids (lactate and short-chain fatty acids), decreased the pH, and induced β-galactosidase and β-glucosidase activities. The genera of the human microbiota that are promoted by FOS and other prebiotics are also stimulated by the Trametes versicolor extract, PSP. Thus, Trametes versicolor, a common East Asian botanical, contains putative prebiotic agents that alter human gut microbiota and pH. This prebiotic-like activity may help explain some of the plethora of the health benefits attributed to this traditional Chinese medicine.

An amino acid composition criterion for membrane active antimicrobials

NASA Astrophysics Data System (ADS)

Schmidt, Nathan; Lai, Ghee Hwee; Mishra, Abhijit; Bong, Dennis; McCray, Paul, Jr.; Selsted, Michael; Ouellette, Andre; Wong, Gerard

2011-03-01

Membrane active antimicrobials (AMPs) are short amphipathic peptides with broad spectrum anti microbial activity. While it is believed that their hydrophobic and cationic moieties are responsible for membrane-based mechanisms of action, membrane disruption by AMPs is manifested in a diversity of outcomes, such as pore formation, blebbing, and budding. This complication, along with others, have made a detailed, molecular understanding of AMPs difficult. We use synchrotron small angle xray scattering to investigate the interaction of model bacterial and eukaryotic cell membranes with archetypes from beta-sheet AMPs (e.g. defensins) and alpha-helical AMPs (e.g. magainins). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane rearrangement and phase behavior induced by these different peptides we will discuss the importance of amino acid composition on AMP design.

Cellular responses to Mycobacterium avium, subsp. paratuberculosis in colostrum-deprived and colostrum-replete holstein calves supplemented with fat-soluble vitamins

USDA-ARS?s Scientific Manuscript database

Immune benefits of colostrum are attributed to passively transferred IgG but also to growth factors, cytokines, antimicrobial peptides, and leukocytes. Non-nutritive compounds in colostrum promote Th2-biased immune responses to early microbial encounters and prevent harmful, inappropriate inflammat...

Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications

PubMed Central

Solstad, Runar Gjerp; Li, Chun; Isaksson, Johan; Johansen, Jostein; Svenson, Johan; Stensvåg, Klara; Haug, Tor

2016-01-01

The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring. PMID:27007817

Treatment of Oral Multispecies Biofilms by an Anti-Biofilm Peptide.

PubMed

Wang, Zhejun; de la Fuente-Núñez, Cesar; Shen, Ya; Haapasalo, Markus; Hancock, Robert E W

2015-01-01

Human oral biofilms are multispecies microbial communities that exhibit high resistance to antimicrobial agents. Dental plaque gives rise to highly prevalent and costly biofilm-related oral infections, which lead to caries or other types of oral infections. We investigated the ability of the recently identified anti-biofilm peptide 1018 to induce killing of bacterial cells present within oral multispecies biofilms. At 10 μg/ml (6.5 μM), peptide 1018 was able to significantly (p<0.05) prevent biofilm formation over 3 days. The activity of the peptide on preformed biofilms was found to be concentration-dependent since more than 60% of the total plaque biofilm cell population was killed by 10 μg/ml of peptide 1018 in 3 days, while at 5 μg/ml 50% of cells were dead and at 1 μg/ml the peptide triggered cell death in around 30% of the total bacterial population, as revealed by confocal microscopy. The presence of saliva did not affect peptide activity, since no statistically significant difference was found in the ability of peptide 1018 to kill oral biofilms using either saliva coated and non-saliva coated hydroxyapatite surfaces. Scanning electron microscopy experiments indicated that peptide 1018 induced cell lysis in plaque biofilms. Furthermore, combined treatment using peptide 1018 and chlorhexidine (CHX) increased the anti-biofilm activity of each compound compared to when these were used alone, resulting in >50% of the biofilm being killed and >35% being dispersed in only 3 minutes. Peptide 1018 may potentially be used by itself or in combination with CHX as a non-toxic and effective anti-biofilm agent for plaque disinfection in clinical dentistry.

PH dependent adhesive peptides

DOEpatents

Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

2010-06-29

A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

Microbial pattern of pressure ulcer in pediatric patients

NASA Astrophysics Data System (ADS)

Paramita, D. A.; Khairina; Lubis, N. Z.

2018-03-01

Pressure ulcer (PU) is a localized trauma to the skin and or tissue beneath which lies in bony prominence due to pressure or pressure that combines with a sharp surface. Several studies have found that PU is a common problem in pediatrics population. Infection at the site of a PU is the most common complication in which the PU may host a resistant microorganism and may turn into a local infection that will be the source of bacteremia in hospitalized patients. To reveal which is the most common microbial species that underlie in pressure ulcer of pediatrics patients.A cross-sectional study was conducted in July-September 2017, involving 18 PU pediatric patients in Haji Adam Malik Hospital. To each subject, swab culture from the ulcer was madein microbial laboratory in Haji Adam Malik Hospital to determine the microbial pattern. This study found that the most common microbial pattern in pressure ulcers of pediatrics patient in Haji Adam Malik Hospital is Acinetobacter baumannii (22.2%).

A motif detection and classification method for peptide sequences using genetic programming.

PubMed

Tomita, Yasuyuki; Kato, Ryuji; Okochi, Mina; Honda, Hiroyuki

2008-08-01

An exploration of common rules (property motifs) in amino acid sequences has been required for the design of novel sequences and elucidation of the interactions between molecules controlled by the structural or physical environment. In the present study, we developed a new method to search property motifs that are common in peptide sequence data. Our method comprises the following two characteristics: (i) the automatic determination of the position and length of common property motifs by calculating the physicochemical similarity of amino acids, and (ii) the quick and effective exploration of motif candidates that discriminates the positives and negatives by the introduction of genetic programming (GP). Our method was evaluated by two types of model data sets. First, the intentionally buried property motifs were searched in the artificially derived peptide data containing intentionally buried property motifs. As a result, the expected property motifs were correctly extracted by our algorithm. Second, the peptide data that interact with MHC class II molecules were analyzed as one of the models of biologically active peptides with buried motifs in various lengths. Twofold MHC class II binding peptides were identified with the rule using our method, compared to the existing scoring matrix method. In conclusion, our GP based motif searching approach enabled to obtain knowledge of functional aspects of the peptides without any prior knowledge.

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by antimicrobial peptides (AMPs) and plant essential oils.

PubMed

Zouhir, Abdelmajid; Jridi, Taoufik; Nefzi, Adel; Ben Hamida, Jeannette; Sebei, Khaled

2016-12-01

Drug-resistant bacterial infections cause considerable patient mortality and morbidity. The annual frequency of deaths from methicillin-resistant Staphylococcus aureus (MRSA) has surpassed those caused by human immunodeficiency virus/acquired immune deficiency syndrome. The antimicrobial peptides (AMPs), plant essential oils (EOs) and their combinations have proven to be quite effective in killing a wide selection of bacterial pathogens including MRSA. This review summarizes the studies in the use of AMPs, plant EOs and their combinations for coping with MRSA bacteria, and to formulate new prospects for future studies on this topic. The sources of scientific literature such as PubMed, library search, Google Scholar, Science Direct and electronic databases such as 'The Antimicrobial Peptide Database', 'Collection of Anti-Microbial Peptides' and 'YADAMP'. Physicochemical data of anti-MRSA peptides were determined by Scientific DataBase Maker software. Of the 118 peptides, 88 exhibited an activity against MRSA with the highest activity of minimum inhibitory concentration values. Various plant EOs have been effective against MRSA. Remarkably, lemongrass EOs completely inhibited all MRSA growth on the plate. Lemon myrtle, Mountain savory, Cinnamon bark and Melissa EOs showed a significant inhibition. Several of these AMPs, EOs and their combinations were effective against MRSA. Their activities have implications for the development of new drugs for medical use.

Technical difficulties and solutions of direct transesterification process of microbial oil for biodiesel synthesis.

PubMed

Yousuf, Abu; Khan, Maksudur Rahman; Islam, M Amirul; Wahid, Zularisam Ab; Pirozzi, Domenico

2017-01-01

Microbial oils are considered as alternative to vegetable oils or animal fats as biodiesel feedstock. Microalgae and oleaginous yeast are the main candidates of microbial oil producers' community. However, biodiesel synthesis from these sources is associated with high cost and process complexity. The traditional transesterification method includes several steps such as biomass drying, cell disruption, oil extraction and solvent recovery. Therefore, direct transesterification or in situ transesterification, which combines all the steps in a single reactor, has been suggested to make the process cost effective. Nevertheless, the process is not applicable for large-scale biodiesel production having some difficulties such as high water content of biomass that makes the reaction rate slower and hurdles of cell disruption makes the efficiency of oil extraction lower. Additionally, it requires high heating energy in the solvent extraction and recovery stage. To resolve these difficulties, this review suggests the application of antimicrobial peptides and high electric fields to foster the microbial cell wall disruption.

Molecular recognition of microbial lipid-based antigens by T cells.

PubMed

Gras, Stephanie; Van Rhijn, Ildiko; Shahine, Adam; Le Nours, Jérôme

2018-05-01

The immune system has evolved to protect hosts from pathogens. T cells represent a critical component of the immune system by their engagement in host defence mechanisms against microbial infections. Our knowledge of the molecular recognition by T cells of pathogen-derived peptidic antigens that are presented by the major histocompatibility complex glycoproteins is now well established. However, lipids represent an additional, distinct chemical class of molecules that when presented by the family of CD1 antigen-presenting molecules can serve as antigens, and be recognized by specialized subsets of T cells leading to antigen-specific activation. Over the past decades, numerous CD1-presented self- and bacterial lipid-based antigens have been isolated and characterized. However, our understanding at the molecular level of T cell immunity to CD1 molecules presenting microbial lipid-based antigens is still largely unexplored. Here, we review the insights and the molecular basis underpinning the recognition of microbial lipid-based antigens by T cells.

Antimicrobial activity and interactions of cationic peptides derived from Galleria mellonella cecropin D-like peptide with model membranes.

PubMed

Oñate-Garzón, José; Manrique-Moreno, Marcela; Trier, Steven; Leidy, Chad; Torres, Rodrigo; Patiño, Edwin

2017-03-01

Antimicrobial peptides are effector molecules of the innate immune system against invading pathogens. The cationic charge in their structures has a strong correlation with antimicrobial activity, being responsible for the initial electrostatic interaction between peptides and the anionic microbial surface. This paper contains evidence that charge modification in the neutral peptide Gm cecropin D-like (WT) improved the antimicrobial activity of the modified peptides. Two cationic peptides derived from WT sequence named as ΔM1 and ΔM2, with net charge of +5 and +9, respectively, showed at least an eightfold increase in their antimicrobial activity in comparison to WT. The mechanism of action of these peptides was investigated using small unilamellar vesicles (SUVs) as model membranes. To study permeabilization effects of the peptides on cell membranes, entrapped calcein liposomes were used and the results showed that all peptides induced calcein release from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) SUVs, whereas in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), POPC/POPG and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG SUVs, only ΔM1 and ΔM2 induced a notable permeabilization. In addition, interactions of these peptides with phospholipids at the level of the glycerol backbone and hydrophobic domain were studied through observed changes in generalized polarization and fluorescence anisotropy using probes such as Laurdan and DPH, respectively. The results suggest that peptides slightly ordered the bilayer structure at the level of glycerol backbone and on the hydrophobic core in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) SUVs, whereas in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/DMPG SUVs, only ΔM1 and ΔM2 peptides increased the order of bilayers. Thus, peptides would be inducing clustering of phospholipids creating phospholipid domains with a higher phase transition temperature.

Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide

PubMed Central

Eckert, Randal ; He, Jian; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2006-01-01

Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora. PMID:17060534

Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells.

PubMed

Wang, Yufei; Whittall, Trevor; McGowan, Edward; Younson, Justine; Kelly, Charles; Bergmeier, Lesley A; Singh, Mahavir; Lehner, Thomas

2005-03-15

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

PubMed Central

2013-01-01

Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

PubMed

Pandey, Neeraj; Sachan, Annapurna; Chen, Qi; Ruebling-Jass, Kristin; Bhalla, Ritu; Panguluri, Kiran Kumar; Rouviere, Pierre E; Cheng, Qiong

2013-05-02

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

Neuropeptides, Microbiota, and Behavior.

PubMed

Holzer, P

2016-01-01

The gut microbiota and the brain interact with each other through multiple bidirectional signaling pathways in which neuropeptides and neuroactive peptide messengers play potentially important mediator roles. Currently, six particular modes of a neuropeptide link are emerging. (i) Neuropeptides and neurotransmitters contribute to the mutual microbiota-host interaction. (ii) The synthesis of neuroactive peptides is influenced by microbial control of the availability of amino acids. (iii) The activity of neuropeptides is tempered by microbiota-dependent autoantibodies. (iv) Peptide signaling between periphery and brain is modified by a regulatory action of the gut microbiota on the blood-brain barrier. (v) Within the brain, gut hormones released under the influence of the gut microbiota turn into neuropeptides that regulate multiple aspects of brain activity. (vi) Cerebral neuropeptides participate in the molecular, behavioral, and autonomic alterations which the brain undergoes in response to signals from the gut microbiota. © 2016 Elsevier Inc. All rights reserved.

Influence of common mucosal co-factors on HIV infection in the female genital tract.

PubMed

Ferreira, Victor H; Kafka, Jessica K; Kaushic, Charu

2014-06-01

Women constitute almost half of HIV-infected population globally, and the female genital tract (FGT) accounts for approximately 40% of all new HIV infections worldwide. The FGT is composed of upper and lower parts, distinct in their morphological and functional characteristics. Co-factors in the genital microenvironment, such as presence of hormones, semen, and other sexually transmitted infections, can facilitate or deter HIV infection and play a critical role in determining susceptibility to HIV. In this review, we examine some of these co-factors and their potential influence. Presence of physical and chemical barriers such as epithelial tight junctions, mucus, and anti-microbial peptides can actively block and inhibit viral replication, presenting a significant deterrent to HIV. Upon exposure, HIV and other pathogens first encounter the genital epithelium: cells that express a wide repertoire of pattern recognition receptors that can recognize and directly initiate innate immune responses. These and other interactions in the genital tract can lead to direct and indirect inflammation and enhance the number of local target cells, immune activation, and microbial translocation, all of which promote HIV infection and replication. Better understanding of the dynamics of HIV transmission in the female genital tract would be invaluable for improving the design of prophylactic strategies against HIV. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chemical and Physical Signatures for Microbial Forensics

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

Cliff, John B.; Kreuzer, Helen W.; Ehrhardt, Christopher J.

Chemical and physical signatures for microbial forensics John Cliff and Helen Kreuzer-Martin, eds. Humana Press Chapter 1. Introduction: Review of history and statement of need. Randy Murch, Virginia Tech Chapter 2. The Microbe: Structure, morphology, and physiology of the microbe as they relate to potential signatures of growth conditions. Joany Jackman, Johns Hopkins University Chapter 3. Science for Forensics: Special considerations for the forensic arena - quality control, sample integrity, etc. Mark Wilson (retired FBI): Western Carolina University Chapter 4. Physical signatures: Light and electron microscopy, atomic force microscopy, gravimetry etc. Joseph Michael, Sandia National Laboratory Chapter 5. Lipids: FAME,more » PLFA, steroids, LPS, etc. James Robertson, Federal Bureau of Investigation Chapter 6. Carbohydrates: Cell wall components, cytoplasm components, methods Alvin Fox, University of South Carolina School of Medicine David Wunschel, Pacific Northwest National Laboratory Chapter 7. Peptides: Peptides, proteins, lipoproteins David Wunschel, Pacific Northwest National Laboratory Chapter 8. Elemental content: CNOHPS (treated in passing), metals, prospective cell types John Cliff, International Atomic Energy Agency Chapter 9. Isotopic signatures: Stable isotopes C,N,H,O,S, 14C dating, potential for heavy elements. Helen Kreuzer-Martin, Pacific Northwest National Laboratory Michaele Kashgarian, Lawrence Livermore National Laboratory Chapter 10. Extracellular signatures: Cellular debris, heme, agar, headspace, spent media, etc Karen Wahl, Pacific Northwest National Laboratory Chapter 11. Data Reduction and Integrated Microbial Forensics: Statistical concepts, parametric and multivariate statistics, integrating signatures Kristin Jarman, Pacific Northwest National Laboratory« less

Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus.

PubMed

Grafskaia, Ekaterina N; Polina, Nadezhda F; Babenko, Vladislav V; Kharlampieva, Daria D; Bobrovsky, Pavel A; Manuvera, Valentin A; Farafonova, Tatyana E; Anikanov, Nikolay A; Lazarev, Vassili N

2018-04-01

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

The Neuroendocrine Peptide Catestatin Is a Cutaneous Antimicrobial and Induced in the Skin after Injury

PubMed Central

Radek, Katherine A.; Lopez-Garcia, Belen; Hupe, Melanie; Niesman, Ingrid R.; Elias, Peter M.; Taupenot, Laurent; Mahata, Sushil K.; O’Connor, Daniel T.; Gallo, Richard L.

2009-01-01

Epithelia establish a microbial barrier against infection through the production of antimicrobial peptides (AMPs). In this study, we investigated whether catestatin (Cst), a peptide derived from the neuroendocrine protein chromogranin A (CHGA), is a functional AMP and is present in the epidermis. We show that Cst is antimicrobial against relevant skin microbes, including Gram-positive and Gram-negative bacteria, yeast, and fungi. The antimicrobial mechanism of Cst was found to be similar to other AMPs, as it was dependent on bacterial charge and growth conditions, and induced membrane disruption. The potential relevance of Cst against skin pathogens was supported by the observation that CHGA was expressed in keratinocytes. In human skin, CHGA was found to be proteolytically processed into the antimicrobial fragment Cst, thus enabling its AMP function. Furthermore, Cst expression in murine skin increased in response to injury and infection, providing potential for increased protection against infection. These data demonstrate that a neuroendocrine peptide has antimicrobial function against a wide assortment of skin pathogens and is upregulated upon injury, thus demonstrating a direct link between the neuroendocrine and cutaneous immune systems. PMID:18185531

Human Antimicrobial Peptides and Proteins

PubMed Central

Wang, Guangshun

2014-01-01

As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs. PMID:24828484

Peptide Biomarkers as Evidence of Perchlorate Biodegradation▿ †

PubMed Central

Bansal, Reema; Crawford, Ronald L.; Paszczynski, Andrzej J.

2011-01-01

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways. PMID:21115710

Peptides with Dual Antimicrobial and Anticancer Activities

NASA Astrophysics Data System (ADS)

Felício, Mário R.; Silva, Osmar N.; Gonçalves, Sônia; Santos, Nuno C.; Franco, Octávio L.

2017-02-01

In recent years, the number of people suffering from cancer and multi-resistant infections has increased, such that both diseases are already seen as current and future major causes of death. Moreover, chronic infections are one of the main causes of cancer, due to the instability in the immune system that allows cancer cells to proliferate. Likewise, the physical debility associated with cancer or with anticancer therapy itself often paves the way for opportunistic infections. It is urgent to develop new therapeutic methods, with higher efficiency and lower side effects. Antimicrobial peptides (AMPs) are found in the innate immune system of a wide range of organisms. Identified as the most promising alternative to conventional molecules used nowadays against infections, some of them have been shown to have dual activity, both as antimicrobial and anticancer peptides (ACPs). Highly cationic and amphipathic, they have demonstrated efficacy against both conditions, with the number of nature-driven or synthetically designed peptides increasing year by year. With similar properties, AMPs that can also act as ACPs are viewed as future chemotherapeutic drugs, with the advantage of low propensity to resistance, which started this paradigm in the pharmaceutical market. These peptides have already been described as molecules presenting killing mechanisms at the membrane level, but also acting towards intracellular targets, which increases their success comparatively to specific one-target drugs. This review will approach the desirable characteristics of small peptides that demonstrated dual activity against microbial infections and cancer, as well as the peptides engaged in clinical trials.

Characterization of Three Venom Peptides from the Spitting Spider Scytodes thoracica

PubMed Central

Ariki, Nathanial K.; Muñoz, Lisa E.; Armitage, Elizabeth L.; Goodstein, Francesca R.; George, Kathryn G.; Smith, Vanessa L.; Vetter, Irina; Herzig, Volker; King, Glenn F.; Loening, Nikolaus M.

2016-01-01

We present the solution-state NMR structures and preliminary functional characterizations of three venom peptides identified from the spitting spider Scytodes thoracica. Despite little sequence identity to other venom peptides, structural characterization reveals that these peptides contain an inhibitor cystine knot motif common to many venom peptides. These are the first structures for any peptide or protein from spiders of the Scytodidae family. Many venom peptides target neuronal ion channels or receptors. However, we have not been able to determine the target of these Scytodes peptides so we can only state with certainty the channels and receptors that they do not target. PMID:27227898

Policing the intestinal epithelial barrier: Innate immune functions of intraepithelial lymphocytes.

PubMed

Hu, Madeleine D; Jia, Luo; Edelblum, Karen L

2018-03-01

This review will explore the contribution of IELs to mucosal innate immunity and highlight the similarities in IEL functional responses to bacteria, viruses and protozoan parasite invasion. IELs rapidly respond to microbial invasion by activating host defense responses, including the production of mucus and antimicrobial peptides to prevent microbes from reaching the epithelial surface. During active infection, IELs promote epithelial cytolysis, cytokine and chemokine production to limit pathogen invasion, replication and dissemination. Commensal-induced priming of IEL effector function or continuous surveillance of the epithelium may be important contributing factors to the rapidity of response. Impaired microbial recognition, dysregulated innate immune signaling or microbial dysbiosis may limit the protective function of IELs and increase susceptibility to disease. Further understanding of the mechanisms regulating IEL surveillance and sentinel function may provide insight into the development of more effective targeted therapies designed to reinforce the mucosal barrier.

Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

PubMed

Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V

2013-11-19

As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

Dendrimer-conjugated peptide vaccine enhances clearance of Chlamydia trachomatis genital infection.

PubMed

Ganda, Ingrid S; Zhong, Qian; Hali, Mirabela; Albuquerque, Ricardo L C; Padilha, Francine F; da Rocha, Sandro R P; Whittum-Hudson, Judith A

2017-07-15

Peptide-based vaccines have emerged in recent years as promising candidates in the prevention of infectious diseases. However, there are many challenges to maintaining in vivo peptide stability and enhancement of peptide immunogenicity to generate protective immunity which enhances clearance of infections. Here, a dendrimer-based carrier system is proposed for peptide-based vaccine delivery, and shows its anti-microbial feasibility in a mouse model of Chlamydia trachomatis. Chlamydiae are the most prevalent sexually transmitted bacteria worldwide, and also the causal agent of trachoma, the leading cause of preventable infectious blindness. In spite of the prevalence of this infectious agent and the many previous vaccine-related studies, there is no vaccine commercially available. The carrier system proposed consists of generation 4, hydroxyl-terminated, polyamidoamine (PAMAM) dendrimers (G4OH), to which a peptide mimic of a chlamydial glycolipid antigen-Peptide 4 (Pep4, AFPQFRSATLLL) was conjugated through an ester bond. The ester bond between G4OH and Pep4 is expected to break down mainly in the intracellular environment for antigen presentation. Pep4 conjugated to dendrimer induced Chlamydia-specific serum antibodies after subcutaneous immunizations. Further, this new vaccine formulation significantly protected immunized animals from vaginal challenge with infectious Chlamydia trachomatis, and it reduced infectious loads and tissue (genital tract) damage. Pep4 conjugated to G4OH or only mixed with peptide provided enhanced protection compared to Pep4 and adjuvant (i.e. alum), suggesting a potential adjuvant effect of the PAMAM dendrimer. Combined, these results demonstrate that hydroxyl-terminated PAMAM dendrimer is a promising polymeric nanocarrier platform for the delivery of peptide vaccines and this approach has potential to be expanded to other infectious intracellular bacteria and viruses of public health significance. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Himaya, S.W.A.; Dewapriya, Pradeep; Kim, Se-Kwon, E-mail: sknkim@pknu.ac.kr

Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclearmore » translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.« less

Nematode Peptides with Host-Directed Anti-inflammatory Activity Rescue Caenorhabditis elegans from a Burkholderia pseudomallei Infection.

PubMed

Lim, Mei-Perng; Firdaus-Raih, Mohd; Nathan, Sheila

2016-01-01

Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.

Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine.

PubMed

Chuan, Yap P; Rivera-Hernandez, Tania; Wibowo, Nani; Connors, Natalie K; Wu, Yang; Hughes, Fiona K; Lua, Linda H L; Middelberg, Anton P J

2013-09-01

Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response. These effects were studied in a mouse model and with modular MuPyV VLPs presenting a group A streptococcus (GAS) peptide antigen, J8i. The data presented here demonstrate that immunization with a modular VLP could induce high levels of J8i-specific antibodies despite a strong pre-existing anti-carrier immune response. Doubling of the J8i antigenic element number per VLP did not enhance J8i immunogenicity at a constant peptide dose. However, the strategy, when used in conjunction with increased VLP dose, could effectively increase the peptide dose up to 10-fold, leading to a significantly higher J8i-specific antibody titer. This study further supports feasibility of the MuPyV modular VLP vaccine platform by showing that, in the absence of adjuvant, modularized GAS antigenic peptide at a dose as low as 150 ng was sufficient to raise a high level of peptide-specific IgGs indicative of bactericidal activity. Copyright © 2013 Wiley Periodicals, Inc.

Fusion peptide P15-CSP shows antibiofilm activity and pro-osteogenic activity when deposited as a coating on hydrophilic but not hydrophobic surfaces.

PubMed

Li, Xian; Contreras-Garcia, Angel; LoVetri, Karen; Yakandawala, Nandadeva; Wertheimer, Michael R; De Crescenzo, Gregory; Hoemann, Caroline D

2015-12-01

In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces. © 2015 Wiley Periodicals, Inc.

Short antimicrobial peptides as cosmetic ingredients to deter dermatological pathogens.

PubMed

Rahnamaeian, Mohammad; Vilcinskas, Andreas

2015-11-01

Antimicrobial peptides (AMPs) are components of the innate immune system in many species of animals. Their diverse spectrum of activity against microbial pathogens, both as innate defense molecules and immunomodulators, makes them attractive candidates for the development of a new generation of antibiotics. Although the potential immunogenicity of AMPs means they are not suitable for injection and their susceptibility to digestive peptidases is likely to reduce their oral efficacy, they are ideal for topical formulations such as lotions, creams, shampoos, and wound dressings and could therefore be valuable products for the cosmetic industry. In this context, short AMPs (<20 amino acids) lacking disulfide bonds combine optimal antimicrobial activity with inexpensive chemical synthesis and are therefore more compatible with large-scale production and the modifications required to ensure stability, low toxicity, and microbial specificity. Proof-of-concept for the application of AMPs as novel anti-infectives has already been provided in clinical trials. This perspective considers the anti-infective properties of short AMPs lacking disulfide bonds, which are active against dermatologically important microflora. We consider the challenges that need to be addressed to facilitate the prophylactic application of AMPs in personal care products.

GAKG-RGEKG an Epitope That Provokes Immune Cross-Reactivity between Prevotella sp. and Human Collagen: Evidence of Molecular Mimicry in Chronic Periodontitis

PubMed Central

2016-01-01

Periodontal disease afflicts 20% of world population. This process usually occurs in the form of being lethargic and chronic, and consequently this disease is known as chronic process. All chronic diseases constantly cause activation of the immune system, and therefore the presentation of microbial peptides which are presented to lymphocytes by professional antigen presenting cells can present microbial peptides very similar to important structures of human economy causing autoimmune diseases, process known as molecular mimicry. Thus, the aim of this study was to verify the presence of molecular mimicry phenomenon between periodontopathogens and human proteins. Blasting microbes of Socransky periodontal complexes against human collagen were performed and then the proteins with similarities were modelled and were screened in the MHI binding virtual methods. The epitopes selected were produced and plasma of chronic periodontal volunteers was obtained and a dot immunobinding assay was performed. Hypothetical protein of Prevotella sp. and human collagen epitopes with high similarities were positive for dot immunobinding assay. With this result it can be suggested that the mimicry phenomena can occur on periodontal disease. PMID:28116146

cDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L.

PubMed

Jin, Fengliang; Sun, Qiang; Xu, Xiaoxia; Li, Linmiao; Gao, Gang; Xu, Yingjie; Yu, Xiaoqiang; Ren, Shunxiang

2012-10-01

Cecropins are linear cationic antibacterial peptides that have potent activities against microorganisms. In the present study, a 480bp full-length cDNA encoding diamondback moth (Plutella xylostella) cecropin 1 (designated as Px-cec1) was obtained using RT-PCR. A Northern blot analysis showed that the Px-cec1 transcript was predominantly expressed in fat bodies, hemocytes, midgut and epidermis with the highest expression level in fat bodies. The expression of Px-cec1 mRNA in fat bodies was significantly increased 24h after microbial challenge, with the highest induced expression by Staphylococcus aureus. A circular dichroism (CD) analysis revealed that the recombinant Px-cec1 mainly contained α-helixes. Antimicrobial assays demonstrated that recombinant Px-cec1 exhibited a broad spectrum of anti-microbial properties against fungi, Gram-positive and Gram-negative bacteria, but it did not exhibit hemolytic activity against human erythrocytes. Furthermore, Px-cec1 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy and transmission electron microscopy. These results demonstrated that Px-cec1 exerts its antibacterial activity by acting on the cell membrane to disrupt bacterial cell structures. Copyright © 2012 Elsevier Inc. All rights reserved.

Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae

PubMed Central

Schenk, Mirjam; Mahapatra, Sebabrata; Le, Phuonganh; Kim, Hee Jin; Choi, Aaron W.; Brennan, Patrick J.; Belisle, John T.

2016-01-01

The innate immune system recognizes microbial pathogens via pattern recognition receptors. One such receptor, NOD2, via recognition of muramyl dipeptide (MDP), triggers a distinct network of innate immune responses, including the production of interleukin-32 (IL-32), which leads to the differentiation of monocytes into dendritic cells (DC). NOD2 has been implicated in the pathogenesis of human leprosy, yet it is not clear whether Mycobacterium leprae, which has a distinct MDP structure, can activate this pathway. We investigated the effect of MDP structure on the innate immune response, finding that infection of monocytes with M. leprae induces IL-32 and DC differentiation in a NOD2-dependent manner. The presence of the proximal l-Ala instead of Gly in the common configuration of the peptide side chain of M. leprae did not affect recognition by NOD2 or cytokine production. Furthermore, amidation of the d-Glu residue did not alter NOD2 activation. These data provide experimental evidence that NOD2 recognizes naturally occurring structural variants of MDP. PMID:27297389

Antimicrobial and Attractant Roles for Chemerin in the Oral Cavity during Inflammatory Gum Disease.

PubMed

Godlewska, Urszula; Brzoza, Piotr; Sroka, Aneta; Majewski, Pawel; Jentsch, Holger; Eckert, Martin; Eick, Sigrun; Potempa, Jan; Zabel, Brian A; Cichy, Joanna

2017-01-01

Periodontal inflammation is one of the most common chronic inflammatory conditions in humans. Despite recent advances in identifying and characterizing oral microbiota dysbiosis in the pathogenesis of gum disease, just how host factors maintain a healthy homeostatic oral microbial community or prevent the development of a pathogenic oral microbiota remains poorly understood. An important determinant of microbiota fate is local antimicrobial proteins. Here, we report that chemoattractant protein chemerin, which we recently identified as a potent endogenous antimicrobial agent in body barriers such as the skin, is present in the oral cavity under homeostatic and inflammatory conditions. Chemerin and a chemerin-derived antimicrobial peptide are bactericidal against select bacteria strategically positioned in dental biofilm. Gingival crevicular samples from patients with gingivitis but not periodontitis contain abundant bioactive chemerin capable of inducing CMKLR1-dependent leukocyte migration. Gingipains secreted by the periodontopathogen P. gingivalis inactivate chemerin. Together, these data suggest that as an antimicrobial agent and leukocyte chemoattractant, chemerin likely contributes to antimicrobial immune defense in the oral cavity.

Revisiting the arthritogenic peptide theory: quantitative not qualitative changes in the peptide repertoire of HLA-B27 allotypes.

PubMed

Schittenhelm, Ralf B; Sian, Terry C C Lim Kam; Wilmann, Pascal G; Dudek, Nadine L; Purcell, Anthony W

2015-03-01

The association of HLA-B27 with spondyloarthropathy is one of the strongest documented for any autoimmune disease. A common hypothesis for this association is the arthritogenic peptide concept. This dictates that differences in the peptide binding preferences of disease-associated and non-disease-associated HLA-B27 allotypes underlie the presentation of bacterial and self-peptides, leading to cross-reactive T cell immunity and subsequent autoimmune attack of affected tissues. The aim of this study was to analyze and compare self-peptides from 8 HLA-B27 allotypes, to increase existing data sets of HLA-B27 ligands, to refine and compare their consensus-binding motifs, and to reveal similarities and differences in the peptide repertoire of the HLA-B27 subtypes. Qualitative differences in the peptides bound to the 8 most frequent HLA-B27 subtypes were determined by tandem mass spectrometry, and quantitative changes in allelic binding specificities were determined by highly sensitive and targeted multiple reaction monitoring mass spectrometry. We identified >7,500 major histocompatibility complex class I peptides derived from the 8 most common HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09). We describe individual binding motifs for these alleles for the 9-12-mer ligands. The peptide repertoires of these closely related alleles showed significant overlap. Allelic polymorphisms resulting in changes in the amino acid composition of the antigen-binding cleft manifested largely as quantitative changes in the peptide cargo of these molecules. Absolute binding preferences of HLA-B27 allotypes do not explain disease association. The arthritogenic peptide theory needs to be reassessed in terms of quantitative changes in self-peptide presentation, T cell selection, and altered conformation of bound peptides. Copyright © 2015 by the American College of Rheumatology.

Co-ordination of incoming and outgoing traffic in antigen-presenting cells by pattern recognition receptors and T cells.

PubMed

Nair, Priyanka; Amsen, Derk; Blander, J Magarian

2011-12-01

Dendritic cells are innate sentinels of the immune system and potent activators of naÏve T cells. Mechanisms must exist to enable these cells to achieve maximal activation of T cells specific for microbial antigens, while avoiding activation of T cells specific for self-antigens. Here we discuss how a combination of signals from pattern recognition receptors and T cells co-ordinates subcellular trafficking of antigen with both major histocompatibility complex class I and class II molecules and T-cell costimulatory molecules, resulting in the preferential presentation of microbial peptides within a stimulatory context. © 2011 John Wiley & Sons A/S.

Microbial incorporation of nitrogen in stream detritus

Treesearch

Diane M. Sanzone; Jennifer L. Tank; Judy L. Meyer; Patrick J. Mulholland; Stuart E.G. Findlay

2001-01-01

We adapted the chloroform fumigation method to determine microbial nitrogen (N) and microbial incorporation of 15N on three common substrates [leaves, wood and fine benthic organic matter (FBOM)] in three forest streams. We compared microbial N and 15 content of samples collected during a 6-week15N-NH...

Modulation of Neutrophil Apoptosis by Antimicrobial Peptides

PubMed Central

Nagaoka, Isao; Suzuki, Kaori; Niyonsaba, François; Tamura, Hiroshi; Hirata, Michimasa

2012-01-01

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Human antimicrobial peptides, α-defensins (human neutrophil peptides, HNPs), human β-defensins (hBDs), and cathelicidin (LL-37) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also function as immunomodulatory molecules by inducing cytokine and chemokine production, and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, we provided the evidence that HNP-1, hBD-3, and LL-37 cannot only destroy bacteria but also potently modulate (suppress) neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/-2, the downregulation of tBid (an proapoptotic protein) and upregulation of Bcl-xL (an antiapoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity, possibly via the actions on the distinct receptors, the P2Y6 nucleotide receptor, the chemokine receptor CCR6, and the low-affinity formyl-peptide receptor FPRL1/the nucleotide receptor P2X7, respectively. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion. PMID:23724322

Impact of commercial precooking of common bean (Phaseolus vulgaris) on the generation of peptides, after pepsin-pancreatin hydrolysis, capable to inhibit dipeptidyl peptidase-IV.

PubMed

Mojica, Luis; Chen, Karen; de Mejía, Elvira González

2015-01-01

The objective of this research was to determine the bioactive properties of the released peptides from commercially available precook common beans (Phaseolus vulgaris). Bioactive properties and peptide profiles were evaluated in protein hydrolysates of raw and commercially precooked common beans. Five varieties (Black, Pinto, Red, Navy, and Great Northern) were selected for protein extraction, protein and peptide molecular mass profiles, and peptide sequences. Potential bioactivities of hydrolysates, including antioxidant capacity and inhibition of α-amylase, α-glucosidase, dipeptidyl peptidase-IV (DPP-IV), and angiotensin converting enzyme I (ACE) were analyzed after digestion with pepsin/pancreatin. Hydrolysates from Navy beans were the most potent inhibitors of DPP-IV with no statistical differences between precooked and raw (IC50 = 0.093 and 0.095 mg protein/mL, respectively). α-Amylase inhibition was higher for raw Red, Navy and Great Northern beans (36%, 31%, 27% relative to acarbose (rel ac)/mg protein, respectively). α-Glucosidase inhibition among all bean hydrolysates did not show significant differences; however, inhibition values were above 40% rel ac/mg protein. IC50 values for ACE were not significantly different among all bean hydrolysates (range 0.20 to 0.34 mg protein/mL), except for Red bean that presented higher IC50 values. Peptide molecular mass profile ranged from 500 to 3000 Da. A total of 11 and 17 biologically active peptide sequences were identified in raw and precooked beans, respectively. Peptide sequences YAGGS and YAAGS from raw Great Northern and precooked Pinto showed similar amino acid sequences and same potential ACE inhibition activity. Processing did not affect the bioactive properties of released peptides from precooked beans. Commercially precooked beans could contribute to the intake of bioactive peptides and promote health. © 2014 Institute of Food Technologists®

Bombesin related peptides/receptors and their promising therapeutic roles in cancer imaging, targeting and treatment

PubMed Central

Moreno, Paola; Ramos-Álvarez, Irene; Moody, Terry W.; Jensen, Robert T.

2016-01-01

Introduction Despite remarkable advances in tumor treatment, many patients still die from common tumors (breast, prostate, lung, CNS, colon, and pancreas), and thus, new approaches are needed. Many of these tumors synthesize bombesin (Bn)-related peptides and over-express their receptors (BnRs), hence functioning as autocrine-growth-factors. Recent studies support the conclusion that Bn-peptides/BnRs are well-positioned for numerous novel antitumor treatments, including interrupting autocrine-growth via the use of over-expressed receptors for imaging and targeting cytotoxic-compounds, either by direct-coupling or combined with nanoparticle-technology. Areas covered The unique ability of common neoplasms to synthesize, secrete, and show a growth/proliferative/differentiating response due to BnR over-expression, is reviewed, both in general and with regard to the most frequently investigated neoplasms (breast, prostate, lung, and CNS). Particular attention is paid to advances in the recent years. Also considered are the possible therapeutic approaches to the growth/differentiation effect of Bn-peptides, as well as the therapeutic implication of the frequent BnR over-expression for tumor-imaging and/or targeted-delivery. Expert opinion Given that Bn-related-peptides/BnRs are so frequently ectopically-expressed by common tumors, which are often malignant and become refractory to conventional treatments, therapeutic interventions using novel approaches to Bn-peptides and receptors are being explored. Of particular interest is the potential of reproducing BnRs in common tumors, such as the recent success of utilizing overexpression of somatostatin-receptors by neuroendocrine-tumors to provide the most sensitive imaging methods and targeted delivery of cytotoxic-compounds. PMID:26981612

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

PubMed Central

Di Grazia, Antonio; Luca, Vincenzo; Segev-Zarko, Li-av T.; Shai, Yechiel

2014-01-01

The growing number of microbial pathogens resistant to available antibiotics is a serious threat to human life. Among them is the bacterium Staphylococcus aureus, which colonizes keratinocytes, the most abundant cell type in the epidermis. Its intracellular accumulation complicates treatments against resulting infections, mainly due to the limited diffusion of conventional drugs into the cells. Temporins A (Ta) and B (Tb) are short frog skin antimicrobial peptides (AMPs). Despite extensive studies regarding their antimicrobial activity, very little is known about their activity on infected cells or involvement in various immunomodulatory functions. Here we show that Tb kills both ATCC-derived and multidrug-resistant clinical isolates of S. aureus within infected HaCaT keratinocytes (80% and 40% bacterial mortality, respectively) at a nontoxic concentration, i.e., 16 μM, whereas a weaker effect is displayed by Ta. Furthermore, the peptides prevent killing of keratinocytes by the invading bacteria. Further studies revealed that both temporins promote wound healing in a monolayer of HaCaT cells, with front speed migrations of 19 μm/h and 12 μm/h for Ta and Tb, respectively. Migration is inhibited by mitomycin C and involves the epidermal growth factor receptor (EGFR) signaling pathway. Finally, confocal fluorescence microscopy indicated that the peptides diffuse into the cells. By combining antibacterial and wound-healing activities, Ta and Tb may act as multifunctional mediators of innate immunity in humans. Particularly, their nonendogenous origin may reduce microbial resistance to them as well as the risk of autoimmune diseases in mammals. PMID:24514087

Methodological flaws introduce strong bias into molecular analysis of microbial populations.

PubMed

Krakat, N; Anjum, R; Demirel, B; Schröder, P

2017-02-01

In this study, we report how different cell disruption methods, PCR primers and in silico analyses can seriously bias results from microbial population studies, with consequences for the credibility and reproducibility of the findings. Our results emphasize the pitfalls of commonly used experimental methods that can seriously weaken the interpretation of results. Four different cell lysis methods, three commonly used primer pairs and various computer-based analyses were applied to investigate the microbial diversity of a fermentation sample composed of chicken dung. The fault-prone, but still frequently used, amplified rRNA gene restriction analysis was chosen to identify common weaknesses. In contrast to other studies, we focused on the complete analytical process, from cell disruption to in silico analysis, and identified potential error rates. This identified a wide disagreement of results between applied experimental approaches leading to very different community structures depending on the chosen approach. The interpretation of microbial diversity data remains a challenge. In order to accurately investigate the taxonomic diversity and structure of prokaryotic communities, we suggest a multi-level approach combining DNA-based and DNA-independent techniques. The identified weaknesses of commonly used methods to study microbial diversity can be overcome by a multi-level approach, which produces more reliable data about the fate and behaviour of microbial communities of engineered habitats such as biogas plants, so that the best performance can be ensured. © 2016 The Society for Applied Microbiology.

Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y.; Drake, Steven K.; Gucek, Marjan; Sacks, David B.; Yu, Yi-Kuo

2018-06-01

Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry.

PubMed

Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y; Drake, Steven K; Gucek, Marjan; Sacks, David B; Yu, Yi-Kuo

2018-06-05

Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html . Graphical Abstract ᅟ.

Antibiotic and synergistic effect of Leu-Lys rich peptide against antibiotic resistant microorganisms isolated from patients with cholelithiasis.

PubMed

Jeong, Nari; Kim, Jin-Young; Park, Seong-Cheol; Lee, Jong-Kook; Gopal, Ramamourthy; Yoo, Suyeon; Son, Byoung Kwan; Hahm, Joon Soo; Park, Yoonkyung; Hahm, Kyung-Soo

2010-09-03

Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5xMIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria. Copyright 2010 Elsevier Inc. All rights reserved.

Antimicrobial Peptides: A Promising Therapeutic Strategy in Tackling Antimicrobial Resistance.

PubMed

Nuti, Ramya; Goud, Nerella S; Saraswati, A Prasanth; Alvala, Ravi; Alvala, Mallika

2017-01-01

Antimicrobial resistance (AMR) has posed a serious threat to global public health and it requires immediate action, preferably long term. Current drug therapies have failed to curb this menace due to the ability of microbes to circumvent the mechanisms through which the drugs act. From the drug discovery point of view, the majority of drugs currently employed for antimicrobial therapy are small molecules. Recent trends reveal a surge in the use of peptides as drug candidates as they offer remarkable advantages over small molecules. Newer synthetic strategies like organometalic complexes, Peptide-polymer conjugates, solid phase, liquid phase and recombinant DNA technology encouraging the use of peptides as therapeutic agents with a host of chemical functions, and tailored for specific applications. In the last decade, many peptide based drugs have been successfully approved by the Food and Drug Administration (FDA). This success can be attributed to their high specificity, selectivity and efficacy, high penetrability into the tissues, less immunogenicity and less tissue accumulation. Considering the enormity of AMR, the use of Antimicrobial Peptides (AMPs) can be a viable alternative to current therapeutics strategies. AMPs are naturally abundant allowing synthetic chemists to develop semi-synthetics peptide molecules. AMPs have a broad spectrum of activity towards microbes and they possess the ability to bypass the resistance induction mechanisms of microbes. The present review focuses on the potential applications of AMPs against various microbial disorders and their future prospects. Several resistance mechanisms and their strategies have also been discussed to highlight the importance in the current scenario. Breakthroughs in AMP designing, peptide synthesis and biotechnology have shown promise in tackling this challenge and has revived the interest of using AMPs as an important weapon in fighting AMR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Novel RNase 3/ECP Peptide for Pseudomonas aeruginosa Biofilm Eradication That Combines Antimicrobial, Lipopolysaccharide Binding, and Cell-Agglutinating Activities

PubMed Central

Prats-Ejarque, Guillem; Villalba, Clara; Albacar, Marcel; González-López, Juan J.; Torrent, Marc; Moussaoui, Mohammed

2016-01-01

Eradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms. PMID:27527084

Binding, folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing.

PubMed

Reid, Keon A; Davis, Caitlin M; Dyer, R Brian; Kindt, James T

2018-03-01

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV d P l PTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a "flip and dip" mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers. Copyright © 2017 Elsevier B.V. All rights reserved.

Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5.

PubMed

Suyama, Keitaro; Taniguchi, Suguru; Tatsubo, Daiki; Maeda, Iori; Nose, Takeru

2016-04-01

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n>40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Microcins from Enterobacteria: On the Edge Between Gram-Positive Bacteriocins and Colicins

NASA Astrophysics Data System (ADS)

Rebuffat, Sylvie

Most bacteria and archaea produce gene-encoded antimicrobial peptides/proteins called bacteriocins, which are secreted by the producing bacteria to compete against other microorganisms in a given niche. They are considered important mediators of intra- and interspecies interactions and therefore a factor in ­maintaining the microbial diversity and stability. They are ribosomally synthesized, and most of them are produced as inactive precursor proteins, which in some cases are further enzymatically modified. Bacteriocins generally exert potent antibacterial activities directed against bacterial species closely related to the producing bacteria. Bacteriocins are abundant and diverse in Gram-negative and Gram-positive bacteria. This chapter focuses on colicins and microcins from enterobacteria (mainly Escherichia coli) and on bacteriocins from lactic acid bacteria (LAB). Microcins are the lower-molecular-mass bacteriocins produced by Gram-negative bacteria with a repertoire of only 14 representatives. They form a very restricted family of bacteriocins, compared to the huge family of LAB bacteriocins that is constituted of several hundreds of peptides, with which microcins share common characteristics. Nevertheless, microcins also show similarities, particularly in their uptake mechanisms, with the higher-molecular-mass colicins, also produced by E. coli strains. On the edge between LAB bacteriocins and colicins, microcins appear to combine highly efficient strategies developed by both Gram-positive and Gram-negative bacteria at different levels, including uptake, translocation, killing of target cells, and immunity of the producing bacteria, making them important actors of bacterial competitions and fascinating models for novel concepts toward antimicrobial strategies and against resistance mechanisms.

Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy.

PubMed

Chen, Wentao; Dong, Jiajia; Li, Suhua; Liu, Yu; Wang, Yujia; Yoon, Leonard; Wu, Peng; Sharpless, K Barry; Kelly, Jeffery W

2016-01-26

Tyrosine O-sulfation is a common protein post-translational modification that regulates many biological processes, including leukocyte adhesion and chemotaxis. Many peptides with therapeutic potential contain one or more sulfotyrosine residues. We report a one-step synthesis for Fmoc-fluorosulfated tyrosine. An efficient Fmoc-based solid-phase peptide synthetic strategy is then introduced for incorporating the fluorosulfated tyrosine residue into peptides of interest. Standard simultaneous peptide-resin cleavage and removal of the acid-labile side-chain protecting groups affords the crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol, serving both as solvent and reactant, transforms the fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antimicrobial preservative use in parenteral products: past and present.

PubMed

Meyer, Brian K; Ni, Alex; Hu, Binghua; Shi, Li

2007-12-01

The following review provides a comprehensive summary of antimicrobial preservatives that are commonly used in licensed parenteral products to date. The information reviewed includes the general properties of the preservatives, the doses and frequency of their use, the classes of the preserved products (peptide, protein, vaccine, and small molecule products), the interactions with other formulation components, and the criteria commonly used for their selection in parental product formulations. It was revealed that phenol and benzyl alcohol are the two most common antimicrobial preservatives used in peptide and protein products, while phenoxyethanol is the most frequently used preservative in vaccines. Benzyl alcohol or a combination of methylparaben and propylparaben are generally found in small molecule parenteral formulations. The key criteria for antimicrobial preservative selection are the preservative's dose, antimicrobial functionality, and effect on the active ingredient. Additionally, the use of spectroscopic techniques (circular dicroism (CD) and fluorescence) and differential scanning calorimetry (DSC) were identified as common techniques used in evaluating an antimicrobial preservative for its impact on the conformational stability of peptide, protein, and vaccine antigens. The future use of preservatives is also discussed, including antimicrobial agents such as peptides, and regulatory requirements for antimicrobial effectiveness testing. (c) 2007 Wiley-Liss, Inc.

The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective.

PubMed

Mousa, Walaa Kamel; Raizada, Manish N

2013-01-01

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides, and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g., Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens.

The Diversity of Anti-Microbial Secondary Metabolites Produced by Fungal Endophytes: An Interdisciplinary Perspective

PubMed Central

Mousa, Walaa Kamel; Raizada, Manish N.

2013-01-01

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides, and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g., Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens. PMID:23543048

Synthetic fermentation of bioactive non-ribosomal peptides without organisms, enzymes or reagents

NASA Astrophysics Data System (ADS)

Huang, Yi-Lin; Bode, Jeffrey W.

2014-10-01

Microbial fermentation can rapidly provide potent compounds that can be easily screened for biological activity, and the active components can be isolated. Its success in drug discovery has inspired extensive efforts to modulate and control the products. In this Article, we document a ‘synthetic fermentation’ of bioactive, unnatural peptides ‘grown’ from small building blocks in water using amide-forming ligations. No organisms, enzymes or reagents are needed. The sequences, structures and compositions of the products can be modulated by adjusting the building blocks and conditions. No specialized knowledge of organic chemistry or handling of toxic material is required to produce complex organic molecules. The ‘fermentations’ can be conducted in arrays and screened for biological activity without isolation or workup. As a proof-of-concept, about 6,000 unnatural peptides were produced from just 23 building blocks, from which a hepatitis C virus NS3/4A protease inhibitor with a half-maximum inhibitory concentration of 1.0 μM was identified and characterized.

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

PubMed

O'Brien-Simpson, Neil M; Pantarat, Namfon; Attard, Troy J; Walsh, Katrina A; Reynolds, Eric C

2016-01-01

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Paleoproteomics of the Dental Pulp: The plague paradigm.

PubMed

Barbieri, Rémi; Mekni, Rania; Levasseur, Anthony; Chabrière, Eric; Signoli, Michel; Tzortzis, Stéfan; Aboudharam, Gérard; Drancourt, Michel

2017-01-01

Chemical decomposition and fragmentation may limit the detection of ancient host and microbial DNA while some proteins can be detected for extended periods of time. We applied paleoproteomics on 300-year-old dental pulp specimens recovered from 16 individuals in two archeological funeral sites in France, comprising one documented plague site and one documented plague-negative site. The dental pulp paleoproteome of the 16 teeth comprised 439 peptides representative of 30 proteins of human origin and 211 peptides representative of 27 proteins of non-human origin. Human proteins consisted of conjunctive tissue and blood proteins including IgA immunoglobulins. Four peptides were indicative of three presumable Yersinia pestis proteins detected in 3/8 dental pulp specimens from the plague-positive site but not in the eight dental pulp specimens collected in the plague-negative site. Paleoproteomics applied to the dental pulp is a new and innovative approach to screen ancient individuals for the detection of blood-borne pathogens and host inflammatory response.

Protein-based stable isotope probing.

PubMed

Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

2010-12-01

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

Identification of a candidate therapeutic autophagy–inducing peptide

PubMed Central

Shoji-Kawata, Sanae; Sumpter, Rhea; Leveno, Matthew; Campbell, Grant R.; Zou, Zhongju; Kinch, Lisa; Wilkins, Angela D.; Sun, Qihua; Pallauf, Kathrin; MacDuff, Donna; Huerta, Carlos; Virgin, Herbert W.; Helms, J. Bernd; Eerland, Ruud; Tooze, Sharon A.; Xavier, Ramnik; Lenschow, Deborah J.; Yamamoto, Ai; King, David; Lichtarge, Olivier; Grishin, Nick V.; Spector, Stephen A.; Kaloyanova, Dora V.; Levine, Beth

2013-01-01

The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat–beclin 1—derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef—is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat–beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases. PMID:23364696

A polyalanine peptide derived from polar fish with anti-infectious activities

NASA Astrophysics Data System (ADS)

Cardoso, Marlon H.; Ribeiro, Suzana M.; Nolasco, Diego O.; de La Fuente-Núñez, César; Felício, Mário R.; Gonçalves, Sónia; Matos, Carolina O.; Liao, Luciano M.; Santos, Nuno C.; Hancock, Robert E. W.; Franco, Octávio L.; Migliolo, Ludovico

2016-02-01

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Increased survival of experimentally evolved antimicrobial peptide-resistant Staphylococcus aureus in an animal host

PubMed Central

Dobson, Adam J; Purves, Joanne; Rolff, Jens

2014-01-01

Antimicrobial peptides (AMPs) have been proposed as new class of antimicrobial drugs, following the increasing prevalence of bacteria resistant to antibiotics. Synthetic AMPs are functional analogues of highly evolutionarily conserved immune effectors in animals and plants, produced in response to microbial infection. Therefore, the proposed therapeutic use of AMPs bears the risk of ‘arming the enemy’: bacteria that evolve resistance to AMPs may be cross-resistant to immune effectors (AMPs) in their hosts. We used a panel of populations of Staphylococcus aureus that were experimentally selected for resistance to a suite of individual AMPs and antibiotics to investigate the ‘arming the enemy’ hypothesis. We tested whether the selected strains showed higher survival in an insect model (Tenebrio molitor) and cross-resistance against other antimicrobials in vitro. A population selected for resistance to the antimicrobial peptide iseganan showed increased in vivo survival, but was not more virulent. We suggest that increased survival of AMP-resistant bacteria almost certainly poses problems to immune-compromised hosts. PMID:25469169

Preparation and characterization of aminoethyl hydroxypropyl starch modified with collagen peptide.

PubMed

Wen, Huigao; Hu, Jin; Ge, Hongyu; Zou, Shengqiong; Xiao, Yao; Li, Ya; Feng, Han; Fan, Lihong

2017-08-01

The preparation of aminoethyl hydroxypropyl starch collagen peptide (AEHPS-COP) was via an enzyme-catalyzed reaction between amino groups in aminoethyl hydroxypropyl starch (AEHPS) and γ-carboxamide groups in collagen peptide (COP) by using microbial transglutaminase (MTGase) as biocatalyst. As an intermediate reactant, AEHPS was synthesized from hydroxypropyl starch (HPS) and 2-chloroethylamine hydrochloride (CEH). The chemical structures of HPS, AEHPS and AEHPS-COP were characterized by Fourier transform infrared spectroscopy (FT-IR) and 13 C nuclear magnetic resonance ( 13 C NMR). The reaction conditions that influenced the degree of substitution (DS) of AEHPS-COP were optimized, which included the reaction temperature, the reaction time, the mass ratio of collagen peptide to aminoethyl hydroxypropyl starch and the pH value. In addition, in vitro antioxidant activities of AEHPS-COP were evaluated through the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Furthermore, the methylthiazol tetrazolium (MTT) assay was applied to investigate the cell viability of AEHPS-COP. The results indicated that the AEHPS-COP exhibited better cell viability to L929 mouse fibroblast cells. Therefore, the AEHPS-COP showed a promising potential application in cosmetic, biomedical and pharmaceutical fields. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.).

PubMed

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.)

PubMed Central

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens. PMID:28056070

Method Analysis of Microbial Resistant Gypsum Products

EPA Science Inventory

Abstract: Several commercially available gypsum products are marketed as microbial-resistant. During previous test method research on a microbial resistant gypsum wallboard study, a common theme from both stakeholders and product vendors was the need for a unified and accepted m...

Understanding the microbiome of diabetic foot osteomyelitis: insights from molecular and microscopic approaches.

PubMed

Johani, K; Fritz, B G; Bjarnsholt, T; Lipsky, B A; Jensen, S O; Yang, M; Dean, A; Hu, H; Vickery, K; Malone, M

2018-05-19

Rigorous visual evidence on whether or not biofilms are involved in diabetic foot osteomyelitis (DFO) is lacking. We employed a suite of molecular and microscopic approaches to investigate the microbiome, and phenotypic state of microorganisms involved in DFO. In 20 consecutive subjects with suspected DFO, we collected intraoperative bone specimens. To explore the microbial diversity present in infected bone we performed next generation DNA sequencing. We used scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) with confocal microscopy to visualize and confirm the presence of biofilms. In 19 of 20 (95%) studied patients presenting with DFO, it was associated with an infected diabetic foot ulcer. By DNA sequencing of infected bone, Corynebacterium sp. was the most commonly identified microorganism, followed by Finegoldia sp., Staphylococcus sp., Streptococcus sp., Porphyromonas sp., and Anaerococcus sp. Six of 20 bone samples (30%) contained only one or two pathogens, while the remaining 14 (70%) had polymicrobial communities. Using a combination of SEM and PNA-FISH, we identified microbial aggregates in biofilms in 16 (80%) bone specimens and found that they were typically coccoid or rod-shaped aggregates. The presence of biofilms in DFO may explain why non-surgical treatment of DFO, relying on systemic antibiotic therapy, may not resolve some chronic infections caused by biofilm-producing strains. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

PubMed

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potential candidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research. Creative Commons Attribution License

Metaproteomics of Microbiota in Naturally Fermented Soybean Paste, Da-jiang.

PubMed

Zhang, Ping; Zhang, Pengfei; Xie, Mengxi; An, Feiyu; Qiu, Boshu; Wu, Rina

2018-05-01

Da-jiang is a typical traditional fermented soybean product in China. At present, the proteins in da-jiang are needed to be explored. The composition and species of microbial proteins in traditional fermented da-jiang were analyzed by metaproteomics based on sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results showed that the number and variety of microbial proteins in the traditional fermented da-jiang from different regions were different. The production site influences the fermentation in da-jiang. Then we analyzed the functions of the microbial proteins identified in da-jiang, and found that they were mainly involved in the process of protein synthesis, glycometabolism and nucleic acid synthesis. In addtion, we compared the proteins composition in different da-jiang. There are 51 common proteins of naturally fermented da-jiang, and 25 common microbial sources. The main commonly microbial sources of fungal proteins are Saccharomyces cerevisiae and Schizosaccharomyces; the main commonly microbial sources of bacterial proteins are Enterococcus faecalis, Leuconostoc mesenteroides, Acinetobacter baumannii, and Bacillus subtilis. These common microbes play the predominant role in da-jiang fermentation. The present results help us to understand the fermentation of da-jiang and improve the quality and safety of final products in the future. The study illustrated metaproteome of microbiota in traditional fermented soybean paste, da-jiang, by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A method of extracting metaproteome from microbiota in da-jiang was attempted. The findings help to understand the fermentation of da-jiang and improve the quality and safety of da-jiang in fermented industry. © 2018 Institute of Food Technologists®.

The C-terminal sequence of several human serine proteases encodes host defense functions.

PubMed

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Walse, Björn; Svensson, Bo; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2011-01-01

Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest. Copyright © 2011 S. Karger AG, Basel.

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae).

PubMed

Conlon, J Michael; Mechkarska, Milena; Kolodziejek, Jolanta; Leprince, Jérôme; Coquet, Laurent; Jouenne, Thierry; Vaudry, Hubert; Nowotny, Norbert; King, Jay D

2015-10-01

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

PubMed

Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

2016-03-15

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Receptor Kinases in Plant-Pathogen Interactions: More Than Pattern Recognition[OPEN

PubMed Central

2017-01-01

Receptor-like kinases (RLKs) and Receptor-like proteins (RLPs) play crucial roles in plant immunity, growth, and development. Plants deploy a large number of RLKs and RLPs as pattern recognition receptors (PRRs) that detect microbe- and host-derived molecular patterns as the first layer of inducible defense. Recent advances have uncovered novel PRRs, their corresponding ligands, and mechanisms underlying PRR activation and signaling. In general, PRRs associate with other RLKs and function as part of multiprotein immune complexes at the cell surface. Innovative strategies have emerged for the rapid identification of microbial patterns and their cognate PRRs. Successful pathogens can evade or block host recognition by secreting effector proteins to “hide” microbial patterns or inhibit PRR-mediated signaling. Furthermore, newly identified pathogen effectors have been shown to manipulate RLKs controlling growth and development by mimicking peptide hormones of host plants. The ongoing studies illustrate the importance of diverse plant RLKs in plant disease resistance and microbial pathogenesis. PMID:28302675

In Vitro Evaluation of Antimicrobial Activity of Alimentary Canal Extracts from the Red Palm Weevil, Rhynchophorus ferrugineus Olivier Larvae.

PubMed

Sewify, Gamal H; Hamada, Hanan M; Alhadrami, Hani A

2017-01-01

The invasive red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae), is considered one of the world's most devastating insect pests to palm trees. It was observed that larvae of this pest are able to inhibit microbial growth on the rearing media when they start feeding and this observation has led us to study the effect of red palm weevils on various microbial species. The antimicrobial effect of extracts from different parts of the alimentary canal on Gram positive bacteria ( Enterococcus faecalis and Staphylococcus aureus ), Gram negative bacteria ( Escherichia coli and Klebsiella spp.), Candida albicans, and Penicillium sp. was tested using the agar well diffusion method. All extracts inhibited the tested microbial species. Foregut extracts had the greatest zones of growth inhibition. Enterococcus faecalis , Staphylococcus aureus, and Penicillium sp. were significantly sensitive to the extracts and had the largest growth inhibition zones. It is concluded that the gut extracts contain potent antimicrobial activity and may provide a new source of antimicrobial peptides.

The role of macrobiota in structuring microbial communities along rocky shores

DOE PAGES

Pfister, Catherine A.; Gilbert, Jack A.; Gibbons, Sean M.

2014-10-16

Rocky shore microbial diversity presents an excellent system to test for microbial habitat specificity or generality, enabling us to decipher how common macrobiota shape microbial community structure. At two coastal locations in the northeast Pacific Ocean, we show that microbial composition was significantly different between inert surfaces, the biogenic surfaces that included rocky shore animals and an alga, and the water column plankton. While all sampled entities had a core of common OTUs, rare OTUs drove differences among biotic and abiotic substrates. For the mussel Mytilus californianus, the shell surface harbored greater alpha diversity compared to internal tissues of themore » gill and siphon. Strikingly, a 7-year experimental removal of this mussel from tidepools did not significantly alter the microbial community structure of microbes associated with inert surfaces when compared with unmanipulated tidepools. However, bacterial taxa associated with nitrate reduction had greater relative abundance with mussels present, suggesting an impact of increased animal-derived nitrogen on a subset of microbial metabolism. Because the presence of mussels did not affect the structure and diversity of the microbial community on adjacent inert substrates, microbes in this rocky shore environment may be predominantly affected through direct physical association with macrobiota.« less

The role of macrobiota in structuring microbial communities along rocky shores

PubMed Central

Gilbert, Jack A.; Gibbons, Sean M.

2014-01-01

Rocky shore microbial diversity presents an excellent system to test for microbial habitat specificity or generality, enabling us to decipher how common macrobiota shape microbial community structure. At two coastal locations in the northeast Pacific Ocean, we show that microbial composition was significantly different between inert surfaces, the biogenic surfaces that included rocky shore animals and an alga, and the water column plankton. While all sampled entities had a core of common OTUs, rare OTUs drove differences among biotic and abiotic substrates. For the mussel Mytilus californianus, the shell surface harbored greater alpha diversity compared to internal tissues of the gill and siphon. Strikingly, a 7-year experimental removal of this mussel from tidepools did not significantly alter the microbial community structure of microbes associated with inert surfaces when compared with unmanipulated tidepools. However, bacterial taxa associated with nitrate reduction had greater relative abundance with mussels present, suggesting an impact of increased animal-derived nitrogen on a subset of microbial metabolism. Because the presence of mussels did not affect the structure and diversity of the microbial community on adjacent inert substrates, microbes in this rocky shore environment may be predominantly affected through direct physical association with macrobiota. PMID:25337459

Status, Antimicrobial Mechanism, and Regulation of Natural Preservatives in Livestock Food Systems.

PubMed

Lee, Na-Kyoung; Paik, Hyun-Dong

2016-01-01

This review discusses the status, antimicrobial mechanisms, application, and regulation of natural preservatives in livestock food systems. Conventional preservatives are synthetic chemical substances including nitrates/nitrites, sulfites, sodium benzoate, propyl gallate, and potassium sorbate. The use of artificial preservatives is being reconsidered because of concerns relating to headache, allergies, and cancer. As the demand for biopreservation in food systems has increased, new natural antimicrobial compounds of various origins are being developed, including plant-derived products (polyphenolics, essential oils, plant antimicrobial peptides (pAMPs)), animal-derived products (lysozymes, lactoperoxidase, lactoferrin, ovotransferrin, antimicrobial peptide (AMP), chitosan and others), and microbial metabolites (nisin, natamycin, pullulan, ε-polylysine, organic acid, and others). These natural preservatives act by inhibiting microbial cell walls/membranes, DNA/RNA replication and transcription, protein synthesis, and metabolism. Natural preservatives have been recognized for their safety; however, these substances can influence color, smell, and toxicity in large amounts while being effective as a food preservative. Therefore, to evaluate the safety and toxicity of natural preservatives, various trials including combinations of other substances or different food preservation systems, and capsulation have been performed. Natamycin and nisin are currently the only natural preservatives being regulated, and other natural preservatives will have to be legally regulated before their widespread use.

Status, Antimicrobial Mechanism, and Regulation of Natural Preservatives in Livestock Food Systems

PubMed Central

Lee, Na-Kyoung; Paik, Hyun-Dong

2016-01-01

This review discusses the status, antimicrobial mechanisms, application, and regulation of natural preservatives in livestock food systems. Conventional preservatives are synthetic chemical substances including nitrates/nitrites, sulfites, sodium benzoate, propyl gallate, and potassium sorbate. The use of artificial preservatives is being reconsidered because of concerns relating to headache, allergies, and cancer. As the demand for biopreservation in food systems has increased, new natural antimicrobial compounds of various origins are being developed, including plant-derived products (polyphenolics, essential oils, plant antimicrobial peptides (pAMPs)), animal-derived products (lysozymes, lactoperoxidase, lactoferrin, ovotransferrin, antimicrobial peptide (AMP), chitosan and others), and microbial metabolites (nisin, natamycin, pullulan, ε-polylysine, organic acid, and others). These natural preservatives act by inhibiting microbial cell walls/membranes, DNA/RNA replication and transcription, protein synthesis, and metabolism. Natural preservatives have been recognized for their safety; however, these substances can influence color, smell, and toxicity in large amounts while being effective as a food preservative. Therefore, to evaluate the safety and toxicity of natural preservatives, various trials including combinations of other substances or different food preservation systems, and capsulation have been performed. Natamycin and nisin are currently the only natural preservatives being regulated, and other natural preservatives will have to be legally regulated before their widespread use. PMID:27621697

PRISM 3: expanded prediction of natural product chemical structures from microbial genomes

PubMed Central

Skinnider, Michael A.; Merwin, Nishanth J.; Johnston, Chad W.

2017-01-01

Abstract Microbial natural products represent a rich resource of pharmaceutically and industrially important compounds. Genome sequencing has revealed that the majority of natural products remain undiscovered, and computational methods to connect biosynthetic gene clusters to their corresponding natural products therefore have the potential to revitalize natural product discovery. Previously, we described PRediction Informatics for Secondary Metabolomes (PRISM), a combinatorial approach to chemical structure prediction for genetically encoded nonribosomal peptides and type I and II polyketides. Here, we present a ground-up rewrite of the PRISM structure prediction algorithm to derive prediction of natural products arising from non-modular biosynthetic paradigms. Within this new version, PRISM 3, natural product scaffolds are modeled as chemical graphs, permitting structure prediction for aminocoumarins, antimetabolites, bisindoles and phosphonate natural products, and building upon the addition of ribosomally synthesized and post-translationally modified peptides. Further, with the addition of cluster detection for 11 new cluster types, PRISM 3 expands to detect 22 distinct natural product cluster types. Other major modifications to PRISM include improved sequence input and ORF detection, user-friendliness and output. Distribution of PRISM 3 over a 300-core server grid improves the speed and capacity of the web application. PRISM 3 is available at http://magarveylab.ca/prism/. PMID:28460067

Optimization of nonribosomal peptides production by a psychrotrophic fungus: Trichoderma velutinum ACR-P1.

PubMed

Sharma, Richa; Singh, Varun P; Singh, Deepika; Yusuf, Farnaz; Kumar, Anil; Vishwakarma, Ram A; Chaubey, Asha

2016-11-01

Trichoderma is an anamorphic filamentous fungal genus with immense potential for production of small valuable secondary metabolites with indispensable biological activities. Microbial dynamics of a psychrotrophic strain Trichoderma velutinum ACR-P1, isolated from unexplored niches of the Shiwalik region, bestowed with rich biodiversity of microflora, was investigated for production of nonribosomal peptides (NRPs) by metabolite profiling by intact-cell mass spectrometry (ICMS) employing matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. Being the first report on NRPs production by T. velutinum, studies on optimization of growth conditions by Response Surface Methodology (RSM) for production of NRPs by ACR-P1 was carried out strategically. Multifold enhancement in the yield of NRPs belonging to subfamily SF4 with medium chain of amino acid residues having m/z 1437.9, 1453.9, and 1452.0 at pH 5.9 at 20 °C and of subfamily SF1 with long-chain amino acid residues having m/z 1770.2, 1784.2, 1800.1, 1802.1, and 1815.1 was achieved at pH 7.0 at 25 °C. Complexities of natural mixtures were thus considerably reduced under respective optimized culture conditions accelerating the production of novel microbial natural products by saving time and resources.

Identification of ageing-associated naturally occurring peptides in human urine

PubMed Central

Nkuipou-Kenfack, Esther; Bhat, Akshay; Klein, Julie; Jankowski, Vera; Mullen, William; Vlahou, Antonia; Dakna, Mohammed; Koeck, Thomas; Schanstra, Joost P.; Zürbig, Petra; Rudolph, Karl L.; Schumacher, Björn; Pich, Andreas; Mischak, Harald

2015-01-01

To assess normal and pathological peptidomic changes that may lead to an improved understanding of molecular mechanisms underlying ageing, urinary peptidomes of 1227 healthy and 10333 diseased individuals between 20 and 86 years of age were investigated. The diseases thereby comprised diabetes mellitus, renal and cardiovascular diseases. Using age as a continuous variable, 116 peptides were identified that significantly (p < 0.05; |ρ|≥0.2) correlated with age in the healthy cohort. The same approach was applied to the diseased cohort. Upon comparison of the peptide patterns of the two cohorts 112 common age-correlated peptides were identified. These 112 peptides predominantly originated from collagen, uromodulin and fibrinogen. While most fibrillar and basement membrane collagen fragments showed a decreased age-related excretion, uromodulin, beta-2-microglobulin and fibrinogen fragments showed an increase. Peptide-based in silico protease analysis was performed and 32 proteases, including matrix metalloproteinases and cathepsins, were predicted to be involved in ageing. Identified peptides, predicted proteases and patient information were combined in a systems biology pathway analysis to identify molecular pathways associated with normal and/or pathological ageing. While perturbations in collagen homeostasis, trafficking of toll-like receptors and endosomal pathways were commonly identified, degradation of insulin-like growth factor-binding proteins was uniquely identified in pathological ageing. PMID:26431327

A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics

PubMed Central

Nesvizhskii, Alexey I.

2010-01-01

This manuscript provides a comprehensive review of the peptide and protein identification process using tandem mass spectrometry (MS/MS) data generated in shotgun proteomic experiments. The commonly used methods for assigning peptide sequences to MS/MS spectra are critically discussed and compared, from basic strategies to advanced multi-stage approaches. A particular attention is paid to the problem of false-positive identifications. Existing statistical approaches for assessing the significance of peptide to spectrum matches are surveyed, ranging from single-spectrum approaches such as expectation values to global error rate estimation procedures such as false discovery rates and posterior probabilities. The importance of using auxiliary discriminant information (mass accuracy, peptide separation coordinates, digestion properties, and etc.) is discussed, and advanced computational approaches for joint modeling of multiple sources of information are presented. This review also includes a detailed analysis of the issues affecting the interpretation of data at the protein level, including the amplification of error rates when going from peptide to protein level, and the ambiguities in inferring the identifies of sample proteins in the presence of shared peptides. Commonly used methods for computing protein-level confidence scores are discussed in detail. The review concludes with a discussion of several outstanding computational issues. PMID:20816881

Engineering antimicrobial peptides with improved antimicrobial and hemolytic activities.

PubMed

Zhao, Jun; Zhao, Chao; Liang, Guizhao; Zhang, Mingzhen; Zheng, Jie

2013-12-23

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

PubMed Central

Amoutzias, Grigoris D.; Chaliotis, Anargyros; Mossialos, Dimitris

2016-01-01

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds. PMID:27092515

Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions.

PubMed

Albergaria, Helena; Arneborg, Nils

2016-03-01

Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

An Integrated Metagenomics/Metaproteomics Investigation of the Microbial Communities and Enzymes in Solid-state Fermentation of Pu-erh tea

PubMed Central

Zhao, Ming; Zhang, Dong-lian; Su, Xiao-qin; Duan, Shuang-mei; Wan, Jin-qiong; Yuan, Wen-xia; Liu, Ben-ying; Ma, Yan; Pan, Ying-hong

2015-01-01

Microbial enzymes during solid-state fermentation (SSF), which play important roles in the food, chemical, pharmaceutical and environmental fields, remain relatively unknown. In this work, the microbial communities and enzymes in SSF of Pu-erh tea, a well-known traditional Chinese tea, were investigated by integrated metagenomics/metaproteomics approach. The dominant bacteria and fungi were identified as Proteobacteria (48.42%) and Aspergillus (94.98%), through pyrosequencing-based analyses of the bacterial 16S and fungal 18S rRNA genes, respectively. In total, 335 proteins with at least two unique peptides were identified and classified into 28 Biological Processes and 35 Molecular Function categories using a metaproteomics analysis. The integration of metagenomics and metaproteomics data demonstrated that Aspergillus was dominant fungus and major host of identified proteins (50.45%). Enzymes involved in the degradation of the plant cell wall were identified and associated with the soft-rotting of tea leaves. Peroxiredoxins, catalase and peroxidases were associated with the oxidation of catechins. In conclusion, this work greatly advances our understanding of the SSF of Pu-erh tea and provides a powerful tool for studying SSF mechanisms, especially in relation to the microbial communities present. PMID:25974221

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis

PubMed Central

Singhal, Neelja; Kumar, Manish; Kanaujia, Pawan K.; Virdi, Jugsharan S.

2015-01-01

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi. PMID:26300860

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.

PubMed

Singhal, Neelja; Kumar, Manish; Kanaujia, Pawan K; Virdi, Jugsharan S

2015-01-01

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

Shotgun metaproteomics of the human distal gut microbiota

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

VerBerkmoes, N.C.; Russell, A.L.; Shah, M.

2008-10-15

The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thusmore » demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes.« less

A new family of cystine knot peptides from the seeds of Momordica cochinchinensis.

PubMed

Chan, Lai Yue; He, Wenjun; Tan, Ninghua; Zeng, Guangzhi; Craik, David J; Daly, Norelle L

2013-01-01

Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3-MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

Microbial Source Module (MSM): Documenting the Science and Software for Discovery, Evaluation, and Integration

EPA Science Inventory

The Microbial Source Module (MSM) estimates microbial loading rates to land surfaces from non-point sources, and to streams from point sources for each subwatershed within a watershed. A subwatershed, the smallest modeling unit, represents the common basis for information consume...

Structural prediction and analysis of VIH-related peptides from selected crustacean species.

PubMed

Nagaraju, Ganji Purna Chandra; Kumari, Nunna Siva; Prasad, Ganji Lakshmi Vara; Rajitha, Balney; Meenu, Madan; Rao, Manam Sreenivasa; Naik, Bannoth Reddya

2009-08-17

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.

Acceleration of yoghurt fermentation time by yeast extract and partial characterisation of the active components.

PubMed

Smith, Esti-Andrine; Myburgh, Jacobus; Osthoff, Gernot; de Wit, Maryna

2014-11-01

Water soluble autolysate of yeast, usually utilised for microbial growth support, was used as additive in yoghurt fermentation. The yeast extract (YE) resulted in a decrease of fermentation time by 21% to reach a pH of 4·6. However, the YE resulted in unacceptable flavour and taste. By size exclusion chromatography, a fraction of the YE was obtained that could account for the observed 21% decrease in fermentation time. The fraction contained molecules of low molecular weight, consisting of minerals, free amino acids and peptides. The acceleration of the yoghurt fermentation was ascribed to the short peptides in the fraction. It is proposed that the application of this extract in industrial yoghurt manufacture would result in savings for both the industry and the consumer.

Decoding the Effect of Isobaric Substitutions on Identifying Missing Proteins and Variant Peptides in Human Proteome.

PubMed

Choong, Wai-Kok; Lih, Tung-Shing Mamie; Chen, Yu-Ju; Sung, Ting-Yi

2017-12-01

To confirm the existence of missing proteins, we need to identify at least two unique peptides with length of 9-40 amino acids of a missing protein in bottom-up mass-spectrometry-based proteomic experiments. However, an identified unique peptide of the missing protein, even identified with high level of confidence, could possibly coincide with a peptide of a commonly observed protein due to isobaric substitutions, mass modifications, alternative splice isoforms, or single amino acid variants (SAAVs). Besides unique peptides of missing proteins, identified variant peptides (SAAV-containing peptides) could also alternatively map to peptides of other proteins due to the aforementioned issues. Therefore, we conducted a thorough comparative analysis on data sets in PeptideAtlas Tiered Human Integrated Search Proteome (THISP, 2017-03 release), including neXtProt (2017-01 release), to systematically investigate the possibility of unique peptides in missing proteins (PE2-4), unique peptides in dubious proteins, and variant peptides affected by isobaric substitutions, causing doubtful identification results. In this study, we considered 11 isobaric substitutions. From our analysis, we found <5% of the unique peptides of missing proteins and >6% of variant peptides became shared with peptides of PE1 proteins after isobaric substitutions.

Characterization of Microbial Population Structures in Recreational Waters and Primary Sources of Fecal Pollution with a Next-Generation Sequencing Approach

EPA Science Inventory

The invention of new approaches to DNA sequencing commonly referred to as next generation sequencing technologies is revolutionizing the study of microbial diversity. In this chapter, we discuss the characterization of microbial population structures in recreational waters and p...

Effect of physicochemical properties of peptides from soy protein on their antimicrobial activity.

PubMed

Xiang, Ning; Lyu, Yuan; Zhu, Xiao; Bhunia, Arun K; Narsimhan, Ganesan

2017-08-01

Antimicrobial peptides (AMPs) kill microbial cells through insertion and damage/permeabilization of the cytoplasmic cell membranes and has applications in food safety and antibiotic replacement. Soy protein is an attractive, abundant natural source for commercial production of AMPs. In this research, explicit solvent molecular dynamics (MD) simulation was employed to investigate the effects of (i) number of total and net charges, (ii) hydrophobicity (iii) hydrophobic moment and (iv) helicity of peptides from soy protein on their ability to bind to lipid bilayer and their transmembrane aggregates to form pores. Interaction of possible AMP segments from soy protein with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPC/POPG) bilayers, a mimic of bacterial cell membrane, was investigated. Pore formation was insensitive to helicity and occurred for hydrophobicity threshold in the range of -0.3-0kcal/mol, hydrophobic moment threshold of 0.3kcal/mol, net charge threshold of 2. Though low hydrophobicity and high number of charges help in the formation of water channel for transmembrane aggregates, insertion of peptides with these properties requires overcome of energy barrier, as shown by potential of mean force calculations, thereby resulting in low antimicrobial activity. Experimental evaluation of antimicrobial activity of these peptides against Gram positive L. monocytogenes and Gram negative E. coli as obtained by spot-on-lawn assay was consistent with simulation results. These results should help in the development of guidelines for selection of peptides with antimicrobial activity based on their physicochemical properties. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

PubMed

Mishra, Biswajit; Leishangthem, Geeta Devi; Gill, Kamaldeep; Singh, Abhay K; Das, Swagata; Singh, Kusum; Xess, Immaculata; Dinda, Amit; Kapil, Arti; Patro, Ishan K; Dey, Sharmistha

2013-02-01

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials. Copyright © 2012 Elsevier B.V. All rights reserved.

Peptide vaccination of patients with metastatic melanoma: improved clinical outcome in patients demonstrating effective immunization.

PubMed

Markovic, Svetomir N; Suman, Vera J; Ingle, James N; Kaur, Judith S; Pitot, Henry C; Loprinzi, Charles L; Rao, Ravi D; Creagan, Edward T; Pittelkow, Mark R; Allred, Jakob B; Nevala, Wendy K; Celis, Esteban

2006-08-01

Therapeutic peptide vaccines for melanoma continue to only demonstrate anecdotal success. We set out to evaluate the impact of low-dose GM-CSF emulsified in Montanide ISA-51 on the immunogenicity of HLA-A2 restricted melanoma differentiation antigen peptide vaccines (MART-1, gp100 and tyrosinase) administered in separate subcutaneous injections. We conducted a randomized phase II clinical trial of HLA-A2+ patients with metastatic melanoma that were immunized every 3 weeks with one of the following vaccine preparations: (A) peptides + Montanide ISA-51; (B) peptides + Montanide ISA-51 + GM-CSF (10 microg); (C) peptides + Montanide ISA-51 + GM-CSF (50 microg). Immunization efficacy was determined by quantification of vaccine specific tetramer positive cytotoxic T cells in peripheral blood. Global assessment of immune competence was ascertained using DTH testing to common recall antigens as well as peripheral blood immunophenotyping. Twenty-five eligible patients were equally distributed across all 3 treatment groups. Only 9 patients demonstrated evidence of immunization. Most commonly, immune response was achieved to the gp100 peptide. The addition of low-dose GM-CSF did not impact immunization efficacy. DTH reactivity to Candida appeared predictive of successful immunization. Successful immunization with the peptide vaccines was associated with improved clinical outcomes. The addition of low dose GM-CSF to peptide vaccines did not enhance immunogenicity. Higher doses of GM-CSF may be needed to achieve this effect and this is a testable hypothesis. Likewise, better patient selection based on immunologic status (DTH reactivity) may be helpful to better understand the clinical impact of therapeutic cancer vaccines.

Predicting the Retention Behavior of Specific O-Linked Glycopeptides.

PubMed

Badgett, Majors J; Boyes, Barry; Orlando, Ron

2017-09-01

O -Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O - N -acetylgalactosamine ( O -GalNAc), O - N -acetylglucosamine ( O -GlcNAc), and O -fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.

Predicting the Retention Behavior of Specific O-Linked Glycopeptides

PubMed Central

Badgett, Majors J.; Boyes, Barry; Orlando, Ron

2017-01-01

O-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O-glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O-N-acetylgalactosamine (O-GalNAc), O-N-acetylglucosamine (O-GlcNAc), and O-fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications. PMID:28785176

Microbes, Immunity, and Behavior: Psychoneuroimmunology Meets the Microbiome

PubMed Central

Dinan, Timothy G; Cryan, John F

2017-01-01

There is now a large volume of evidence to support the view that the immune system is a key communication pathway between the gut and brain, which plays an important role in stress-related psychopathologies and thus provides a potentially fruitful target for psychotropic intervention. The gut microbiota is a complex ecosystem with a diverse range of organisms and a sophisticated genomic structure. Bacteria within the gut are estimated to weigh in excess of 1 kg in the adult human and the microbes within not only produce antimicrobial peptides, short chain fatty acids, and vitamins, but also most of the common neurotransmitters found in the human brain. That the microbial content of the gut plays a key role in immune development is now beyond doubt. Early disruption of the host-microbe interplay can have lifelong consequences, not just in terms of intestinal function but in distal organs including the brain. It is clear that the immune system and nervous system are in continuous communication in order to maintain a state of homeostasis. Significant gaps in knowledge remain about the effect of the gut microbiota in coordinating the immune-nervous systems dialogue. However, studies using germ-free animals, infective models, prebiotics, probiotics, and antibiotics have increased our understanding of the interplay. Early life stress can have a lifelong impact on the microbial content of the intestine and permanently alter immune functioning. That early life stress can also impact adult psychopathology has long been appreciated in psychiatry. The challenge now is to fully decipher the molecular mechanisms that link the gut microbiota, immune, and central nervous systems in a network of communication that impacts behavior patterns and psychopathology, to eventually translate these findings to the human situation both in health and disease. Even at this juncture, there is evidence to pinpoint key sites of communication where gut microbial interventions either with drugs or diet or perhaps fecal microbiota transplantation may positively impact mental health. PMID:27319972

Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review

PubMed Central

Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

2017-01-01

Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed. PMID:28212329

Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review.

PubMed

Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

2017-02-16

Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed.

Improving Peptide Applications Using Nanotechnology.

PubMed

Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

2016-01-01

Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

Effective Design of Multifunctional Peptides by Combining Compatible Functions

PubMed Central

Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A.; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

2016-01-01

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf. PMID:27096600

The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides.

PubMed

Sophocleous, Andreas M; Desai, Kashappa-Goud H; Mazzara, J Maxwell; Tong, Ling; Cheng, Ji-Xin; Olsen, Karl F; Schwendeman, Steven P

2013-12-28

An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4mM octreotide or leuprolide acetate salts in a 0.1M HEPES buffer, pH7.4, with polymer particles or films at 4-37°C for 24h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy, stimulated Raman scattering (SRS) and laser scanning confocal imaging, and microtome sectioning techniques were used to examine peptide penetration into the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST+0.02% sodium azide, 37°C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but also can be internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt.% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for >2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides. © 2013.

The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

PubMed Central

Sophocleous, Andreas M.; Desai, Kashappa-Goud H.; Mazzara, J. Maxwell; Tong, Ling; Cheng, Ji-Xin; Olsen, Karl F.; Schwendeman, Steven P.

2013-01-01

An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4 mM octreotide or leuprolide acetate salts in 0.1 M HEPES buffer, pH 7.4, with polymer particles or films at 4-37 °C for 24 h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy and stimulated Raman scattering (SRS) and laser scanning confocal imaging techniques were used to examine peptide penetration in the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST + 0.02% sodium azide, 37 °C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but can also internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for > 2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides. PMID:24021356

Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

PubMed

Hoppers, A; Stoudenmire, J; Wu, S; Lopanik, N B

2015-02-01

Sessile marine invertebrates engage in a diverse array of beneficial interactions with bacterial symbionts. One feature of some of these relationships is the presence of bioactive natural products that can defend the holobiont from predation, competition or disease. In this study, we investigated the antimicrobial activity and microbial community of a common temperate sponge from coastal North Carolina. The sponge was identified as a member of the genus Haliclona, a prolific source of bioactive natural products, based on its 18S rRNA gene sequence. The crude chemical extract and methanol partition had broad activity against the assayed Gram-negative and Gram-positive pathogenic bacteria. Further fractionation resulted in two groups of compounds with differing antimicrobial activity, primarily against Gram-positive test organisms. There was, however, notable activity against the Gram-negative marine pathogen, Vibrio parahaemolyticus. Microbial community analysis of the sponge and surrounding sea water via denaturing gradient gel electrophoresis (DGGE) indicates that it harbours a distinct group of bacterial associates. The common temperate sponge, Haliclona sp., is a source of multiple antimicrobial compounds and has some consistent microbial community members that may play a role in secondary metabolite production. These data suggest that common temperate sponges can be a source of bioactive chemical and microbial diversity. Further studies may reveal the importance of the microbial associates to the sponge and natural product biosynthesis. © 2014 The Society for Applied Microbiology.

Microbial mitigation-exacerbation continuum: a novel framework for microbiome effects on hosts in the face of stress.

PubMed

David, Aaron S; Thapa-Magar, Khum B; Afkhami, Michelle E

2018-03-01

A key challenge to understanding microbiomes and their role in ecological processes is contextualizing their effects on host organisms, particularly when faced with environmental stress. One influential theory, the Stress Gradient Hypothesis, might predict that the frequency of positive interactions increases with stressful conditions such that microbial taxa would mitigate harmful effects on host performance. Yet, equally plausible is that microbial taxa could exacerbate these effects. Here, we introduce the Mitigation-Exacerbation Continuum as a novel framework to conceptualize microbial mediation of stress. We (1) use this continuum to quantify microbial mediation of stress for six plant species and (2) test the association between these continuum values and natural species' abundance. We factorially manipulated a common stress (allelopathy) and the presence of soil microbes to quantify microbial effects in benign and stressed environments for two critical early life-history metrics, seed germination and seedling biomass. Although we found evidence of both mitigation and exacerbation among the six species, exacerbation was more common. Across species, the degree of microbial-mediated effects on germination explained >80% of the variation of natural field abundances. Our results suggest a critical role of soil microbes in mediating plant stress responses, and a potential microbial mechanism underlying species abundance. © 2018 by the Ecological Society of America.

Microbial diversities (16S and 18S rDNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

EPA Science Inventory

Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, e.g. Legionella pneumophila, via parasitization of free-living amoebae such as Acanthamoebae. Yet knowledge about the microbial composition of DW biofilms developed on common in-premise pl...

Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae.

PubMed

Schenk, Mirjam; Mahapatra, Sebabrata; Le, Phuonganh; Kim, Hee Jin; Choi, Aaron W; Brennan, Patrick J; Belisle, John T; Modlin, Robert L

2016-09-01

The innate immune system recognizes microbial pathogens via pattern recognition receptors. One such receptor, NOD2, via recognition of muramyl dipeptide (MDP), triggers a distinct network of innate immune responses, including the production of interleukin-32 (IL-32), which leads to the differentiation of monocytes into dendritic cells (DC). NOD2 has been implicated in the pathogenesis of human leprosy, yet it is not clear whether Mycobacterium leprae, which has a distinct MDP structure, can activate this pathway. We investigated the effect of MDP structure on the innate immune response, finding that infection of monocytes with M. leprae induces IL-32 and DC differentiation in a NOD2-dependent manner. The presence of the proximal l-Ala instead of Gly in the common configuration of the peptide side chain of M. leprae did not affect recognition by NOD2 or cytokine production. Furthermore, amidation of the d-Glu residue did not alter NOD2 activation. These data provide experimental evidence that NOD2 recognizes naturally occurring structural variants of MDP. Copyright © 2016 Schenk et al.

Antimicrobial and Attractant Roles for Chemerin in the Oral Cavity during Inflammatory Gum Disease

PubMed Central

Godlewska, Urszula; Brzoza, Piotr; Sroka, Aneta; Majewski, Pawel; Jentsch, Holger; Eckert, Martin; Eick, Sigrun; Potempa, Jan; Zabel, Brian A.; Cichy, Joanna

2017-01-01

Periodontal inflammation is one of the most common chronic inflammatory conditions in humans. Despite recent advances in identifying and characterizing oral microbiota dysbiosis in the pathogenesis of gum disease, just how host factors maintain a healthy homeostatic oral microbial community or prevent the development of a pathogenic oral microbiota remains poorly understood. An important determinant of microbiota fate is local antimicrobial proteins. Here, we report that chemoattractant protein chemerin, which we recently identified as a potent endogenous antimicrobial agent in body barriers such as the skin, is present in the oral cavity under homeostatic and inflammatory conditions. Chemerin and a chemerin-derived antimicrobial peptide are bactericidal against select bacteria strategically positioned in dental biofilm. Gingival crevicular samples from patients with gingivitis but not periodontitis contain abundant bioactive chemerin capable of inducing CMKLR1-dependent leukocyte migration. Gingipains secreted by the periodontopathogen P. gingivalis inactivate chemerin. Together, these data suggest that as an antimicrobial agent and leukocyte chemoattractant, chemerin likely contributes to antimicrobial immune defense in the oral cavity. PMID:28424689

Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

PubMed

Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu

2016-01-01

The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide-based antimicrobial biomaterials. Further exploration of these AMPs will allow for diverse biotechnological and clinical applications such as biomedical coating, food storaging, and animal feeding. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Nanoliposome is a Promising Carrier of Protein and Peptide Biomolecule for the Treatment of Cancer.

PubMed

Kumar Giri, Tapan; Giri, Ayan; Kumar Barman, Tapan; Maity, Subhasis

2016-01-01

Nano-liposomes are the newly developed delivery systems for cancer therapy that are finding a position particularly suitable as peptide and protein carriers. These are three-layered self-assembled structures with nanoparticulate carrier systems. The overall pharmacological properties of commonly used protein and peptide in cancer therapy can be improved by the incorporation of protein and peptide into the nano-liposome. The surface modifications can be made liposomes to make compatible with targeting ligands has made these nanocarriers for targeted delivery. This review discusses the method of preparation and characterization of liposome based protein peptide delivery for the treatment of cancer. This review also explores latest work intended for targeted treatment of cancer by nano-liposomal protein and peptide delivery system. This type of delivery is targeting protein and peptide to tumor site by avoiding the reticuloendothelial system. Methods of nano-liposome delivery containing protein and peptide are also highlighted.

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-05-01

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Signals generating anorexia during acute illness.

PubMed

Langhans, Wolfgang

2007-08-01

Anorexia is part of the body's acute-phase response to illness. Microbial products such as lipopolysaccharides (LPS), which are also commonly used to model acute illness, trigger the acute-phase response and cause anorexia mainly through pro-inflammatory cytokines. LPS stimulate cytokine production through the cell-surface structural molecule CD14 and toll-like receptor-4. Cytokines ultimately change neural activity in brain areas controlling food intake and energy balance. The blood-brain barrier endothelial cells (BBB EC) are an important site of cytokine action in this context. BBB EC and perivascular cells (microglia and macrophages) form a complex regulatory interface that modulates neuronal activity by the release of messengers (e.g. PG, NO) in response to peripheral challenges. Serotonergic neurons originating in the raphe nuclei and glucagon-like peptide-1-expressing neurons in the hindbrain may be among the targets of these messengers, because serotonin (5-HT), acting through the 5-HT2C receptor, and glucagon-like peptide-1 have recently emerged as neurochemical mediators of LPS anorexia. The central melanocortin system, which is a downstream target of serotonergic neurons, also appears to be involved in mediation of LPS anorexia. Interestingly, LPS also reduce orexin expression and the activity of orexin neurons in the lateral hypothalamic area of fasted mice. As the eating-stimulatory properties of orexin are apparently related to arousal, the inhibitory effect of LPS on orexin neurons might be involved in LPS-induced inactivity and anorexia. In summary, the immune signalling pathways of LPS-induced, and presumably acute illness-induced, anorexia converge on central neural signalling systems that control food intake and energy balance in healthy individuals.

Roles of Mas-related G protein-coupled receptor X2 on mast cell-mediated host defense, pseudoallergic drug reactions, and chronic inflammatory diseases.

PubMed

Subramanian, Hariharan; Gupta, Kshitij; Ali, Hydar

2016-09-01

Mast cells (MCs), which are granulated tissue-resident cells of hematopoietic lineage, contribute to vascular homeostasis, innate/adaptive immunity, and wound healing. However, MCs are best known for their roles in allergic and inflammatory diseases, such as anaphylaxis, food allergy, rhinitis, itch, urticaria, atopic dermatitis, and asthma. In addition to the high-affinity IgE receptor (FcεRI), MCs express numerous G protein-coupled receptors (GPCRs), which are the largest group of membrane receptor proteins and the most common targets of drug therapy. Antimicrobial host defense peptides, neuropeptides, major basic protein, eosinophil peroxidase, and many US Food and Drug Administration-approved peptidergic drugs activate human MCs through a novel GPCR known as Mas-related G protein-coupled receptor X2 (MRGPRX2; formerly known as MrgX2). Unique features of MRGPRX2 that distinguish it from other GPCRs include their presence both on the plasma membrane and intracellular sites and their selective expression in MCs. In this article we review the possible roles of MRGPRX2 on host defense, drug-induced anaphylactoid reactions, neurogenic inflammation, pain, itch, and chronic inflammatory diseases, such as urticaria and asthma. We propose that host defense peptides that kill microbes directly and activate MCs through MRGPRX2 could serve as novel GPCR targets to modulate host defense against microbial infection. Furthermore, mAbs or small-molecule inhibitors of MRGPRX2 could be developed for the treatment of MC-dependent allergic and inflammatory disorders. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Obesity: An overview of possible role(s) of gut hormones, lipid sensing and gut microbiota.

PubMed

Mishra, Alok Kumar; Dubey, Vinay; Ghosh, Asit Ranjan

2016-01-01

Obesity is one of the major challenges for public health in 21st century, with 1.9 billion people being considered as overweight and 600 million as obese. There are certain diseases such as type 2 diabetes, hypertension, cardiovascular disease, and several forms of cancer which were found to be associated with obesity. Therefore, understanding the key molecular mechanisms involved in the pathogenesis of obesity could be beneficial for the development of a therapeutic approach. Hormones such as ghrelin, glucagon like peptide 1 (GLP-1) peptide YY (PYY), pancreatic polypeptide (PP), cholecystokinin (CCK) secreted by an endocrine organ gut, have an intense impact on energy balance and maintenance of homeostasis by inducing satiety and meal termination. Glucose and energy homeostasis are also affected by lipid sensing in which different organs respond in different ways. However, there is one common mechanism i.e. formation of esterified lipids (long chain fatty acyl CoAs) and the activation of protein kinase C δ (PKC δ) involved in all these organs. The possible role of gut microbiota and obesity has been addressed by several researchers in recent years, indicating the possible therapeutic approach toward the management of obesity by the introduction of an external living system such as a probiotic. The proposed mechanism behind this activity is attributed by metabolites produced by gut microbial organisms. Thus, this review summarizes the role of various physiological factors such as gut hormone and lipid sensing involved in various tissues and organ and most important by the role of gut microbiota in weight management. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

PubMed

Gutiérrez-Chávez, A J; Martínez-Ortega, E A; Valencia-Posadas, M; León-Galván, M F; de la Fuente-Salcido, N M; Bideshi, D K; Barboza-Corona, J E

2016-01-01

Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis.

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

PubMed Central

2013-01-01

Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

Testing the functional significance of microbial community composition.

Treesearch

Michael S. Strickland; Christian Lauber; Noah Fierer; Mark A. Bradford

2009-01-01

A critical assumption underlying terrestrial ecosystem models is that soil microbial communities, when placed in a common environment, will function in an identical manner regardless of the composition...

Structural prediction and analysis of VIH-related peptides from selected crustacean species

PubMed Central

Nagaraju, Ganji Purna Chandra; Kumari, Nunna Siva; Prasad, Ganji Lakshmi Vara; Rajitha, Balney; Meenu, Madan; Rao, Manam Sreenivasa; Naik, Bannoth Reddya

2009-01-01

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family. PMID:20011146

Microbial enzymatic activity and secondary production in sediments affected by the sedimentation pulse following the Deepwater Horizon oil spill

NASA Astrophysics Data System (ADS)

Ziervogel, Kai; Joye, Samantha B.; Arnosti, Carol

2016-07-01

A large fraction of the spilled oil from the Deepwater Horizon (DwH) blowout in April 2010 reached the seafloor via sinking oil aggregates (oil snow) in a massive sedimentation that continued until late summer 2010 (;Dirty blizzard;). We measured heterotrophic microbial metabolic rates as well as porewater and sedimentary geochemical parameters at sites proximate to and distant from the wellhead to investigate microbial responses to the "Dirty Blizzard". Lipase activity and rates of bacterial protein production were highest and leucine-aminopeptidase activity was lowest in 0-2 cm sediment layers at the sites proximate to the wellhead. These results suggest that the presence of the oil snow stimulated benthic microbial enzymatic hydrolysis of oil-derived organic matter that was depleted in peptide substrates at the time of our sampling. The strong gradients in porewater DOC, NH4+, and HPO43- concentrations in the upper 6 cm of the sediments near the wellhead likewise indicate elevated heterotrophic responses to recently-sedimented organic matter. In addition to enhanced microbial activities in the 0-2 cm sediment layers, we found peaks of total organic carbon and elevated microbial metabolic rates down to 10 cm at the sites closest to the wellhead. Our results indicate distinct benthic metabolic responses of heterotrophic microbial communities, even three months after the ending of the "Dirty Blizzard". Compared to other deep-sea environments, however, metabolic rates associated with the recently deposited particulate matter around the wellhead were only moderately enhanced. Oil contaminants at the seafloor may therefore have prolonged residence times, enhancing the potential for longer-term ecological consequences in deep-sea environments.

Animal Venom Peptides: Potential for New Antimicrobial Agents.

PubMed

Primon-Barros, Muriel; José Macedo, Alexandre

2017-01-01

Microbial infections affect people worldwide, causing diseases with significant impact on public health, indicating the need for research and development of new antimicrobial agents. Animal venoms represent a vast and largely unexploited source of biologically active molecules with attractive candidates for the development of novel therapeutics. Venoms consist of complex mixtures of molecules, including antimicrobial peptides (AMPs). Since the discovery of AMPs, they have been studied as promising new antimicrobial drugs. Amongst the remarkable sources of AMPs with known antimicrobial activities are ants, bees, centipedes, cone snails, scorpions, snakes, spiders, and wasps. The antimicrobial tests against bacteria, protozoans, fungi and viruses using 170 different peptides isolated directly from crude venoms or cDNA libraries of venom glands are listed and discussed in this review, as well as hemolytic ativity. The potential of venoms as source of new compounds, including AMPs, is extensively discussed. Currently, there are six FDA-approved drugs and many others are undergoing preclinical and clinical trials. The search for antimicrobial "weapons" makes the AMPs from venoms promising candidates. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer.

PubMed

Lau, Derick; Guo, Linlang; Liu, Ruiwu; Marik, Jan; Lam, Kit

2006-06-01

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

Microbial reduction of vanadium (V) in groundwater: Interactions with coexisting common electron acceptors and analysis of microbial community.

PubMed

Liu, Hui; Zhang, Baogang; Yuan, Heyang; Cheng, Yutong; Wang, Song; He, Zhen

2017-12-01

Vanadium (V) pollution in groundwater has posed serious risks to the environment and public health. Anaerobic microbial reduction can achieve efficient and cost-effective remediation of V(V) pollution, but its interactions with coexisting common electron acceptors such as NO 3 - , Fe 3+ , SO 4 2- and CO 2 in groundwater remain unknown. In this study, the interactions between V(V) reduction and reduction of common electron acceptors were examined with revealing relevant microbial community and identifying dominant species. The results showed that the presence of NO 3 - slowed down the removal of V(V) in the early stage of the reaction but eventually led to a similar reduction efficiency (90.0% ± 0.4% in 72-h operation) to that in the reactor without NO 3 - . The addition of Fe 3+ , SO 4 2- , or CO 2 decreased the efficiency of V(V) reduction. Furthermore, the microbial reduction of these coexisting electron acceptors was also adversely affected by the presence of V(V). The addition of V(V) as well as the extra dose of Fe 3+ , SO 4 2- and CO 2 decreased microbial diversity and evenness, whereas the reactor supplied with NO 3 - showed the increased diversity. High-throughput 16S rRNA gene pyrosequencing analysis indicated the accumulation of Geobacter, Longilinea, Syntrophobacter, Spirochaeta and Anaerolinea, which might be responsible for the reduction of multiple electron acceptors. The findings of this study have demonstrated the feasibility of anaerobic bioremediation of V(V) and the possible influence of coexisting electron acceptors commonly found in groundwater. Copyright © 2017 Elsevier Ltd. All rights reserved.

A study of the proteorhodopsin primary photoreaction by low-temperature FTIR difference and ultrafast transient infrared spectroscopy

NASA Astrophysics Data System (ADS)

Amsden, Jason J.

Proteorhodopsin (PR), a newly discovered microbial rhodopsin found in marine proteobacteria, functions as a light-driven proton pump similar to bacteriorhodopsin (BR). PR-containing bacteria account for ˜13% of the microorganisms in the oceans' photic zone and are responsible for a significant fraction of the biosphere's solar energy conversion. We study the initial response of proteorhodopsin to photon absorption using a combination of low-temperature (80 K) Fourier transform infrared (FTIR) difference spectroscopy and ultrafast transient infrared (TIR) spectroscopy. Low-temperature FTIR difference spectroscopy combined with site-directed mutagenesis and isotope labeling is used to detect and characterize changes occurring in the conformation of the retinal chromophore, protein, and internal water molecules of green-absorbing PR (GPR) and blue-absorbing PR (BPR) during the initial phototransition. Measurements on cryogenically trapped intermediates do not accurately reflect all native structural changes occurring in PR and other microbial rhodopsins on ultrafast time scales at room temperature. Recent studies demonstrate that photoactive proteins such as photoactive yellow protein, myoglobin, and green-fluorescent protein, can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal chromophore photoisomerization may impulsively drive structural changes in nearby protein groups. Here, I test this possibility by investigating the earliest protein and chromophore structural changes occurring in GPR using ultrafast TIR spectroscopy with ˜200 fs time resolution combined with non-perturbing isotope labeling. On the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling, the all-trans to 13-cis retinal chromophore isomerization occurs with a 500-700 fs time constant and the amide II mode of one or more peptide bonds in the protein backbone downshifts in frequency on the same 500--700 fs time scale. This downshift represents the weakening of the hydrogen bonding in one or more peptide bonds in the peptide backbone. Preliminary data on BR indicates that this amide II downshift is a universal feature in microbial rhodopsins. In addition, the protein changes we observe on a subpicosecond time-scale may be involved in storage and transfer of the absorbed photon energy subsequently utilized for proton transport.

Incorporating sequence information into the scoring function: a hidden Markov model for improved peptide identification.

PubMed

Khatun, Jainab; Hamlett, Eric; Giddings, Morgan C

2008-03-01

The identification of peptides by tandem mass spectrometry (MS/MS) is a central method of proteomics research, but due to the complexity of MS/MS data and the large databases searched, the accuracy of peptide identification algorithms remains limited. To improve the accuracy of identification we applied a machine-learning approach using a hidden Markov model (HMM) to capture the complex and often subtle links between a peptide sequence and its MS/MS spectrum. Our model, HMM_Score, represents ion types as HMM states and calculates the maximum joint probability for a peptide/spectrum pair using emission probabilities from three factors: the amino acids adjacent to each fragmentation site, the mass dependence of ion types and the intensity dependence of ion types. The Viterbi algorithm is used to calculate the most probable assignment between ion types in a spectrum and a peptide sequence, then a correction factor is added to account for the propensity of the model to favor longer peptides. An expectation value is calculated based on the model score to assess the significance of each peptide/spectrum match. We trained and tested HMM_Score on three data sets generated by two different mass spectrometer types. For a reference data set recently reported in the literature and validated using seven identification algorithms, HMM_Score produced 43% more positive identification results at a 1% false positive rate than the best of two other commonly used algorithms, Mascot and X!Tandem. HMM_Score is a highly accurate platform for peptide identification that works well for a variety of mass spectrometer and biological sample types. The program is freely available on ProteomeCommons via an OpenSource license. See http://bioinfo.unc.edu/downloads/ for the download link.

Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics.

PubMed

Tang, Xian-Bing; Si, Shu-Yi; Zhang, Yue-Qin

2004-09-01

To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E. coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Molecular Insights into Arctic Soil Organic Matter Degradation under Warming

DOE PAGES

Chen, Hongmei; Yang, Ziming; Chu, Rosalie K.; ...

2018-03-23

Molecular composition of the Arctic soil organic carbon (SOC) and its susceptibility to microbial degradation are uncertain due to heterogeneity and unknown SOC compositions. By using ultrahigh-resolution mass spectrometry, we determined the susceptibility and compositional changes of extractable dissolved organic matter (EDOM) in an anoxic warming incubation experiment (up to 122 days) with a tundra soil from Alaska (United States). EDOM was extracted with 10 mM NH 4HCO 3 from both the organic- and mineral-layer soils during incubation at both -2 and 8°C. Based on their O:C and H:C ratios, EDOM molecular formulas were qualitatively grouped into nine biochemical classesmore » of compounds, among which lignin-like compounds dominated both the organic and the mineral soils and were the most stable, whereas amino sugars, peptides, and carbohydrate-like compounds were the most biologically labile. These results corresponded with shifts in EDOM elemental composition in which the ratios of O:C and N:C decreased, while the average C content in EDOM, molecular mass, and aromaticity increased after 122 days of incubation. This research demonstrates that certain EDOM components, such as amino sugars, peptides, and carbohydrate-like compounds, are disproportionately more susceptible to microbial degradation than others in the soil, and these results should be considered in SOC degradation models to improve predictions of Arctic climate feedbacks.« less

PRISM 3: expanded prediction of natural product chemical structures from microbial genomes.

PubMed

Skinnider, Michael A; Merwin, Nishanth J; Johnston, Chad W; Magarvey, Nathan A

2017-07-03

Microbial natural products represent a rich resource of pharmaceutically and industrially important compounds. Genome sequencing has revealed that the majority of natural products remain undiscovered, and computational methods to connect biosynthetic gene clusters to their corresponding natural products therefore have the potential to revitalize natural product discovery. Previously, we described PRediction Informatics for Secondary Metabolomes (PRISM), a combinatorial approach to chemical structure prediction for genetically encoded nonribosomal peptides and type I and II polyketides. Here, we present a ground-up rewrite of the PRISM structure prediction algorithm to derive prediction of natural products arising from non-modular biosynthetic paradigms. Within this new version, PRISM 3, natural product scaffolds are modeled as chemical graphs, permitting structure prediction for aminocoumarins, antimetabolites, bisindoles and phosphonate natural products, and building upon the addition of ribosomally synthesized and post-translationally modified peptides. Further, with the addition of cluster detection for 11 new cluster types, PRISM 3 expands to detect 22 distinct natural product cluster types. Other major modifications to PRISM include improved sequence input and ORF detection, user-friendliness and output. Distribution of PRISM 3 over a 300-core server grid improves the speed and capacity of the web application. PRISM 3 is available at http://magarveylab.ca/prism/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular Insights into Arctic Soil Organic Matter Degradation under Warming

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

Chen, Hongmei; Yang, Ziming; Chu, Rosalie K.

Molecular composition of the Arctic soil organic carbon (SOC) and its susceptibility to microbial degradation are uncertain due to heterogeneity and unknown SOC compositions. By using ultrahigh-resolution mass spectrometry, we determined the susceptibility and compositional changes of extractable dissolved organic matter (EDOM) in an anoxic warming incubation experiment (up to 122 days) with a tundra soil from Alaska (United States). EDOM was extracted with 10 mM NH 4HCO 3 from both the organic- and mineral-layer soils during incubation at both -2 and 8°C. Based on their O:C and H:C ratios, EDOM molecular formulas were qualitatively grouped into nine biochemical classesmore » of compounds, among which lignin-like compounds dominated both the organic and the mineral soils and were the most stable, whereas amino sugars, peptides, and carbohydrate-like compounds were the most biologically labile. These results corresponded with shifts in EDOM elemental composition in which the ratios of O:C and N:C decreased, while the average C content in EDOM, molecular mass, and aromaticity increased after 122 days of incubation. This research demonstrates that certain EDOM components, such as amino sugars, peptides, and carbohydrate-like compounds, are disproportionately more susceptible to microbial degradation than others in the soil, and these results should be considered in SOC degradation models to improve predictions of Arctic climate feedbacks.« less

Antimicrobial Activity and Cell Selectivity of Synthetic and Biosynthetic Cationic Polymers

PubMed Central

Venkatesh, Mayandi; Barathi, Veluchamy Amutha; Goh, Eunice Tze Leng; Anggara, Raditya; Fazil, Mobashar Hussain Urf Turabe; Ng, Alice Jie Ying; Harini, Sriram; Aung, Thet Tun; Fox, Stephen John; Liu, Shouping; Barkham, Timothy Mark Sebastian; Loh, Xian Jun

2017-01-01

ABSTRACT The mammalian and microbial cell selectivity of synthetic and biosynthetic cationic polymers has been investigated. Among the polymers with peptide backbones, polymers containing amino side chains display greater antimicrobial activity than those with guanidine side chains, whereas ethylenimines display superior activity over allylamines. The biosynthetic polymer ε-polylysine (εPL) is noncytotoxic to primary human dermal fibroblasts at concentrations of up to 2,000 μg/ml, suggesting that the presence of an isopeptide backbone has greater cell selectivity than the presence of α-peptide backbones. Both εPL and linear polyethylenimine (LPEI) exhibit bactericidal properties by depolarizing the cytoplasmic membrane and disrupt preformed biofilms. εPL displays broad-spectrum antimicrobial properties against antibiotic-resistant Gram-negative and Gram-positive strains and fungi. εPL elicits rapid bactericidal activity against both Gram-negative and Gram-positive bacteria, and its biocompatibility index is superior to those of cationic antiseptic agents and LPEI. εPL does not interfere with the wound closure of injured rabbit corneas. In a rabbit model of bacterial keratitis, the topical application of εPL (0.3%, wt/vol) decreases the bacterial burden and severity of infections caused by Pseudomonas aeruginosa and Staphylococcus aureus strains. In vivo imaging studies confirm that εPL-treated corneas appeared transparent and nonedematous compared to untreated infected corneas. Taken together, our results highlight the potential of εPL in resolving topical microbial infections. PMID:28784676

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.

PubMed

Dolashka, Pavlina; Moshtanska, Vesela; Borisova, Valika; Dolashki, Aleksander; Stevanovic, Stefan; Dimanov, Tzvetan; Voelter, Wolfgang

2011-07-01

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. Copyright © 2011 Elsevier Inc. All rights reserved.

Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿

PubMed Central

He, Jian; Eckert, Randal; Pharm, Thanh; Simanian, Maurice D.; Hu, Chuhong; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2007-01-01

Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents. PMID:17296741

Targeted quantification of functional enzyme dynamics in environmental samples for microbially mediated biogeochemical processes: Targeted quantification of functional enzyme dynamics

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

Li, Minjing; Gao, Yuqian; Qian, Wei-Jun

Microbially mediated biogeochemical processes are catalyzed by enzymes that control the transformation of carbon, nitrogen, and other elements in environment. The dynamic linkage between enzymes and biogeochemical species transformation has, however, rarely been investigated because of the lack of analytical approaches to efficiently and reliably quantify enzymes and their dynamics in soils and sediments. Herein, we developed a signature peptide-based technique for sensitively quantifying dissimilatory and assimilatory enzymes using nitrate-reducing enzymes in a hyporheic zone sediment as an example. Moreover, the measured changes in enzyme concentration were found to correlate with the nitrate reduction rate in a way different frommore » that inferred from biogeochemical models based on biomass or functional genes as surrogates for functional enzymes. This phenomenon has important implications for understanding and modeling the dynamics of microbial community functions and biogeochemical processes in environments. Our results also demonstrate the importance of enzyme quantification for the identification and interrogation of those biogeochemical processes with low metabolite concentrations as a result of faster enzyme-catalyzed consumption of metabolites than their production. The dynamic enzyme behaviors provide a basis for the development of enzyme-based models to describe the relationship between the microbial community and biogeochemical processes.« less

The importance of using the optimal plastic and glassware in studies involving peptides

PubMed Central

Goebel-Stengel, Miriam; Stengel, Andreas; Taché, Yvette; Reeve, Joseph R.

2011-01-01

Background The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be utilized. Objective To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research by testing the recovery of radiolabeled 125I-endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. Methods 125I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin releasing factor, glucagon-like peptide-1 (GLP-1), insulin, leptin, nesfatin-1, peptide YY) representing a wide spectrum in net charge, size, end groups and modifications were incubated for 48h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of 125I-peptides was determined by γ-counting. Results Important differences in 125I-peptide binding capacities to various types of surfaces exist. Siliconization decreased while addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing 125I-peptides and BSA in the tubes best suited for individual peptides rendered >89% recovery for all peptides. Ghrelin specifically displaced 125I-ghrelin from borosilicate glass while GLP-1 and Fmoc-arginine did not. Conclusion Choosing the appropriate experimental container avoids unpredictable peptide loss resulting in inaccurate measurements and false conclusions. PMID:21315060

Atomic Force Microscopy Study of the Interactions of Indolicidin with Model Membranes and DNA.

PubMed

Fojan, Peter; Gurevich, Leonid

2017-01-01

The cell membrane is the first barrier and quite often the primary target that antimicrobial peptides (AMPs) have to destroy or penetrate to fulfill their mission. Upon penetrating through the membrane, the peptides can further attack intracellular targets, in particular DNA. Studying the interaction of an antimicrobial peptide with a cell membrane and DNA holds keys to understanding its killing mechanisms. Commonly, these interactions are studied by using optical or scanning electron microscopy and appropriately labeled peptides. However, labeling can significantly affect the hydrophobicity, conformation, and size of the peptide, hence altering the interaction significantly. Here, we describe the use of atomic force microscopy (AFM) for a label-free study of the interactions of peptides with model membranes under physiological conditions and DNA as a possible intracellular target.

Factors Affecting the Production of Aromatic Immonium Ions in MALDI 157 nm Photodissociation Studies

NASA Astrophysics Data System (ADS)

DeGraan-Weber, Nick; Ashley, Daniel C.; Keijzer, Karlijn; Baik, Mu-Hyun; Reilly, James P.

2016-05-01

Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.

Microflora of root filled teeth with apical periodontitis in Latvian patients.

PubMed

Mindere, Anda; Kundzina, Rita; Nikolajeva, Vizma; Eze, Daina; Petrina, Zaiga

2010-01-01

The aim of the present study was to investigate the microbial flora of root filled teeth with apical periodontitis and to determine the prevalence of β-lactamase producing strains in isolated bacteria in Latvian patients. 33 root filled teeth with asymptomatic persisting periapical lesions were selected for the present study. During nonsurgical endodontic retreatment, the root filling material was removed and canals were sampled. Determination of microbial species was based on series of biochemical tests using identification kits. All strains of bacteria were tested for β-lactamase production by using chromogenic nitrocefin-impregnated slides. Bacteria were found in 32 (97%) of initial specimens from the teeth. The number of isolated microbial strains in the specimens ranged from one to six (mean 2.7). 79% of the isolated microbial species were Gram-positive bacteria. The most common isolates were Streptococcus (27%), Actinomyces (27%), Staphylococcus (18%), Enterococcus (18%) and Lactobacillus (18%) spp. Yeasts were found as four isolates in 3 cases (9%). β-lactamase-producing bacterial strains were detected in 12 specimens, 36% of the patients. The most common enzyme-producing bacteria belonged to Actinomyces and Staphylococcus spp. The microbial flora in previously treated root canals with apical periodontitis is limited to a small number of predominantly Gram-positive microbial species. The most common isolates are Streptococcus, Actinomyces, Staphylococcus, Enterococcus and Lactobacillus spp. A moderately high prevalence of β-lactamase producing bacterial strains was detected in patients with root filled teeth with apical periodontitis.

Stream microbial diversity in response to environmental changes: review and synthesis of existing research

PubMed Central

Zeglin, Lydia H.

2015-01-01

The importance of microbial activity to ecosystem function in aquatic ecosystems is well established, but microbial diversity has been less frequently addressed. This review and synthesis of 100s of published studies on stream microbial diversity shows that factors known to drive ecosystem processes, such as nutrient availability, hydrology, metal contamination, contrasting land-use and temperature, also cause heterogeneity in bacterial diversity. Temporal heterogeneity in stream bacterial diversity was frequently observed, reflecting the dynamic nature of both stream ecosystems and microbial community composition. However, within-stream spatial differences in stream bacterial diversity were more commonly observed, driven specifically by different organic matter (OM) compartments. Bacterial phyla showed similar patterns in relative abundance with regard to compartment type across different streams. For example, surface water contained the highest relative abundance of Actinobacteria, while epilithon contained the highest relative abundance of Cyanobacteria and Bacteroidetes. This suggests that contrasting physical and/or nutritional habitats characterized by different stream OM compartment types may select for certain bacterial lineages. When comparing the prevalence of physicochemical effects on stream bacterial diversity, effects of changing metal concentrations were most, while effects of differences in nutrient concentrations were least frequently observed. This may indicate that although changing nutrient concentrations do tend to affect microbial diversity, other environmental factors are more likely to alter stream microbial diversity and function. The common observation of connections between ecosystem process drivers and microbial diversity suggests that microbial taxonomic turnover could mediate ecosystem-scale responses to changing environmental conditions, including both microbial habitat distribution and physicochemical factors. PMID:26042102

Informatic analysis reveals Legionella as a source of novel natural products.

PubMed

Johnston, Chad W; Plumb, Jonathan; Li, Xiang; Grinstein, Sergio; Magarvey, Nathan A

2016-06-01

Microbial natural products are a crucial source of bioactive molecules and unique chemical scaffolds. Despite their importance, rediscovery of known natural products from established productive microbes has led to declining interest, even while emergent genomic data suggest that the majority of microbial natural products remain to be discovered. Now, new sources of microbial natural products must be defined in order to provide chemical scaffolds for the next generation of small molecules for therapeutic, agricultural, and industrial purposes. In this work, we use specialized bioinformatic programs, genetic knockouts, and comparative metabolomics to define the genus Legionella as a new source of novel natural products. We show that Legionella spp. hold a diverse collection of biosynthetic gene clusters for the production of polyketide and nonribosomal peptide natural products. To confirm this bioinformatic survey, we create targeted mutants of L. pneumophila and use comparative metabolomics to identify a novel polyketide surfactant. Using spectroscopic techniques, we show that this polyketide possesses a new chemical scaffold, and firmly demonstrate that this unexplored genus is a source for novel natural products.

Characterization and Imaging of Antibody-Coated Gold Nanoparticles for Targeted Treatment of Microbial Keratitis

NASA Astrophysics Data System (ADS)

Mahan, Matthew

Microbial keratitis (MK) is an infection of the cornea by pathogenic organisms that causes inflammation and irritation. It can lead to full or partial blindness if left untreated. Current clinical treatment methods rely on high frequency application of topical drugs which are subject to the issues of patient compliance and microbial resistance. In this work, gold nanoparticles (AuNP) were proposed as an alternative treatment method in light-based therapies. Particle formulation methods were investigated and assessed using transmission electron microscopy (TEM) and ultraviolet/visible spectroscopy (UV-Vis). AuNP of 20 nm diameter were used as platforms to attach monoclonal antibodies anti-FLAG or anti-F1 to enhance their cell-targeting ability as well as polyethylene glycol to reduce non-specific binding and protein adsorption. These functionalized particles were qualitatively assessed using UV-Vis. The antibody-functionalized AuNP were then assessed for their ability to attach directly to Pseudomonas aeruginosa, expressing FLAG peptide, or Aspergillus fumigatus, expressing the F1 receptor. Attachment was imaged using dark field microscopy, transmission electron microscopy, and fluorescence microscopy.

Immunity in Drosophila melanogaster--from microbial recognition to whole-organism physiology.

PubMed

Buchon, Nicolas; Silverman, Neal; Cherry, Sara

2014-12-01

Since the discovery of antimicrobial peptide responses 40 years ago, the fruit fly Drosophila melanogaster has proven to be a powerful model for the study of innate immunity. Early work focused on innate immune mechanisms of microbial recognition and subsequent nuclear factor-κB signal transduction. More recently, D. melanogaster has been used to understand how the immune response is regulated and coordinated at the level of the whole organism. For example, researchers have used this model in studies investigating interactions between the microbiota and the immune system at barrier epithelial surfaces that ensure proper nutritional and immune homeostasis both locally and systemically. In addition, studies in D. melanogaster have been pivotal in uncovering how the immune response is regulated by both endocrine and metabolic signalling systems, and how the immune response modifies these systems as part of a homeostatic circuit. In this Review, we briefly summarize microbial recognition and antiviral immunity in D. melanogaster, and we highlight recent studies that have explored the effects of organism-wide regulation of the immune response and, conversely, the effects of the immune response on organism physiology.

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

PubMed

Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

2015-05-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

PubMed Central

Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

2015-01-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

Antimicrobial peptide-like genes in Nasonia vitripennis: a genomic perspective

PubMed Central

2010-01-01

Background Antimicrobial peptides (AMPs) are an essential component of innate immunity which can rapidly respond to diverse microbial pathogens. Insects, as a rich source of AMPs, attract great attention of scientists in both understanding of the basic biology of the immune system and searching molecular templates for anti-infective drug design. Despite a large number of AMPs have been identified from different insect species, little information in terms of these peptides is available from parasitic insects. Results By using integrated computational approaches to systemically mining the Hymenopteran parasitic wasp Nasonia vitripennis genome, we establish the first AMP repertoire whose members exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types, including insect and fungal defensin-like peptides (DLPs) with the cysteine-stabilized α-helical and β-sheet (CSαβ) fold; Pro- or Gly-rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the inhibitor cystine knot (ICK) fold; and a linear α-helical peptide. Inducible expression pattern of seven N. vitripennis AMP genes were verified, and two representative peptides were synthesized and functionally identified to be antibacterial. In comparison with Apis mellifera (Hymenoptera) and several non-Hymenopteran model insects, N. vitripennis has evolved a complex antimicrobial immune system with more genes and larger protein precursors. Three classical strategies that are likely responsible for the complexity increase have been recognized: 1) Gene duplication; 2) Exon duplication; and 3) Exon-shuffling. Conclusion The present study established the N. vitripennis peptidome associated with antimicrobial immunity by using a combined computational and experimental strategy. As the first AMP repertoire of a parasitic wasp, our results offer a basic platform for further studying the immunological and evolutionary significances of these newly discovered AMP-like genes in this class of insects. PMID:20302637

The molecular mechanism for interaction of ceruloplasmin and myeloperoxidase

NASA Astrophysics Data System (ADS)

Bakhautdin, Bakytzhan; Bakhautdin, Esen Göksöy

2016-04-01

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.

Toward structure prediction of cyclic peptides.

PubMed

Yu, Hongtao; Lin, Yu-Shan

2015-02-14

Cyclic peptides are a promising class of molecules that can be used to target specific protein-protein interactions. A computational method to accurately predict their structures would substantially advance the development of cyclic peptides as modulators of protein-protein interactions. Here, we develop a computational method that integrates bias-exchange metadynamics simulations, a Boltzmann reweighting scheme, dihedral principal component analysis and a modified density peak-based cluster analysis to provide a converged structural description for cyclic peptides. Using this method, we evaluate the performance of a number of popular protein force fields on a model cyclic peptide. All the tested force fields seem to over-stabilize the α-helix and PPII/β regions in the Ramachandran plot, commonly populated by linear peptides and proteins. Our findings suggest that re-parameterization of a force field that well describes the full Ramachandran plot is necessary to accurately model cyclic peptides.

Structural basis for precursor protein-directed ribosomal peptide macrocyclization

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

Li, Kunhua; Condurso, Heather L.; Li, Gengnan

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight intomore » the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.« less

Synthesis, molecular docking and anticancer studies of peptides and iso-peptides.

PubMed

Jabeen, Farukh; Panda, Siva S; Kondratyuk, Tamara P; Park, Eun-Jung; Pezzuto, John M; Ihsan-ul-Haq; Hall, C Dennis; Katritzky, Alan R

2015-08-01

Chiral peptides and iso-peptides were synthesized in excellent yield by using benzotriazole mediated solution phase synthesis. Benzotriazole acted both as activating and leaving group, eliminating frequent use of protection and subsequent deprotection. The procedure was based on the hypothesis that epimerization should be suppressed in solution due to a faster coupling rate than SPPS. All the synthesized peptides complied with Lipinski's Ro5 except for the rotatable bonds. Inhibition of cell proliferation of cancer cell lines is one of the most commonly used methods to study the effectiveness of any anticancer agents. Synthesized peptides and iso-peptides were tested against three cancer cell lines (MCF-7, MDA-MB 231) to determine their anti-proliferative potential. NFkB was also determined. Molecular docking studies were also carried out to complement the experimental results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Improving oral bioavailability of cyclic peptides by N-methylation.

PubMed

Räder, Andreas F B; Reichart, Florian; Weinmüller, Michael; Kessler, Horst

2018-06-01

The renaissance of peptides in pharmaceutical industry results from their importance in many biological functions. However, low metabolic stability and the lack of oral availability of most peptides is a certain limitation. Whereas metabolic instability may be often overcome by development of small cyclic peptides containing d-amino acids, the very low oral availability of most peptides is a serious limitation for some medicinal applications. The situation is complicated because a twofold optimization - biological activity and oral availability - is required to overcome this problem. Moreover, most simple "rules" for achieving oral availability are not general and are applicable only to limited cases. Many structural modifications for increasing biological activities and metabolic stabilities of cyclic peptides have been described, of which N-alkylation is probably the most common. This mini-review focuses on the effects of N-methylation of cyclic peptides in strategies to optimize bioavailabilities. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Towards the Improved Discovery and Design of Functional Peptides: Common Features of Diverse Classes Permit Generalized Prediction of Bioactivity

PubMed Central

Mooney, Catherine; Haslam, Niall J.; Pollastri, Gianluca; Shields, Denis C.

2012-01-01

The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides. We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4–20 amino acids) and one focused on long peptides ( amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure. We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive. PMID:23056189

Microbial aetiology and sensitivity of asymptomatic bacteriuria among ante-natal mothers in Mulago hospital, Uganda.

PubMed

Andabati, G; Byamugisha, J

2010-12-01

Asymptomatic bacteriuria in pregnancy is associated with potential urinary and obstetric complications. However the prevalence aetiology and antimicrobial sensitivity patterns of asymptomatic bacteriurea among women attending ante-natal care in our Hospital is not known. To determine the prevalence and identify the aetiological agents associated with asymptomatic bacteriurea in antenatal mothers in Mulago Hospital. We also intended to determine the anti-microbial sensitivity patterns of the common uropathogen in this population We performed culture and anti-microbial sensitivity tests on urine samples from 218 consecutive ante-natal mothers in Mulago Hospital. All participants did not have any clinical symptoms attributable to urinary tract infection. Twenty nine (13.3%) of the samples had significant bacterial growth and E.coli was the commonest isolate (51.2%). There was a high level (20-62%) of anti-bacterial resistance to the commonly used antibiotics. Asymptomatic bacteriuria is common among ante-natal mothers in Mulago. E. Coli that is resistant to the most commonly used antibiotics is the commonest isolate."

Cell surface engineering of microorganisms towards adsorption of heavy metals.

PubMed

Li, Peng-Song; Tao, Hu-Chun

2015-06-01

Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed.

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction

PubMed Central

Uegaki, Koichi; Kumanogoh, Haruko; Mizui, Toshiyuki; Hirokawa, Takatsugu; Ishikawa, Yasuyuki; Kojima, Masami

2017-01-01

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism. PMID:28498321

Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations.

PubMed

Heljo, P; Ross, A; Zarraga, I E; Pappenberger, A; Mahler, H-C

2015-10-01

Antimicrobial preservatives are known to interact with proteins and potentially affect their stability in aqueous solutions. In this systematic study, the interactions of a model peptide with three commonly used preservatives, benzyl alcohol, phenol and m-cresol, were evaluated. The impact on peptide oligomerization was studied using GC-MALS, SEC-MALS and DLS, antimicrobial efficiency of different formulations were studied using the Ph. Eur. antimicrobial efficacy test, and the molecular adsorption of preservative molecules on reversible peptide oligomers was monitored using NMR. The hydrodynamic radius and molar mass of the peptide oligomers was shown to clearly increase in the presence of m-cresol but less significantly with phenol and benzyl alcohol. The increase in size was most likely caused by peptide self-interactions becoming more attractive, leading to reversible oligomerization. On the other hand, increasing the concentration of peptide in multi-dose formulations led to reduced molecular mobility and decreased antimicrobial efficacy of all preservatives. Peptide-preservative interactions not only affect peptide self-interactions, but also antimicrobial efficiency of the preservatives and are thus of significant relevance. Adsorption of preservatives on oligomeric states of peptides is proposed as a mechanism to explain this reduced antimicrobial efficacy.

Protonation States in molecular dynamics simulations of peptide folding and binding.

PubMed

Ben-Shimon, Avraham; Shalev, Deborah E; Niv, Masha Y

2013-01-01

Peptides are important signaling modules, acting both as individual hormones and as parts of larger molecules, mediating their protein-protein interactions. Many peptidic and peptidomimetic drugs have reached the marketplace and opportunities for peptide-based drug discovery are on the rise. pH-dependent behavior of peptides is well documented in the context of misfolding diseases and peptide translocation. Changes in the protonation states of peptide residues often have a crucial effect on a peptide's structure, dynamics and function, which may be exploited for biotechnological applications. The current review surveys the increasing levels of sophistication in the treatment of protonation states in computational studies involving peptides. Specifically we describe I) the common practice of assigning a single protonation state and using it throughout the dynamic simulation, II) approaches that consider multiple protonation states and compare computed observables to experimental ones, III) constant pH molecular dynamics methods that couple changes in protonation states with conformational dynamics "on the fly". Applications of conformational dynamics treatment of peptides in the context of binding, folding and interactions with the membrane are presented, illustrating the growing body of work in this field and highlighting the importance of careful handling of protonation states of peptidic residues.

Immunogenicity of peptides of measles virus origin and influence of adjuvants.

PubMed

Halassy, Beata; Mateljak, Sanja; Bouche, Fabienne B; Pütz, Mike M; Muller, Claude P; Frkanec, Ruza; Habjanec, Lidija; Tomasić, Jelka

2006-01-12

Epitope-based peptide antigens have been under development for protection against measles virus. The immunogenicity of five peptides composed of the same B cell epitope (BCE) (H236-250 of the measles virus hemagglutinin), and different T cell epitopes of measles virus fusion protein (F421-435, F256-270, F288-302) and nucleoprotein (NP335-345) was studied in mice (subcutaneous immunisation). The adjuvant effects of peptidoglycan monomer (PGM), Montanide ISA 720 and 206 were also investigated. Results showed basic differences in peptide immunogenicity that were consistent with already described structural differences. PGM elevated peptide-specific IgG when applied together with four of five tested peptides. A strong synergistic effect was observed after co-immunisation of mice with a mixture containing all five chimeric peptides in small and equal amounts. Results revealed for the first time that immunisation with several peptides having the common BCE generated significantly higher levels of both anti-peptide and anti-BCE IgG in comparison to those obtained after immunisation with a single peptide in much higher quantity. Further improvement of immune response was obtained after incorporation of such a peptide mixture into oil-based adjuvants.

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update.

PubMed

Muller, Jean-Marc; Debaigt, Colin; Goursaud, Stéphanie; Montoni, Alicia; Pineau, Nicolas; Meunier, Annie-Claire; Janet, Thierry

2007-09-01

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.

Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens.

PubMed

Hicks, R P; Abercrombie, J J; Wong, R K; Leung, K P

2013-01-01

A series of 36 synthetic antimicrobial peptides containing unnatural amino acids were screened to determine their effectiveness to treat Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens, which are known to commonly infect chronic wounds. The primary amino acid sequences of these peptides incorporate either three or six dipeptide units consisting of the unnatural amino acids Tetrahydroisoquinolinecarboxylic acid (Tic) and Octahydroindolecarboxylic acid (Oic). The Tic-Oic dipeptide units are separated by SPACER amino acids with specific physicochemical properties that control how these peptides interact with bacterial cell membranes of different chemical compositions. These peptides exhibited minimum inhibitory concentrations (MIC) against these pathogens in the range from >100 to 6.25 μg/mL. The observed diversity of MIC values for these peptides against the various bacterial strains are consistent with our hypothesis that the complementarity of the physicochemical properties of the peptide and the lipid of the bacteria's cell membrane determines the resulting antibacterial activity of the peptide. Published by Elsevier Ltd.

Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

PubMed

Matsumoto, Emiko; Fujita, Yuko; Okada, Yohei; Kauppinen, Esko I; Kamiya, Hidehiro; Chiba, Kazuhiro

2015-09-01

C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic

PubMed Central

Kristensen, Kasper; Henriksen, Jonas R.; Andresen, Thomas L.

2015-01-01

Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects. PMID:25932639

Recombinant production of antimicrobial peptides in Escherichia coli: a review.

PubMed

Li, Yifeng

2011-12-01

Antimicrobial peptides are of great interest due to their potential application as novel antibiotics. Large quantities of highly purified peptides are required to meet the needs of basic research and clinical trials. Compared with isolation from natural sources and chemical synthesis, recombinant approach offers the most cost-effective means for large-scale peptide manufacture. Among the systems available for heterologous protein production, Escherichia coli has been the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, a strategy necessary to mask these peptides' lethal effect towards the host and protect them from proteolytic degradation. The present article reviews commonly used fusion partners (e.g., solubility-enhancing, aggregation-promoting and self-cleavable carriers, etc.), cleavage methods and optimization options for antimicrobial peptides production in E. coli. In addition, the various approaches developed to generate recombinant human antimicrobial peptide LL-37, which offer excellent examples demonstrating effective production strategies, were briefly discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Trophic interactions induce spatial self-organization of microbial consortia on rough surfaces.

PubMed

Wang, Gang; Or, Dani

2014-10-24

The spatial context of microbial interactions common in natural systems is largely absent in traditional pure culture-based microbiology. The understanding of how interdependent microbial communities assemble and coexist in limited spatial domains remains sketchy. A mechanistic model of cell-level interactions among multispecies microbial populations grown on hydrated rough surfaces facilitated systematic evaluation of how trophic dependencies shape spatial self-organization of microbial consortia in complex diffusion fields. The emerging patterns were persistent irrespective of initial conditions and resilient to spatial and temporal perturbations. Surprisingly, the hydration conditions conducive for self-assembly are extremely narrow and last only while microbial cells remain motile within thin aqueous films. The resulting self-organized microbial consortia patterns could represent optimal ecological templates for the architecture that underlie sessile microbial colonies on natural surfaces. Understanding microbial spatial self-organization offers new insights into mechanisms that sustain small-scale soil microbial diversity; and may guide the engineering of functional artificial microbial consortia.

Novel Structures of Self-Associating Stapled Peptides

PubMed Central

Bhattacharya, Shibani; Zhang, Hongtao; Cowburn, David; Debnath, Asim K.

2012-01-01

Hydrocarbon stapling of peptides is a powerful technique to transform linear peptides into cell-permeable helical structures that can bind to specific biological targets. In this study, we have used high resolution solution NMR techniques complemented by Dynamic Light Scattering to characterize extensively a family of hydrocarbon stapled peptides with known inhibitory activity against HIV-1 capsid assembly to evaluate the various factors that modulate activity. The helical peptides share a common binding motif but differ in charge, the length and position of the staple. An important outcome of the study was to show the peptides share a propensity to self-associate into organized polymeric structures mediated predominantly by hydrophobic interactions between the olefinic chain and the aromatic side-chains from the peptide. We have also investigated in detail the structural significance of the length and position of the staple, and of olefinic bond isomerization in stabilizing the helical conformation of the peptides as potential factors driving polymerization. This study presents the numerous challenges of designing biologically active stapled peptides and the conclusions have broad implications for optimizing a promising new class of compounds in drug discovery. PMID:22170623

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients.

PubMed

Thong, Kwai-Lin; Tang, Swee-Seong; Tan, Wen-Siang; Devi, Shamala

2007-01-01

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

PubMed

Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

2015-09-09

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

Alanine rich peptide from Populus trichocarpa inhibit growth of Staphylococcus aureus via targetting its extracellular domain of Sensor Histidine Kinase YycGex protein.

PubMed

Al Akeel, Raid; Mateen, Ayesha; Syed, Rabbani; Alqahtani, Mohammed S; Alqahtani, Ali S

2018-05-22

Due to growing concern towards microbial resistance, ongoing search for developing novel bioactive compounds such as peptides is on rise. The aim of this study was to evaluate antimicrobial effect of Populus trichocarpa extract, chemically identify the active peptide fraction and finds its target in Staphylococcus aureus. In this study the active fraction of P. trichocarpa crude extract was purified and characterized using MS/MS. This peptide PT13 antimicrobial activity was confirmed by in-vitro agar based disk diffusion and in-vivo infection model of G. mellonella. The proteomic expression analysis of S. aureus under influence of PT13 was studied using LTQ-Orbitrap-MS in-solution digestion and identity of target protein was acquired with their quantified expression using label-free approach of Progenesis QI software. Docking study was performed with peptide PT13 and its target YycG protein using CABS-dock. The active fraction PT13 sequence was identified as KVPVAAAAAAAAAVVASSMVVAAAK, with 25 amino acid including 13 alanine having M/Z 2194.2469. PT13 was uniformly inhibited growth S. aureus SA91 and MIC was determined 16 μg/mL for SA91 S. aureus strain. Sensor histidine kinase (YycG) was most significant target found differentially expressed under influence of PT13. G. mellonella larvae were killed rapidly due to S aureus infection, whereas death in protected group was insignificant in compare to control. The docking models showed ten docking models with RMSD value 1.89 for cluster 1 and RMSD value 3.95 for cluster 2 which is predicted to be high quality model. Alanine rich peptide could be useful in constructing as antimicrobial peptide for targeting extracellular Domain of Sensor Histidine Kinase YycG from S. aureus used in the study. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fusarium verticillioides genes conferring xenobiotic detoxification

USDA-ARS?s Scientific Manuscript database

Phytochemicals, microbial metabolites, and agrochemicals can individually or collectively impact the diversity and frequency of microbial species occurring in agricultural field environments. Resistance to such chemicals by plant pathogenic fungi is common and potentially devastating to crop yield a...

Correlating low-similarity peptide sequences and allergenic epitopes.

PubMed

Kanduc, D

2008-01-01

Although a high number of allergenic peptide epitopes has been experimentally identified and defined, the molecular basis and the precise mechanisms underlying peptide allergenicity are unknown. This issue was analyzed exploring the relationship between peptide allergenicity and sequence similarity to the human proteome. The structured analysis of the data reported in literature put into evidence that the most part of IgE-binding epitopes are (or harbor) pentapeptide unit(s) with no/low similarity to the human proteome, this way suggesting that no or low sequence similarity to the host proteome might represent a minimum common denominator identifying allergenic peptides. The present literature analysis might be of relevance in devising and designing short amino acid modules to be used for blocking pathogenic IgE.

Plant resource history effects on contemporary microbial processes

NASA Astrophysics Data System (ADS)

Bradford, Mark

2010-05-01

Background/Questions/Methods Soil microbial communities play a pivotal role in providing ecosystem services, given that they are key drivers of biogeochemical processes such as carbon and nitrogen cycling. As species-rich communities, made-up of populations with short generation times, it is commonly assumed that there is a high degree of functional redundancy within soil communities with respect to broad-physiological processes, such as organic carbon decomposition. This assumption underlies the majority of terrestrial ecosystem models, where relationships between processes and controlling factors are parameterized using statistical relationships generated from measurements across space. However, microbial communities display biogeographic patterns, even at fine scales and it is possible that these patterns extend to influence their function. I first present experiments that combine common garden and reciprocal transplant approaches to test whether microbial communities sourced from distinct habitats across the contiguous United States exhibit functional similarity (i.e. redundancy) or dissimilarity in common environments. The environments are experimental microcosms composed of leaf litters differing markedly in recalcitrance. Following inoculation with the microbial communities, decomposition rates are followed across 300 days. Next, using a similar experimental microcosm design, I present experiments that test whether a common resource history might cause communities, which are initially dissimilar, to converge functionally. Distinct microbial communities are introduced into a fresh litter environment every 100 days for 300 days total. Finally, I present an experiment to test whether functional convergence (either partial or complete) is associated with a reduction in function in alternate environments (essentially a functional ‘trade-off'). In this last experiment decomposition rates are measured for 100 days in alternate environments following 300 days of successive exposure to a single litter type. Results/Conclusions I show in the first experiment that rates of carbon dioxide production from litter decomposition are strongly dependent on the microbial inoculum, with differences in the microbial community alone accounting for substantial (up to 85%) variation in total carbon mineralized. Communities that share a common history with a given foliar litter exhibit higher decomposition rates when compared to communities foreign to that habitat, suggestive of local adaptation. In the second experiment, decomposition rates (measured as cumulative carbon dioxide values per 100-day run) converge partially in the second 100-day run, and cumulative values for all six inocula increase toward the highest values observed in the initial 100-day run. Convergence continues but at a reduced rate in the third 100-day run; yet increases in function appear to asymptote. The increasing similarity in cumulative values between inocula, observed in the successive 100-day runs, is consistent with partial functional convergence of communities exposed to a common environment. In the final experiment, microbial inocula crossed to an alternate environment (e.g. those communities maintained in a grass environment then inoculated into a hardwood litter environment) results in cumulative values in the alternate environments that are approximately half those observed for communities inoculated into the same environment. Collectively, our results suggest that the implicit assumption in ecosystem models (i.e. microbial communities in the same environment are functionally equivalent) is incorrect. The increasing similarity across time in function in common environments of inocula sourced from different resource habitats shows the potential for function to converge. Yet this convergence is only partial and is associated with "apparent' trade-offs in the ability to decompose substrates from the original environments from which the inocula were sourced. To predict accurately how biogeochemical processes will respond to global change may require consideration of the community composition and/or adaptation of microbial communities to past resource environments and the consequences of their (partial) adaptation to new ones.

Microbial responses to microgravity and other low-shear environments.

PubMed

Nickerson, Cheryl A; Ott, C Mark; Wilson, James W; Ramamurthy, Rajee; Pierson, Duane L

2004-06-01

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.

Microbial Responses to Microgravity and Other Low-Shear Environments

NASA Technical Reports Server (NTRS)

Nickerson, Cheryl A.; Ott, C. Mark; Wilson, James W.; Ramamurthy, Rajee; Pierson, Duane L.

2004-01-01

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.

L1 Peptide-Conjugated Fibrin Hydrogels Promote Salivary Gland Regeneration.

PubMed

Nam, K; Wang, C-S; Maruyama, C L M; Lei, P; Andreadis, S T; Baker, O J

2017-07-01

Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.

Computational study of solution behavior of magainin 2 monomers.

PubMed

Petkov, P; Marinova, R; Kochev, V; Ilieva, N; Lilkova, E; Litov, L

2018-03-27

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity - the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.

Peptide Identification by Database Search of Mixture Tandem Mass Spectra*

PubMed Central

Wang, Jian; Bourne, Philip E.; Bandeira, Nuno

2011-01-01

In high-throughput proteomics the development of computational methods and novel experimental strategies often rely on each other. In certain areas, mass spectrometry methods for data acquisition are ahead of computational methods to interpret the resulting tandem mass spectra. Particularly, although there are numerous situations in which a mixture tandem mass spectrum can contain fragment ions from two or more peptides, nearly all database search tools still make the assumption that each tandem mass spectrum comes from one peptide. Common examples include mixture spectra from co-eluting peptides in complex samples, spectra generated from data-independent acquisition methods, and spectra from peptides with complex post-translational modifications. We propose a new database search tool (MixDB) that is able to identify mixture tandem mass spectra from more than one peptide. We show that peptides can be reliably identified with up to 95% accuracy from mixture spectra while considering only a 0.01% of all possible peptide pairs (four orders of magnitude speedup). Comparison with current database search methods indicates that our approach has better or comparable sensitivity and precision at identifying single-peptide spectra while simultaneously being able to identify 38% more peptides from mixture spectra at significantly higher precision. PMID:21862760

Microbial diversity in the floral nectar of seven Epipactis (Orchidaceae) species

PubMed Central

Jacquemyn, Hans; Lenaerts, Marijke; Tyteca, Daniel; Lievens, Bart

2013-01-01

Abstract Floral nectar of animal-pollinated plants is commonly infested with microorganisms, yet little is known about the microorganisms inhabiting the floral nectar of orchids. In this study, we investigated microbial communities occurring in the floral nectar of seven Epipactis (Orchidaceae) species. Culturable bacteria and yeasts were isolated and identified by partially sequencing the small subunit (SSU) ribosomal RNA (rRNA) gene and the D1/D2 domains of the large subunit (LSU) rRNA gene, respectively. Using three different culture media, we found that bacteria were common inhabitants of the floral nectar of Epipactis. The most widely distributed bacterial operational taxonomic units (OTUs) in nectar of Epipactis were representatives of the family of Enterobacteriaceae, with an unspecified Enterobacteriaceae bacterium as the most common. In contrast to previous studies investigating microbial communities in floral nectar, very few yeast species (mainly of the genus Cryptococcus) were observed, and most of them occurred in very low densities. Total OTU richness (i.e., the number of bacterial and yeast OTUs per orchid species) varied between 4 and 20. Cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species. This study extends previous efforts to identify microbial communities in floral nectar and indicates that the floral nectar of the orchids investigated mainly contained bacterial communities with moderate phylogenetic diversity. PMID:23836678

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway.

PubMed

Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-12-12

In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H 2 O drinking water; (2) mock: H 2 O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg -1 )+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg -1 )+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg -1 )+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg -1 )+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake, inflammatory reaction and the formation of fatty liver by activating JAK2 signal transduction through the JAK2/STAT3 and JAK2/AMPK pathways in the high-fructose-induced fatty liver animal model. Therefore, kefir peptides may have the potential for clinical application for the prevention or treatment of clinical metabolic syndrome.

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway

PubMed Central

Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-01-01

Objective: In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. Results: An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H2O drinking water; (2) mock: H2O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg−1)+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg−1)+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg−1)+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg−1)+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Conclusion: Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake, inflammatory reaction and the formation of fatty liver by activating JAK2 signal transduction through the JAK2/STAT3 and JAK2/AMPK pathways in the high-fructose-induced fatty liver animal model. Therefore, kefir peptides may have the potential for clinical application for the prevention or treatment of clinical metabolic syndrome. PMID:27941940

Host-defense peptides from skin secretions of the octoploid frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary relationships.

PubMed

Mechkarska, Milena; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; Michalak, Katarzyna; Michalak, Pawel; Conlon, J Michael

2014-09-01

The primary structures of host-defense peptides have proved useful in elucidating the evolution history of frogs. Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from the octoploid frogs, Xenopus vestitus (Kivu clawed frog) and Xenopus wittei (De Witte's clawed frog) in the family Pipidae. Structural characterization demonstrated that the X. vestitus peptides belong to the magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 4 peptides), xenopsin-precursor fragment (XPF; 1 peptide), and caerulein-precursor fragment (CPF; 5 peptides) families. The X. wittei peptides comprise magainin (4 peptides), PGLa (1 peptide), XPF (2 peptides), and CPF (7 peptides). In addition, secretions from both species contain caerulein, identical to the peptide from Xenopus laevis, but X. wittei secretions contains the novel peptide [R4K]xenopsin. The variability in the numbers of paralogs in each peptide family indicates a selective silencing of the host-defense peptide genes following the polyploidization events. The primary structures of the peptides provide insight into phylogenetic relationships among the octoploid Xenopus frogs. The data support a sister-group relationship between X. vestitus and Xenopus lenduensis, suggestive of bifurcating speciation after allopolyploidization, whereas X. wittei is more closely related to the Xenopus amieti-Xenopus andrei group suggesting a common tetraploid ancestor. Consistent with previous data, the CPF peptides showed the highest growth inhibitory activity against bacteria with CPF-W6 (GIGSLLAKAAKLAAGLV.NH2) combining high antimicrobial potency against Staphylococcus aureus (MIC=4 μM) with relatively low hemolytic activity (LC50=190 μM). Copyright © 2014 Elsevier Inc. All rights reserved.

The Modern Synthesis in the Light of Microbial Genomics.

PubMed

Booth, Austin; Mariscal, Carlos; Doolittle, W Ford

2016-09-08

We review the theoretical implications of findings in genomics for evolutionary biology since the Modern Synthesis. We examine the ways in which microbial genomics has influenced our understanding of the last universal common ancestor, the tree of life, species, lineages, and evolutionary transitions. We conclude by advocating a piecemeal toolkit approach to evolutionary biology, in lieu of any grand unified theory updated to include microbial genomics.

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

NASA Astrophysics Data System (ADS)

Wang, Yukun; Chen, Charles H.; Hu, Dan; Ulmschneider, Martin B.; Ulmschneider, Jakob P.

2016-11-01

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures--formed by a single sequence--may be a key feature in preventing bacterial resistance and could explain why sequence-function relationships in AMPs remain elusive.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

PubMed

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

PubMed

Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

2015-04-21

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

MapReduce implementation of a hybrid spectral library-database search method for large-scale peptide identification.

PubMed

Kalyanaraman, Ananth; Cannon, William R; Latt, Benjamin; Baxter, Douglas J

2011-11-01

A MapReduce-based implementation called MR-MSPolygraph for parallelizing peptide identification from mass spectrometry data is presented. The underlying serial method, MSPolygraph, uses a novel hybrid approach to match an experimental spectrum against a combination of a protein sequence database and a spectral library. Our MapReduce implementation can run on any Hadoop cluster environment. Experimental results demonstrate that, relative to the serial version, MR-MSPolygraph reduces the time to solution from weeks to hours, for processing tens of thousands of experimental spectra. Speedup and other related performance studies are also reported on a 400-core Hadoop cluster using spectral datasets from environmental microbial communities as inputs. The source code along with user documentation are available on http://compbio.eecs.wsu.edu/MR-MSPolygraph. ananth@eecs.wsu.edu; william.cannon@pnnl.gov. Supplementary data are available at Bioinformatics online.

Bioactive Coatings for Orthopaedic Implants—Recent Trends in Development of Implant Coatings

PubMed Central

Zhang, Bill G. X.; Myers, Damian E.; Wallace, Gordon G.; Brandt, Milan; Choong, Peter F. M.

2014-01-01

Joint replacement is a major orthopaedic procedure used to treat joint osteoarthritis. Aseptic loosening and infection are the two most significant causes of prosthetic implant failure. The ideal implant should be able to promote osteointegration, deter bacterial adhesion and minimize prosthetic infection. Recent developments in material science and cell biology have seen the development of new orthopaedic implant coatings to address these issues. Coatings consisting of bioceramics, extracellular matrix proteins, biological peptides or growth factors impart bioactivity and biocompatibility to the metallic surface of conventional orthopaedic prosthesis that promote bone ingrowth and differentiation of stem cells into osteoblasts leading to enhanced osteointegration of the implant. Furthermore, coatings such as silver, nitric oxide, antibiotics, antiseptics and antimicrobial peptides with anti-microbial properties have also been developed, which show promise in reducing bacterial adhesion and prosthetic infections. This review summarizes some of the recent developments in coatings for orthopaedic implants. PMID:25000263

Antimicrobial Peptides from Fruits and Their Potential Use as Biotechnological Tools—A Review and Outlook

PubMed Central

Meneguetti, Beatriz T.; Machado, Leandro dos Santos; Oshiro, Karen G. N.; Nogueira, Micaella L.; Carvalho, Cristiano M. E.; Franco, Octávio L.

2017-01-01

Bacterial resistance is a major threat to plant crops, animals and human health, and over the years this situation has increasingly spread worldwide. Due to their many bioactive compounds, plants are promising sources of antimicrobial compounds that can potentially be used in the treatment of infections caused by microorganisms. As well as stem, flowers and leaves, fruits have an efficient defense mechanism against pests and pathogens, besides presenting nutritional and functional properties due to their multifunctional molecules. Among such compounds, the antimicrobial peptides (AMPs) feature different antimicrobials that are capable of disrupting the microbial membrane and of acting in binding to intra-cytoplasmic targets of microorganisms. They are therefore capable of controlling or halting the growth of microorganisms. In summary, this review describes the major classes of AMPs found in fruits, their possible use as biotechnological tools and prospects for the pharmaceutical industry and agribusiness. PMID:28119671

Antimicrobial peptides extend lifespan in Drosophila

PubMed Central

Mori, Tetsushi; Carrera, Pilar; Schroer, Jonas; Takeyama, Haruko

2017-01-01

Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. We expressed single AMPs in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. We found that activation of single AMPs, including Drosocin, resulted in a significant extension of Drosophila lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal Drosocin induction protected the animals against infections with the natural Drosophila pathogen Pseudomonas entomophila, whereas a germ-reduced environment prevented the lifespan extending effect of Drosocin. Our study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing. PMID:28520752

Antimicrobial peptides extend lifespan in Drosophila.

PubMed

Loch, Gerrit; Zinke, Ingo; Mori, Tetsushi; Carrera, Pilar; Schroer, Jonas; Takeyama, Haruko; Hoch, Michael

2017-01-01

Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. We expressed single AMPs in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. We found that activation of single AMPs, including Drosocin, resulted in a significant extension of Drosophila lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal Drosocin induction protected the animals against infections with the natural Drosophila pathogen Pseudomonas entomophila, whereas a germ-reduced environment prevented the lifespan extending effect of Drosocin. Our study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing.

Labile and recalcitrant organic matter utilization by river biofilm under increasing water temperature.

PubMed

Ylla, Irene; Romaní, Anna M; Sabater, Sergi

2012-10-01

Microbial biofilms in rivers contribute to the decomposition of the available organic matter which typically shows changes in composition and bioavailability due to their origin, seasonality, and watershed characteristics. In the context of global warming, enhanced biofilm organic matter decomposition would be expected but this effect could be specific when either a labile or a recalcitrant organic matter source would be available. A laboratory experiment was performed to mimic the effect of the predicted increase in river water temperature (+4 °C above an ambient temperature) on the microbial biofilm under differential organic matter sources. The biofilm microbial community responded to higher water temperature by increasing bacterial cell number, respiratory activity (electron transport system) and microbial extracellular enzymes (extracellular enzyme activity). At higher temperature, the phenol oxidase enzyme explained a large fraction of respiratory activity variation suggesting an enhanced microbial use of degradation products from humic substances. The decomposition of hemicellulose (β-xylosidase activity) seemed to be also favored by warmer conditions. However, at ambient temperature, the enzymes highly responsible for respiration activity variation were β-glucosidase and leu-aminopeptidase, suggesting an enhanced microbial use of polysaccharides and peptides degradation products. The addition of labile dissolved organic carbon (DOC; dipeptide plus cellobiose) caused a further augmentation of heterotrophic biomass and respiratory activity. The changes in the fluorescence index and the ratio Abs(250)/total DOC indicated that higher temperature accelerated the rates of DOC degradation. The experiment showed that the more bioavailable organic matter was rapidly cycled irrespective of higher temperature while degradation of recalcitrant substances was enhanced by warming. Thus, pulses of carbon at higher water temperature might have consequences for DOC processing.

Contrasting genomic properties of free-living and particle-attached microbial assemblages within a coastal ecosystem

PubMed Central

Smith, Maria W.; Zeigler Allen, Lisa; Allen, Andrew E.; Herfort, Lydie; Simon, Holly M.

2013-01-01

The Columbia River (CR) is a powerful economic and environmental driver in the US Pacific Northwest. Microbial communities in the water column were analyzed from four diverse habitats: (1) an estuarine turbidity maximum (ETM), (2) a chlorophyll maximum of the river plume, (3) an upwelling-associated hypoxic zone, and (4) the deep ocean bottom. Three size fractions, 0.1–0.8, 0.8–3, and 3–200 μm were collected for each habitat in August 2007, and used for DNA isolation and 454 sequencing, resulting in 12 metagenomes of >5 million reads (>1.6 Gbp). To characterize the dominant microorganisms and metabolisms contributing to coastal biogeochemistry, we used predicted peptide and rRNA data. The 3- and 0.8-μm metagenomes, representing particulate fractions, were taxonomically diverse across habitats. The 3-μm size fractions contained a high abundance of eukaryota with diatoms dominating the hypoxic water and plume, while cryptophytes were more abundant in the ETM. The 0.1-μm metagenomes represented mainly free-living bacteria and archaea. The most abundant archaeal hits were observed in the deep ocean and hypoxic water (19% of prokaryotic peptides in the 0.1-μm metagenomes), and were homologous to Nitrosopumilus maritimus (ammonia-oxidizing Thaumarchaeota). Bacteria dominated metagenomes of all samples. In the euphotic zone (estuary, plume and hypoxic ocean), the most abundant bacterial taxa (≥40% of prokaryotic peptides) represented aerobic photoheterotrophs. In contrast, the low-oxygen, deep water metagenome was enriched with sequences for strict and facultative anaerobes. Interestingly, many of the same anaerobic bacterial families were enriched in the 3-μm size fraction of the ETM (2–10X more abundant relative to the 0.1-μm metagenome), indicating possible formation of anoxic microniches within particles. Results from this study provide a metagenome perspective on ecosystem-scale metabolism in an upwelling-influenced river-dominated coastal margin. PMID:23750156

Peptides and the new endocrinology

NASA Astrophysics Data System (ADS)

Schwyzer, Robert

1982-01-01

The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms.

PubMed

Gião, M S; Wilks, S A; Azevedo, N F; Vieira, M J; Keevil, C W

2009-01-01

Legionella pneumophila is a waterborne pathogen that is mainly transmitted by the inhalation of contaminated aerosols. In this article, the influence of several physico-chemical parameters relating to the supply of potable water was studied using a L. pneumophila peptide nucleic acid (PNA) specific probe to quantify total L. pneumophila in addition to standard culture methods. A two-stage chemostat was used to form the heterotrophic biofilms, with biofilm generating vessels fed with naturally occurring L. pneumophila. The substratum was the commonly used potable water pipe material, uPVC. It proved impossible to recover cultivable L. pneumophila due to overgrowth by other microorganisms and/or the loss of cultivability of this pathogen. Nevertheless, results obtained for total L. pneumophila cells in biofilms using a specific PNA probe showed that for the two temperatures studied (15 and 20 degrees C), there were no significant differences when shear stress was increased. However, when a source of carbon was added there was a significant increase in numbers at 20 degrees C. A comparison of the two temperatures showed that at 15 degrees C, the total cell numbers for L. pneumophila were generally higher compared with the total microbial flora, suggesting that lower temperatures support the inclusion of L. pneumophila in drinking water biofilms. The work reported in this article suggests that standard culture methods are not accurate for the evaluation of water quality in terms of L. pneumophila. This raises public health concerns since culture methods are still considered to be the gold standard for assessing the presence of this opportunistic pathogen in water.

Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells.

PubMed

Fantini, Jacques; Garmy, Nicolas; Yahi, Nouara

2006-09-12

Protein-glycolipid interactions mediate the attachment of various pathogens to the host cell surface as well as the association of numerous cellular proteins with lipid rafts. Thus, it is of primary importance to identify the protein domains involved in glycolipid recognition. Using structure similarity searches, we could identify a common glycolipid-binding domain in the three-dimensional structure of several proteins known to interact with lipid rafts. Yet the three-dimensional structure of most raft-targeted proteins is still unknown. In the present study, we have identified a glycolipid-binding domain in the amino acid sequence of a bacterial adhesin (Helicobacter pylori adhesin A, HpaA). The prediction was based on the major properties of the glycolipid-binding domains previously characterized by structural searches. A short (15-mer) synthetic peptide corresponding to this putative glycolipid-binding domain was synthesized, and we studied its interaction with glycolipid monolayers at the air-water interface. The synthetic HpaA peptide recognized LacCer but not Gb3. This glycolipid specificity was in line with that of the whole bacterium. Molecular modeling studies gave some insights into this high selectivity of interaction. It also suggested that Phe147 in HpaA played a key role in LacCer recognition, through sugar-aromatic CH-pi stacking interactions with the hydrophobic side of the galactose ring of LacCer. Correspondingly, the replacement of Phe147 with Ala strongly affected LacCer recognition, whereas substitution with Trp did not. Our method could be used to identify glycolipid-binding domains in microbial and cellular proteins interacting with lipid shells, rafts, and other specialized membrane microdomains.

Activity of Novel Synthetic Peptides against Candida albicans

PubMed Central

Lum, Kah Yean; Tay, Sun Tee; Le, Cheng Foh; Lee, Vannajan Sanghiran; Sabri, Nadia Hanim; Velayuthan, Rukumani Devi; Hassan, Hamimah; Sekaran, Shamala Devi

2015-01-01

Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8–16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64–128 mg/L to 16–64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited. PMID:25965506

Activity of Novel Synthetic Peptides against Candida albicans.

PubMed

Lum, Kah Yean; Tay, Sun Tee; Le, Cheng Foh; Lee, Vannajan Sanghiran; Sabri, Nadia Hanim; Velayuthan, Rukumani Devi; Hassan, Hamimah; Sekaran, Shamala Devi

2015-05-12

Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8-16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64-128 mg/L to 16-64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited.

Using Data Independent Acquisition (DIA) to Model High-responding Peptides for Targeted Proteomics Experiments*

PubMed Central

Searle, Brian C.; Egertson, Jarrett D.; Bollinger, James G.; Stergachis, Andrew B.; MacCoss, Michael J.

2015-01-01

Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although selected reaction monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated by sequence-specific physiochemical properties; one major challenge is in selecting representative peptides to target as a proxy for protein abundance. Here we present PREGO, a software tool that predicts high-responding peptides for SRM experiments. PREGO predicts peptide responses with an artificial neural network trained using 11 minimally redundant, maximally relevant properties. Crucial to its success, PREGO is trained using fragment ion intensities of equimolar synthetic peptides extracted from data independent acquisition experiments. Because of similarities in instrumentation and the nature of data collection, relative peptide responses from data independent acquisition experiments are a suitable substitute for SRM experiments because they both make quantitative measurements from integrated fragment ion chromatograms. Using an SRM experiment containing 12,973 peptides from 724 synthetic proteins, PREGO exhibits a 40–85% improvement over previously published approaches at selecting high-responding peptides. These results also represent a dramatic improvement over the rules-based peptide selection approaches commonly used in the literature. PMID:26100116

Validation of molecularly imprinted polymers for side chain selective phosphopeptide enrichment.

PubMed

Chen, Jing; Shinde, Sudhirkumar; Subedi, Prabal; Wierzbicka, Celina; Sellergren, Börje; Helling, Stefan; Marcus, Katrin

2016-11-04

Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO 2 ) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO 2 enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO 2 failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pY- peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

Absolute Quantification of Norovirus Capsid Protein in Food, Water, and Soil Using Synthetic Peptides with Electrospray and MALDI Mass Spectrometry

PubMed Central

Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

2015-01-01

Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrixassisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

Toxicity assessment using different bioassays and microbial biosensors.

PubMed

Hassan, Sedky H A; Van Ginkel, Steven W; Hussein, Mohamed A M; Abskharon, Romany; Oh, Sang-Eun

2016-01-01

Toxicity assessment of water streams, wastewater, and contaminated sediments, is a very important part of environmental pollution monitoring. Evaluation of biological effects using a rapid, sensitive and cost effective method can indicate specific information on ecotoxicity assessment. Recently, different biological assays for toxicity assessment based on higher and lower organisms such as fish, invertebrates, plants and algal cells, and microbial bioassays have been used. This review focuses on microbial biosensors as an analytical device for environmental, food, and biomedical applications. Different techniques which are commonly used in microbial biosensing include amperometry, potentiometry, conductometry, voltammetry, microbial fuel cells, fluorescence, bioluminescence, and colorimetry. Examples of the use of different microbial biosensors in assessing a variety of environments are summarized. Copyright © 2016 Elsevier Ltd. All rights reserved.

MALDI versus ESI: The Impact of the Ion Source on Peptide Identification.

PubMed

Nadler, Wiebke Maria; Waidelich, Dietmar; Kerner, Alexander; Hanke, Sabrina; Berg, Regina; Trumpp, Andreas; Rösli, Christoph

2017-03-03

For mass spectrometry-based proteomic analyses, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are the commonly used ionization techniques. To investigate the influence of the ion source on peptide detection in large-scale proteomics, an optimized GeLC/MS workflow was developed and applied either with ESI/MS or with MALDI/MS for the proteomic analysis of different human cell lines of pancreatic origin. Statistical analysis of the resulting data set with more than 72 000 peptides emphasized the complementary character of the two methods, as the percentage of peptides identified with both approaches was as low as 39%. Significant differences between the resulting peptide sets were observed with respect to amino acid composition, charge-related parameters, hydrophobicity, and modifications of the detected peptides and could be linked to factors governing the respective ion yields in ESI and MALDI.

Self-Assembly of Peptides at the Air/Water Interface

NASA Astrophysics Data System (ADS)

Sayar, Mehmet

2013-03-01

Peptides are commonly used as building blocks for design and development of novel materials with a variety of application areas ranging from drug design to biotechnology. The precise control of molecular architecture and specific nature of the nonbonded interactions among peptides enable aggregates with well defined structural and functional properties. The interaction of peptides with interfaces leads to dramatic changes in their conformational and aggregation behavior. In this talk, I will discuss our research on the interplay of intermolecular forces and influence of interfaces. In the first part the amphiphilic nature of short peptide oligomers and their behavior at the air/water interface will be discussed. The surface driving force and its decomposition will be analyzed. In the second part aggregation of peptides in bulk water and at an interface will be discussed. Different design features which can be tuned to control aggregation behavior will be analyzed.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

PubMed

Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

2013-10-01

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

Microbial Transglutaminase Used in Bread Preparation at Standard Bakery Concentrations Does Not Increase Immunodetectable Amounts of Deamidated Gliadin.

PubMed

Heil, Andreas; Ohsam, Jürgen; van Genugten, Bernard; Diez, Oscar; Yokoyama, Keiichi; Kumazawa, Yoshiyuki; Pasternack, Ralf; Hils, Martin

2017-08-16

The effect of standard bakery concentrations of microbial transglutaminase (MTG) in wheat bread preparation on the immunoreactivity of sera of celiac disease (CD) patients was investigated. Immunoblotting using monoclonal antibodies specific to unmodified and/or deamidated gliadin showed no differences between control bread and MTG bread. Deamidation of gliadin could not be detected at standard MTG concentrations. Sera of CD patients were characterized using anti-gliadin and anti-deamidated gliadin peptide (DGP) enzyme-linked immunosorbent assay and grouped into DGP high- and low-titer pools. The recognition pattern obtained after using both CD sera pools for immunoblotting did not reveal differences between control and MTG-treated bread protein extracts. Our results indicate that MTG treatment of wheat bread prepared with typical MTG concentrations used in standard bakery processes does not lead to immunodetectable amounts of CD immunotoxic deamidated gliadins.

Learning from eponyms: George F. Odland and Odland bodies

PubMed Central

Joshi, Rajiv

2014-01-01

Odland bodies (lamellar) bodies are small sub-cellular structures of size 200-300 nm that are present in the upper spinous and granular cell layers of the epidermis. These act as processing and repository areas for lipids that contribute to the epidermal permeability barrier. They also contain proteases, cathepsin D, kallikrein and other proteins including corneo-desmosins. Recent information also credits them with a role in the local innate immune response as they contain beta 2 defensins, which are anti-microbial peptides with potent activity against Gram-negative bacteria and candida. Odland bodies are important for maintaining homeostasis of the epidermis and are involved in epidermal permeability barrier function, desquamation of keratinocytes, formation of the cornified envelope and in local anti-microbial immunity. This article reviews the structure and functions of these bodies with a brief biography of George F. Odland who first described these bodies in 1960 and whose name is eponymically associated with them. PMID:25165659

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins.

PubMed

Lenarčič, Tea; Albert, Isabell; Böhm, Hannah; Hodnik, Vesna; Pirc, Katja; Zavec, Apolonija B; Podobnik, Marjetka; Pahovnik, David; Žagar, Ema; Pruitt, Rory; Greimel, Peter; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Zienkiewicz, Agnieszka; Gömann, Jasmin; Mortimer, Jenny C; Fang, Lin; Mamode-Cassim, Adiilah; Deleu, Magali; Lins, Laurence; Oecking, Claudia; Feussner, Ivo; Mongrand, Sébastien; Anderluh, Gregor; Nürnberger, Thorsten

2017-12-15

Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium

PubMed Central

Hill, David R; Huang, Sha; Nagy, Melinda S; Yadagiri, Veda K; Fields, Courtney; Mukherjee, Dishari; Bons, Brooke; Dedhia, Priya H; Chin, Alana M; Tsai, Yu-Hwai; Thodla, Shrikar; Schmidt, Thomas M; Walk, Seth

2017-01-01

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine. PMID:29110754

Sulfonation of Tyrosine as a Method To Improve Biodistribution of Peptide-Based Radiotracers: Novel 18F-Labeled Cyclic RGD Analogues.

PubMed

Haskali, Mohammad B; Denoyer, Delphine; Noonan, Wayne; Culinane, Carleen; Rangger, Christine; Pouliot, Normand; Haubner, Roland; Roselt, Peter D; Hicks, Rodney J; Hutton, Craig A

2017-04-03

Control of the biodistribution of radiolabeled peptides has proven to be a major challenge in their application as imaging agents for positron emission tomography (PET). Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[ 18 F]fluoropropionate, and the biodistribution of the radiolabeled peptides was compared with that of their nonsulfonated, clinically relevant counterparts, [ 18 F]GalactoRGD and [ 18 F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da toward the MW, compared with 189 Da for both the "Galacto" and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabeled peptides.

Using peptide array to identify binding motifs and interaction networks for modular domains.

PubMed

Li, Shawn S-C; Wu, Chenggang

2009-01-01

Specific protein-protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein-protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein-protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein-protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.

Recent insights in the use of nanocarriers for the oral delivery of bioactive proteins and peptides.

PubMed

Batista, Patrícia; Castro, Pedro M; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Manuela

2018-03-01

Bioactive proteins and peptides have been used with either prophylactic or therapeutic purposes, presenting inherent advantages as high specificity and biocompatibility. Nanocarriers play an important role in the stabilization of proteins and peptides, offering enhanced buccal permeation and protection while crossing the gastrointestinal tract. Moreover, preparation of nanoparticles as oral delivery systems for proteins/peptides may include tailored formulation along with functionalization aiming bioavailability enhancement of carried proteins or peptides. Oral delivery systems, namely buccal delivery systems, represent an interesting alternative route to parenteric delivery systems to carry proteins and peptides, resulting in higher comfort of administration and, therefore, compliance to treatment. This paper outlines an extensive overview of the existing publications on proteins/peptides oral nanocarriers delivery systems, with special focus on buccal route. Manufacturing aspects of most commonly used nanoparticles for oral delivery (e.g. polymeric nanoparticles using synthetic or natural polymers and lipid nanoparticles) advantages and limitations and potential applications of nanoparticles as proteins/peptides delivery systems will also be thoroughly addressed. Copyright © 2018 Elsevier Inc. All rights reserved.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

PubMed

Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

2016-03-10

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Impacts of Sampling and Handling Procedures on DNA- and RNA-based Microbial Characterization and Quantification of Groundwater and Saturated Soil

DTIC Science & Technology

2012-07-01

use of molecular biological techniques (MBTs) has allowed microbial ecologists and environmental engineers to determine microbial community...metabolic genes). The most common approaches used in bioremediation research are those based on the polymerase chain reaction (PCR) amplification of... bioremediation . Because of its sensitivity compared to direct hybridization/probing, PCR is increasingly used to analyze groundwater samples and soil samples

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

Watrous, Jeramie D.; Roach, Patrick J.; Alexandrov, Theodore

Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a "holy grail" in microbiology. This work describes a highly sensitive, broadly applicable, and costeffective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes withinmore » bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097–1100]. The antifungal effect of strain SHC52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is amonochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.« less

Defining the microbial transcriptional response to colitis through integrated host and microbiome profiling

PubMed Central

Ilott, Nicholas Edward; Bollrath, Julia; Danne, Camille; Schiering, Chris; Shale, Matthew; Adelmann, Krista; Krausgruber, Thomas; Heger, Andreas; Sims, David; Powrie, Fiona

2016-01-01

The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess modifications to both bacterial community structure and transcriptional activity in a mouse model of colitis. By using transcriptomic analysis of colonic tissue and luminal RNA derived from the host, we have also characterised how host transcription relates to the microbial transcriptional response in inflammation. In colitis, increased abundance and transcription of diverse microbial gene families involved in responses to nutrient deprivation, antimicrobial peptide production and oxidative stress support an adaptation of multiple commensal genera to withstand a diverse set of environmental stressors in the inflammatory environment. These data are supported by a transcriptional signature of activated macrophages and granulocytes in the gut lumen during colitis, a signature that includes the transcription of the key antimicrobial genes S100a8 and S100a9 (calprotectin). Genes involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase were identified as changing to a greater extent at the level of transcription than would be predicted by DNA abundance changes, implicating a role for increased oxygen tension and/or host-derived reactive oxygen species in driving transcriptional changes in commensal microbes. PMID:27003245

Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study.

PubMed

Quagliariello, Andrea; Aloisio, Irene; Bozzi Cionci, Nicole; Luiselli, Donata; D'Auria, Giuseppe; Martinez-Priego, Llúcia; Pérez-Villarroya, David; Langerholc, Tomaž; Primec, Maša; Mičetić-Turk, Dušanka; Di Gioia, Diana

2016-10-22

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridium sensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota . Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Chemical characterization of microbial-dominated soil organic matter in the Garwood Valley, Antarctica

NASA Astrophysics Data System (ADS)

Feng, Xiaojuan; Simpson, André J.; Gregorich, Edward G.; Elberling, Bo; Hopkins, David W.; Sparrow, Ashley D.; Novis, Philip M.; Greenfield, Lawrence G.; Simpson, Myrna J.

2010-11-01

Despite its harsh environmental conditions, terrestrial Antarctica contains a relatively large microbial biomass. Natural abundance carbon and nitrogen stable isotope signatures of organic materials in the dry valleys indicate mixed provenance of the soil organic matter (SOM) with varying proportions of contributions from lichens, mosses, lake-derived algae and cyanobacteria. Here we employed two complementary analytical techniques, biomarker measurements by gas chromatography/mass spectrometry and solution-state 1H nuclear magnetic resonance spectroscopy, to provide further information at a molecular-level about the composition and possible source of SOM in the Garwood Valley, Antarctica. The predominance of branched alkanes and short-chain lipids in the solvent extracts indicates that the primary contribution to the SOM was microbial-derived. Chemical structures in the NaOH extracts from soils were also dominated by amide, peptides, and a CH 3-dominating aliphatic region that were characteristic of microbial signatures. Furthermore, the SOM in the Garwood Valley contained compounds that were different from those in the cyanobacteria-dominated mat from a nearby lake (including monoethyl alkanes and enriched side-chain protons). This observation suggests that easily degradable carbon sources from the nearby lake did not dominate the SOM, which is consistent with a fast turnover of the mat-derived organic matter found in the valley. This study highlights the important role of native soil microbes in the carbon transformation and biogeochemistry in terrestrial Antarctica.

α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

PubMed Central

Checco, James W.; Lee, Erinna F.; Evangelista, Marco; Sleebs, Nerida J.; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J.; Eddinger, Geoffrey A.; Belair, David G.; Wilson, Julia L.; Eller, Chelcie H.; Raines, Ronald T.; Murphy, William L.; Smith, Brian J.; Gellman, Samuel H.; Fairlie, W. Douglas

2015-01-01

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of L-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues (“α/β-peptides”) manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous “α-peptides”. This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a “stapled” Bim BH3 α-peptide, which contains a hydrocarbon crosslink to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain crosslinking to produce synergistic benefits. PMID:26317395

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

PubMed Central

Arnison, Paul G.; Bibb, Mervyn J.; Bierbaum, Gabriele; Bowers, Albert A.; Bugni, Tim S.; Bulaj, Grzegorz; Camarero, Julio A.; Campopiano, Dominic J.; Challis, Gregory L.; Clardy, Jon; Cotter, Paul D.; Craik, David J.; Dawson, Michael; Dittmann, Elke; Donadio, Stefano; Dorrestein, Pieter C.; Entian, Karl-Dieter; Fischbach, Michael A.; Garavelli, John S.; Göransson, Ulf; Gruber, Christian W.; Haft, Daniel H.; Hemscheidt, Thomas K.; Hertweck, Christian; Hill, Colin; Horswill, Alexander R.; Jaspars, Marcel; Kelly, Wendy L.; Klinman, Judith P.; Kuipers, Oscar P.; Link, A. James; Liu, Wen; Marahiel, Mohamed A.; Mitchell, Douglas A.; Moll, Gert N.; Moore, Bradley S.; Müller, Rolf; Nair, Satish K.; Nes, Ingolf F.; Norris, Gillian E.; Olivera, Baldomero M.; Onaka, Hiroyasu; Patchett, Mark L.; Piel, Joern; Reaney, Martin J. T.; Rebuffat, Sylvie; Ross, R. Paul; Sahl, Hans-Georg; Schmidt, Eric W.; Selsted, Michael E.; Severinov, Konstantin; Shen, Ben; Sivonen, Kaarina; Smith, Leif; Stein, Torsten; Süssmuth, Roderich D.; Tagg, John R.; Tang, Gong-Li; Truman, Andrew W.; Vederas, John C.; Walsh, Christopher T.; Walton, Jonathan D.; Wenzel, Silke C.; Willey, Joanne M.; van der Donk, Wilfred A.

2014-01-01

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed. PMID:23165928

Anti-microbial peptide facilitated cytosolic delivery of metallic gold nanomaterials

NASA Astrophysics Data System (ADS)

Kapur, Anshika; Wang, Wentao; Diaz Hernandez, Juan; Medina, Scott; Schneider, Joel P.; Mattoussi, Hedi

2018-02-01

The unique photophysical properties of gold nanomaterials combined with progress in developing effective surfacefunctionalization strategies has motivated researchers to employ them as tools for use in biomedical imaging, biosensing, diagnostics, photothermal therapy, and as drug and gene delivery vehicles. However, a major challenge limiting these advancements has been the unavailability of effective strategies to deliver these and other nanocrystals into the cytoplasm of live cells. In this study, we demonstrate that the use of a chemically-synthesized anti-microbial peptide, SVS-1, can promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. For this, colloidally stable AuNP and AuNRs, surface ligated with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2 were prepared. The amine groups allow dual, covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dye onto the nanocrystal surfaces. We use fluorescence microscopy to demonstrate nanocrystal staining throughout the cytoplasmic volume of the cells incubated with these conjugates. More importantly, we have conducted additional endocytosis inhibition experiments where cells were incubated with the conjugates at 4°C. Here too, the imaging data have shown significant levels of nanocrystal uptake, further verifying that physical translocation of these conjugates takes place through the cell membrane independent of endocytosis. These findings are promising and can provide critical support for the widespread applications of nanomaterials in the field of biology.

Antisense antibiotics: a brief review of novel target discovery and delivery.

PubMed

Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing

2010-06-01

The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

Multi-species Identification of Polymorphic Peptide Variants via Propagation in Spectral Networks*

PubMed Central

Bandeira, Nuno

2016-01-01

Peptide and protein identification remains challenging in organisms with poorly annotated or rapidly evolving genomes, as are commonly encountered in environmental or biofuels research. Such limitations render tandem mass spectrometry (MS/MS) database search algorithms ineffective as they lack corresponding sequences required for peptide-spectrum matching. We address this challenge with the spectral networks approach to (1) match spectra of orthologous peptides across multiple related species and then (2) propagate peptide annotations from identified to unidentified spectra. We here present algorithms to assess the statistical significance of spectral alignments (Align-GF), reduce the impurity in spectral networks, and accurately estimate the error rate in propagated identifications. Analyzing three related Cyanothece species, a model organism for biohydrogen production, spectral networks identified peptides from highly divergent sequences from networks with dozens of variant peptides, including thousands of peptides in species lacking a sequenced genome. Our analysis further detected the presence of many novel putative peptides even in genomically characterized species, thus suggesting the possibility of gaps in our understanding of their proteomic and genomic expression. A web-based pipeline for spectral networks analysis is available at http://proteomics.ucsd.edu/software. PMID:27609420

Will sacubitril-valsartan diminish the clinical utility of B-type natriuretic peptide testing in acute cardiac care?

PubMed

Mair, Johannes; Lindahl, Bertil; Giannitsis, Evangelos; Huber, Kurt; Thygesen, Kristian; Plebani, Mario; Möckel, Martin; Müller, Christian; Jaffe, Allan S

2017-06-01

Since the approval of sacubitril-valsartan for the treatment of chronic heart failure with reduced ejection fraction, a commonly raised suspicion is that a wider clinical use of this new drug may diminish the clinical utility of B-type natriuretic peptide testing as sacubitril may interfere with B-type natriuretic peptide clearance. In this education paper we critically assess this hypothesis based on the pathophysiology of the natriuretic peptide system and the limited published data on the effects of neprilysin inhibition on natriuretic peptide plasma concentrations in humans. As the main clinical application of B-type natriuretic peptide testing in acute cardiac care is and will be the rapid rule-out of suspected acute heart failure there is no significant impairment to be expected for B-type natriuretic peptide testing in the acute setting. However, monitoring of chronic heart failure patients on sacubitril-valsartan treatment with B-type natriuretic peptide testing may be impaired. In contrast to N-terminal-proBNP, the current concept that the lower the B-type natriuretic peptide result in chronic heart failure patients, the better the prognosis during treatment monitoring, may no longer be true.

X-ray crystal structures of Staphylococcus aureus collagen adhesin and sortases

NASA Astrophysics Data System (ADS)

Zong, Yinong

For many gram-positive bacteria, adhesion to host tissues is the first critical step in developing an infection. The adhesion is mediated by a superfamily of bacterial surface proteins, called MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which in most cases are covalently attached to the cell wall peptidoglycan. Collagen adhesin (CNA) from Staphylococcus aureus, one of the MSCRAMMs, is responsible for bacterial binding to collagen molecules. CNA and other MSCRAMMs are anchored to the cell wall by a transpeptidase, sortase. The knowledge about how bacterial surface proteins adhere to host molecules and how they are sorted onto the cell wall is crucial for the design of novel antibiotics against bacterial infections. The crystal structures of CNA31--344 (residue 31 to 334), a truncation of CNA's collagen binding region, and CNA31--344 in complex with a collagen peptide were determined. CNA31--344 contains two domains, and between them is a big hole formed by a loop connecting the two domains. In the structure of CNA31--344-collagen complex, the collagen peptide is locked in the hole formed by the two domains of CNA 31--344. We reason that the two domains of CNA31--344 are open in the physiological condition, and close up when binding to collagen. This binding mechanism may be common for other bacterial collagen adhesins. There are two known sortases in Staphylococcus aureus. Sortase A is responsible for anchoring most MSCRAMMs that have a LPXTG (X represents any amino acid) sorting motif and sortase B for a bacterial ion acquisition protein. The crystal structures of both sortases indicate that they share a common catalytic mechanism. Unlike typical cysteine transpeptidases, sortases may use a novel Cys-Arg catalytic dyad instead of a Cys-His pair. All other sortases found in gram-positive bacteria may have similar active site architecture and employ the same catalytic dyad because the critical residues are all conserved among them. The structures of sortases in complex with their substrates and inhibitors are also useful to explain their substrate specificity and catalytic kinetics.

Characterizing Microbial Community and Geochemical Dynamics at Hydrothermal Vents Using Osmotically Driven Continuous Fluid Samplers

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

Robidart, Julie C.; Callister, Stephen J.; Song, Peng F.

2013-05-07

Microbes play a key role in mediating all aquatic biogeochemical cycles, and ongoing efforts are aimed at better understanding the relationships between microbial phylogenetic and physiological diversity, and habitat physical and chemical characteristics. Establishing such relationships is facilitated by sampling and studying microbiology and geochemistry at the appropriate spatial and temporal scales, to access information on the past and current environmental state that contributes to observed microbial abundances and activities. A modest number of sampling systems exist to date, few of which can be used in remote, harsh environments such as hydrothermal vents, where the ephemeral nature of venting underscoresmore » the necessity for higher resolution sampling. We have developed a robust, continuous fluid sampling system for co-registered microbial and biogeochemical analyses. The osmosis-powered bio-osmosampling system (BOSS) use no electricity, collects fluids with daily resolution or better, can be deployed in harsh, inaccessible environments and can sample fluids continuously for up to five years. Here we present a series of tests to examine DNA, RNA and protein stability over time, as well as material compatability, via lab experiments. We also conducted two field deployments at deep-sea hydrothermal vents to assess changes in microbial diversity and protein expression as a function of the physico-chemical environment. Our data reveal significant changes in microbial community composition co-occurring with relatively modest changes in the geochemistry. These data additionally provide new insights into the distribution of an enigmatic sulfur oxidizing symbiont in its free-living state. Data from the second deployment reveal differences in the representation of peptides over time, underscoring the utility of the BOSS in meta-proteomic studies. In concert, these data demonstrate the efficacy of this approach, and illustrate the value of using this method to study microbial and geochemical phenomena.« less

MICROBIAL VOLATILE ORGANIC COMPOUND EMISSION RATES AND EXPOSURE MODEL

EPA Science Inventory

This paper presents the results from a study that examined microbial volatile organic compound (MVOC) emissions from six fungi and one bacterial species (Streptomyces spp.) commonly found in indoor environments. Data are presented on peak emission rates from inoculated agar plate...

Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland wheat

USDA-ARS?s Scientific Manuscript database

Natural antibiotics are thought to function in microbial defense, fitness, competitiveness, biocontrol, communication and gene regulation activity, and antibiotic-producing species are commonly found in microbial communities throughout nature. However, the frequency and amount of antibiotic producti...

THE NEAR-EQUILIBRIUM OF MICROBIALLY MEDIATED REDOX COUPLES IN REDUCING GROUNDWATER ENVIRONMENTS

EPA Science Inventory

Redox couples are commonly held to be in disequilibrium among each other in most natural waters. To evaluate this view for microbially mediated, reducing, groundwater environments, monitoring data were examined for several couples under conditions ranging from nitrate-detectable...

Soil microbial communities and metabolic function of a Northern Alabama forest ecosystem

USDA-ARS?s Scientific Manuscript database

Thinning, prescribed burning, and their combinations, are common forest management practices to restore degraded forest communities and to prevent uncontrollable wildfires. However, their impacts on soil microbial communities, which are vital to global element cycling, are traditionally overlooked. ...

Small bugs, big business: the economic power of the microbe.

PubMed

Demain, A L

2000-10-01

The versatility of microbial biosynthesis is enormous. The most industrially important primary metabolites are the amino acids, nucleotides, vitamins, solvents, and organic acids. Millions of tons of amino acids are produced each year with a total multibillion dollar market. Many synthetic vitamin production processes are being replaced by microbial fermentations. In addition to the multiple reaction sequences of fermentations, microorganisms are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine chemical industry in the production of single-isomer intermediates. Microbially produced secondary metabolites are extremely important to our health and nutrition. As a group, they have tremendous economic importance. The antibiotic market amounts to almost 30 billion dollars and includes about 160 antibiotics and derivatives such as the beta-lactam peptide antibiotics, the macrolide polyketide erythromycin, tetracyclines, aminoglycosides and others. Other important pharmaceutical products produced by microrganisms are hypocholesterolemic agents, enzyme inhibitors, immunosuppressants and antitumor compounds, some having markets of over 1 billion dollars per year. Agriculturally important secondary metabolites include coccidiostats, animal growth promotants, antihelmintics and biopesticides. The modern biotechnology industry has made a major impact in the business world, biopharmaceuticals (recombinant protein drugs, vaccines and monoclonal antibodies) having a market of 15 billion dollars. Recombinant DNA technology has also produced a revolution in agriculture and has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as means of increasing titers and yields of microbial procresses and in discovery of new drugs. Today, microbiology is a major participant in global industry. The best is yet to come as microbes move into the environmental and energy sectors.

Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics.

PubMed

Bergauer, Kristin; Fernandez-Guerra, Antonio; Garcia, Juan A L; Sprenger, Richard R; Stepanauskas, Ramunas; Pachiadaki, Maria G; Jensen, Ole N; Herndl, Gerhard J

2018-01-16

The phylogenetic composition of the heterotrophic microbial community is depth stratified in the oceanic water column down to abyssopelagic layers. In the layers below the euphotic zone, it has been suggested that heterotrophic microbes rely largely on solubilized particulate organic matter as a carbon and energy source rather than on dissolved organic matter. To decipher whether changes in the phylogenetic composition with depth are reflected in changes in the bacterial and archaeal transporter proteins, we generated an extensive metaproteomic and metagenomic dataset of microbial communities collected from 100- to 5,000-m depth in the Atlantic Ocean. By identifying which compounds of the organic matter pool are absorbed, transported, and incorporated into microbial cells, intriguing insights into organic matter transformation in the deep ocean emerged. On average, solute transporters accounted for 23% of identified protein sequences in the lower euphotic and ∼39% in the bathypelagic layer, indicating the central role of heterotrophy in the dark ocean. In the bathypelagic layer, substrate affinities of expressed transporters suggest that, in addition to amino acids, peptides and carbohydrates, carboxylic acids and compatible solutes may be essential substrates for the microbial community. Key players with highest expression of solute transporters were Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria, accounting for 40%, 11%, and 10%, respectively, of relative protein abundances. The in situ expression of solute transporters indicates that the heterotrophic prokaryotic community is geared toward the utilization of similar organic compounds throughout the water column, with yet higher abundances of transporters targeting aromatic compounds in the bathypelagic realm. Copyright © 2018 the Author(s). Published by PNAS.

Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics

PubMed Central

Bergauer, Kristin; Fernandez-Guerra, Antonio; Garcia, Juan A. L.; Sprenger, Richard R.; Stepanauskas, Ramunas; Pachiadaki, Maria G.; Herndl, Gerhard J.

2018-01-01

The phylogenetic composition of the heterotrophic microbial community is depth stratified in the oceanic water column down to abyssopelagic layers. In the layers below the euphotic zone, it has been suggested that heterotrophic microbes rely largely on solubilized particulate organic matter as a carbon and energy source rather than on dissolved organic matter. To decipher whether changes in the phylogenetic composition with depth are reflected in changes in the bacterial and archaeal transporter proteins, we generated an extensive metaproteomic and metagenomic dataset of microbial communities collected from 100- to 5,000-m depth in the Atlantic Ocean. By identifying which compounds of the organic matter pool are absorbed, transported, and incorporated into microbial cells, intriguing insights into organic matter transformation in the deep ocean emerged. On average, solute transporters accounted for 23% of identified protein sequences in the lower euphotic and ∼39% in the bathypelagic layer, indicating the central role of heterotrophy in the dark ocean. In the bathypelagic layer, substrate affinities of expressed transporters suggest that, in addition to amino acids, peptides and carbohydrates, carboxylic acids and compatible solutes may be essential substrates for the microbial community. Key players with highest expression of solute transporters were Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria, accounting for 40%, 11%, and 10%, respectively, of relative protein abundances. The in situ expression of solute transporters indicates that the heterotrophic prokaryotic community is geared toward the utilization of similar organic compounds throughout the water column, with yet higher abundances of transporters targeting aromatic compounds in the bathypelagic realm. PMID:29255014

Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes.

PubMed

Bateman, E A L; Ardern-Jones, M R; Ogg, G S

2008-11-01

Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. To identify commonly recognized CD4(+) T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA-restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 ELISpots. Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA-DQB1(*)06 and -DPB1(*)0401 were characterized in detail. Significantly higher ex vivo IL-4 and lower IFN-gamma responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL-10 responses were significantly lower in those with AD in the individuals with HLA-DPB1(*)0401. We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4(+) T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

PubMed

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F; Sette, Alessandro

2015-11-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires

PubMed Central

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M.; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F.; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F.; Sette, Alessandro

2016-01-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif. PMID:26399241

Activity assessment of microbial fibrinolytic enzymes.

PubMed

Kotb, Essam

2013-08-01

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

Biodiversity of genes encoding anti-microbial traits within plant associated microbes

PubMed Central

Mousa, Walaa K.; Raizada, Manish N.

2015-01-01

The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produces a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins, and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant-associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. We review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous coding sequences (CDS). We conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters. PMID:25914708

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium

PubMed Central

Silva, Tânia; Moreira, Ana C.; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G. M.; Rodrigues, Pedro N.; Bastos, Margarida

2017-01-01

ABSTRACT Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17–30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17–30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17–30 did not localize to M. avium-harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17–30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis, M. leprae, M. avium, etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17–30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17–30 on macrophages, which may be useful under other conditions in which macrophage activation is needed. PMID:28875176

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

PubMed

Silva, Tânia; Moreira, Ana C; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2017-01-01

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium -harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis , M. leprae , M. avium , etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17-30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17-30 on macrophages, which may be useful under other conditions in which macrophage activation is needed.

Identification of cross-reactive B-cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI-TOF MS.

PubMed

Candreva, Ángela María; Ferrer-Navarro, Mario; Bronsoms, Silvia; Quiroga, Alejandra; Curciarello, Renata; Cauerhff, Ana; Petruccelli, Silvana; Docena, Guillermo Horacio; Trejo, Sebastián Alejandro

2017-08-01

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOF MS analysis. On a second approach, the peptide mixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Peptide Repertoire of HLA-B27 may include Ligands with Lysine at P2 Anchor Position.

PubMed

Yair-Sabag, Shira; Tedeschi, Valentina; Vitulano, Carolina; Barnea, Eilon; Glaser, Fabian; Melamed Kadosh, Dganit; Taurog, Joel D; Fiorillo, Maria Teresa; Sorrentino, Rosa; Admon, Arie

2018-05-01

The HLA-B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA-B*27 alleles and the inflammatory rheumatic disease ankylosing spondylitis (AS), for which a comprehensive biological explanation is still lacking. This study aims to expand the known limits of the HLA-B*27 peptidome to facilitate selection and testing of new peptides, possibly involved in the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS-associated HLA-B*27:05 allele, the nonassociated HLA-B*27:09 allele, or their cysteine 67 to serine mutants (C67S), are analyzed on a very large scale. In addition, the peptidomes of HLA-B*27:05 and HLA-B*27:05-C67S are analyzed from the spleens of rats transgenic for these alleles. The results indicate that C67S mutation increases the percentage of peptides with glutamine or lysine at their P2 position (P2-Lys), in both HLA-B*27:05 and HLA-B*27:09. Furthermore, a small fraction of HLA-B*27 peptides contains lysine at their second position (P2), in addition to the more commonly found peptides with arginine (P2-Arg) or the less common glutamine (P2-Gln) located at this anchor position. Overall these data indicate that peptides with P2-Lys should be considered as real ligands of HLA-B*27 molecules and taken into account while looking for putative peptides implicated in the AS. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A quantitative microbial risk assessment for center pivot irrigation of dairy wastewaters

USDA-ARS?s Scientific Manuscript database

In the western United States where livestock wastewaters are commonly land applied, there are concerns over individuals being exposed to airborne pathogens. In response, a quantitative microbial risk assessment (QMRA) was performed to estimate infectious risks from inhaling pathogens aerosolized dur...

The secret world of endophytes in perspective

USDA-ARS?s Scientific Manuscript database

This work in Fungal Ecology is focused on the group of plant symbionts that have been termed collectively ‘microbial endophytes’. Broadly, microbial endophytes are commonly considered to be any of a diverse group of bacteria, cyanobacteria, or fungi that colonize internal tissues of plants. After ...

Rainfall-induced fecal indicator organisms transport from manured fields: Model sensitivity analysis

EPA Science Inventory

Microbial quality of surface waters attracts attention due to food- and waterborne disease outbreaks. Fecal indicator organisms (FIOs) are commonly used for the microbial pollution level evaluation. Models predicting the fate and transport of FIOs are required to design and evalu...

Lotka-Volterra pairwise modeling fails to capture diverse pairwise microbial interactions

PubMed Central

Momeni, Babak; Xie, Li; Shou, Wenying

2017-01-01

Pairwise models are commonly used to describe many-species communities. In these models, an individual receives additive fitness effects from pairwise interactions with each species in the community ('additivity assumption'). All pairwise interactions are typically represented by a single equation where parameters reflect signs and strengths of fitness effects ('universality assumption'). Here, we show that a single equation fails to qualitatively capture diverse pairwise microbial interactions. We build mechanistic reference models for two microbial species engaging in commonly-found chemical-mediated interactions, and attempt to derive pairwise models. Different equations are appropriate depending on whether a mediator is consumable or reusable, whether an interaction is mediated by one or more mediators, and sometimes even on quantitative details of the community (e.g. relative fitness of the two species, initial conditions). Our results, combined with potential violation of the additivity assumption in many-species communities, suggest that pairwise modeling will often fail to predict microbial dynamics. DOI: http://dx.doi.org/10.7554/eLife.25051.001 PMID:28350295

A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide.

PubMed

Taylor, Steven W; Clarke, Nigel J; Chen, Zhaohui; McPhaul, Michael J

2016-04-01

Measurements of fasting levels of insulin and C-peptide are useful in documenting insulin resistance and may help predict development of diabetes mellitus. However, the specific insulin and C-peptide levels associated with specific degrees of insulin resistance have not been defined, owing to marked variability among immunoassays and lack of standardization. Herein, we describe a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for intact insulin and C-peptide. Insulin and C-peptide were enriched from patient sera using monoclonal antibodies immobilized on magnetic beads and processed on a robotic liquid handler. Eluted peptides were analyzed by LC-MS/MS. Bovine insulin and a stable isotopically-labeled (13C/15N) C-peptide were utilized as internal standards. The assay had an analytical measurement range of 3 to 320 μIU/ml (18 to 1920 pmol/l) for insulin and 0.11 to 27.2 ng/ml (36 to 9006 pmol/l) for C-peptide. Intra- and inter-day assay variation was less than 11% for both peptides. Of the 5 insulin analogs commonly prescribed to treat diabetes, only the recombinant drug insulin lispro caused significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide. We developed and validated a high-throughput, quantitative, multiplexed LC-MS/MS assay for intact insulin and C-peptide. Copyright © 2016 Elsevier B.V. All rights reserved.

Microbial Activity in Aquatic Environments Measured by Dimethyl Sulfoxide Reduction and Intercomparison with Commonly Used Methods

PubMed Central

Griebler, Christian; Slezak, Doris

2001-01-01

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN3, KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 × 10−17 ± 0.12 × 10−17 mol of DMS per produced cell (mean ± standard error; R2 = 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (the R2 values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (the R2 values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity. PMID:11133433

Microbial activity in aquatic environments measured by dimethyl sulfoxide reduction and intercomparison with commonly used methods.

PubMed

Griebler, C; Slezak, D

2001-01-01

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN(3), KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 x 10(-17) +/- 0. 12 x 10(-17) mol of DMS per produced cell (mean +/- standard error; R(2) = 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (the R(2) values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (the R(2) values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

PubMed

Tomasinsig, Linda; Skerlavaj, Barbara; Scarsini, Michele; Guida, Filomena; Piccinini, Renata; Tossi, Alessandro; Zanetti, Margherita

2012-02-01

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data.

PubMed

Braaksma, Machtelt; Martens-Uzunova, Elena S; Punt, Peter J; Schaap, Peter J

2010-10-19

The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

PubMed Central

2010-01-01

Background The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. Results A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. Conclusions We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions. PMID:20959013

Altering the Composition of Caseicins A and B as a Means of Determining the Contribution of Specific Residues to Antimicrobial Activity ▿

PubMed Central

Norberg, Sarah; O'Connor, Paula M.; Stanton, Catherine; Ross, R. Paul; Hill, Colin; Fitzgerald, Gerald F.; Cotter, Paul D.

2011-01-01

Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR) are antimicrobial peptides generated through the bacterial fermentation of sodium caseinate, and on the basis of this and previous studies, they are active against many Gram-negative pathogens (Cronobacter sakazakii, Cronobacter muytjensii, Salmonella enterica serovar Typhimurium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas fluorescens) as well as the Gram-positive organism Staphylococcus aureus. Here we describe further studies with the aim of establishing the importance of specific (charged and nonpolar aliphatic) residues within the caseicin peptides and the effects that they have on the bacteria listed above. In order to achieve our objective, we created four derivatives of each caseicin (A1 to A4 and B1 to B4) in which specific residues were altered, and results obtained with these derivatives were compared to wild-type caseicin activity. Although conversion of cationic residues to alanine in caseicins B1 (R8A change), A1 (K2A), A2 (H3A), and A3 (K2A-H3A) generally resulted in their activity against microbial targets being reduced or unaltered, C. sakazakii DPC6440 was unusual in that it displayed enhanced sensitivity to three peptides (caseicins A1, A3, and B2) in which positively charged residues had been eliminated. While the replacement of leucine with alanine in selected variants (B3 and B4) resulted in reduced activity against a number of strains of Cronobacter and, in some cases, S. Typhimurium, these changes enhanced the activities of these peptides against DPC6440 and a number of S. aureus strains. It is thus apparent that the importance of specific residues within the caseicin peptides is dependent on the strain being targeted. PMID:21296933

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

PubMed

Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

2016-08-19

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. Copyright © 2016 Elsevier Inc. All rights reserved.

Sonorensin: an Antimicrobial Peptide, Belonging to the Heterocycloanthracin Subfamily of Bacteriocins, from a New Marine Isolate, Bacillus sonorensis MT93

PubMed Central

Chopra, Lipsy; Singh, Gurdeep; Choudhary, Vikas

2014-01-01

Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized. PMID:24610839

Sonorensin: an antimicrobial peptide, belonging to the heterocycloanthracin subfamily of bacteriocins, from a new marine isolate, Bacillus sonorensis MT93.

PubMed

Chopra, Lipsy; Singh, Gurdeep; Choudhary, Vikas; Sahoo, Debendra K

2014-05-01

Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized.

Pep2Path: automated mass spectrometry-guided genome mining of peptidic natural products.

PubMed

Medema, Marnix H; Paalvast, Yared; Nguyen, Don D; Melnik, Alexey; Dorrestein, Pieter C; Takano, Eriko; Breitling, Rainer

2014-09-01

Nonribosomally and ribosomally synthesized bioactive peptides constitute a source of molecules of great biomedical importance, including antibiotics such as penicillin, immunosuppressants such as cyclosporine, and cytostatics such as bleomycin. Recently, an innovative mass-spectrometry-based strategy, peptidogenomics, has been pioneered to effectively mine microbial strains for novel peptidic metabolites. Even though mass-spectrometric peptide detection can be performed quite fast, true high-throughput natural product discovery approaches have still been limited by the inability to rapidly match the identified tandem mass spectra to the gene clusters responsible for the biosynthesis of the corresponding compounds. With Pep2Path, we introduce a software package to fully automate the peptidogenomics approach through the rapid Bayesian probabilistic matching of mass spectra to their corresponding biosynthetic gene clusters. Detailed benchmarking of the method shows that the approach is powerful enough to correctly identify gene clusters even in data sets that consist of hundreds of genomes, which also makes it possible to match compounds from unsequenced organisms to closely related biosynthetic gene clusters in other genomes. Applying Pep2Path to a data set of compounds without known biosynthesis routes, we were able to identify candidate gene clusters for the biosynthesis of five important compounds. Notably, one of these clusters was detected in a genome from a different subphylum of Proteobacteria than that in which the molecule had first been identified. All in all, our approach paves the way towards high-throughput discovery of novel peptidic natural products. Pep2Path is freely available from http://pep2path.sourceforge.net/, implemented in Python, licensed under the GNU General Public License v3 and supported on MS Windows, Linux and Mac OS X.

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

PubMed

Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

2016-12-01

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Recent advances in protein and Peptide drug delivery: a special emphasis on polymeric nanoparticles.

PubMed

Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K

2014-01-01

Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly em- ployed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transder mal, and ocular administrations.

Recent Advances in Protein and Peptide Drug Delivery: A Special Emphasis on Polymeric Nanoparticles

PubMed Central

Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K.

2015-01-01

Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly employed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transdermal, and ocular administrations. PMID:25106908

BG-4, a novel bioactive peptide from Momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages

USDA-ARS?s Scientific Manuscript database

Background: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. Purpose: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated f...

Drug forecast - the peptide deformylase inhibitors as antibacterial agents.

PubMed

Guay, David R P

2007-08-01

The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC(90) values ranging from 1-8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted.

Antimicrobial peptide hLF1-11 directs granulocyte-macrophage colony-stimulating factor-driven monocyte differentiation toward macrophages with enhanced recognition and clearance of pathogens.

PubMed

van der Does, Anne M; Bogaards, Sylvia J P; Ravensbergen, Bep; Beekhuizen, Henry; van Dissel, Jaap T; Nibbering, Peter H

2010-02-01

The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.

Peptides and Ageing.

PubMed

Khavinson, Vladimir Kh

2002-01-01

A technology has been developed for manufacturing of biologically active complex peptide preparations from extracts of different tissues. In particular, the pineal preparation (Epithalamin) augments the in vitro outgrowth of explants from the pineal gland but not from other tissues, the latter being stimulated by peptide preparations from respective tissues. Epithalamin increases melatonin production by the pineal gland of rats, improves immunological parameters in rats and mice, produces anticarcinogenic effects in different experimental models, stimulates antioxidant defenses, and restores the reproductive function in old rats. These effects are combined in the ability of Epithalamin to increase the lifespan in rats, mice, and fruit flies. Many of these effects are reproduced in clinical trials, which have demonstrated the geroprotector activity of Epithalamin in humans. Among the effects of the thymic preparation Thymalin, those related to its ability to stimulate immunity are the most prominent. This ability is associated with anticarcinogenic and geroprotector activities. Clinical trials of the peptide preparations obtained from other organs including the prostate, the cerebral cortex, and the eye retina, have demonstrated beneficial effects reflected by the improvement of the conditions of respective organs. Based on the data about the amino acid compositions of the peptide preparations, novel principles of the design of biologically active short peptides possessing tissue-specific activities has been developed. Dipeptides specific for the thymus and tetrapeptides specific for the heart, liver, brain cortex, and pineal glands stimulate the in vitro outgrowth of explants of respective organs. Interestingly, for eye retina and the pineal gland, a common tetrapeptide Ala-Glu-Asp-Gly (Epitalon) has been designed, probably reflecting the common embryonal origin of these two organs. Epitalon reproduces the effects of Epithalamin including those related to its geroprotector activity. In particular, Epitalon increases the lifespan of mice and fruit flies and restores the circadian rhythms of melatonin and cortisol production in old rhesus monkeys. At the same time, Epitalon prolongs the functional integrity of the eye retina in Campbell rats with hereditary Retinitis Pigmentosa and improves the visual functions in patients with pigmental retinal degeneration. Changes in gene expression were observed to be produced by the short peptide preparations. Therefore, the effects of Epitalon are suggested to be mediated by transcriptional machinery common for the pineal gland and the retina and, probably, for regulation of melatonin production in fruit flies. Based on three decades of studies of the peptide preparations, the peptide theory of ageing has been put forward. According this theory, ageing is an evolutionary determined biological process of changes in gene expression resulting in impaired synthesis of regulatory and tissue-specific peptides in organs and tissues, which provokes their structural and functional changes and the development of diseases. Correspondingly, correction of such disorders by means of stimulation of peptide production in the organism or through their delivery can promote the normalisation of disturbed body functions.

Tools and perspectives for a unified approach to understanding microbial ecology in the critical zone

NASA Astrophysics Data System (ADS)

Gallery, R. E.; Aronson, E. L.; Fairbanks, D.; Murphy, M. A.; Rich, V. I.; Hart, S. C.

2015-12-01

Microbial communities that control nutrient transformation and storage in ecosystems are themselves influenced by landscape topography and vegetative cover. Globally, disturbances such as fires and insect outbreaks are increasing in frequency and severity with enormous impacts on global carbon cycling. The resiliency of soil microbial communities to these heterogeneous disturbances determines rates of nutrient transformations as well as ecosystem structure and recovery. Natural and anthropogenic disturbances are a common thread throughout Critical Zone Observatories and ecosystems in general. Using the 2013 Thompson Ridge Fire in the Jemez River Basin CZO as a case study, we examine the effect of a wildfire disturbance regime on successional changes in soil microbiota and ecosystem fluxes across a landscape with high topographic variation. We find that, layered over the topographic controls of hotspots of biogeochemical activity, fire alters organic substrate quality, microbial biomass, community structure, and activity. For example, fire increases soil pH, which is commonly found as an explanatory variable describing bacterial community structure. Soil microbes excrete exoenzymes to decompose polymers and acquire nutrients, and these activities can indicate changing microbial function or soil quality. In these mixed conifer forests, we find shifts from carbon to nitrogen-dominated exoenzyme activities in burned forests with alkaline soils, suggesting shifts of microbial taxa and function that correspond with recovering soil microbial biomass. More generally we ask - what combination of tools and perspectives is needed to fully understand soil microbial ecology and biogeochemistry of the critical zone? Results from an NSF Science Across Virtual Institutes (SAVI) CZO Network Biogeochemistry Workshop highlight the importance of incorporating a standard suite of microbial activity and community assays along with soil biogeochemical and flux measurements to enable comparisons across the broader CZO network. These characterizations would provide regional microbial function and biodiversity data in a standardized framework that can be used to enable more effective management and valuation of critical zone services and inform projections under global change scenarios.

cDNA cloning, characterization and expression analysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

PubMed

Shanthi, S; Vaseeharan, B

2012-03-20

A new member of antimicrobial peptide genes of the penaeidin family, penaeidin 3, was cloned from the haemocytes of Indian white shrimp Fenneropeneaus indicus (F. indicus), by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR) methods. The complete nucleotide sequence of cDNA clone of Indian white shrimp F. indicus Penaeidin 3 (Fi-Pen3) was 243bp long and has an open reading frame which encodes 80 amino acid peptide. The homology analysis of Fi-Pen3 sequence with other Penaeidins 3 shows higher similarity with Penaeus monodon (92%). The theoretical 3D structure generated through ab initio modelling indicated the presence of two-disulphide bridges in the alpha-helix. The signal peptide sequence of Fi-Pen3 is almost entirely homologous to that of other Penaeidin 3 of crustaceans, while differing relatively in the N-terminal domain of the mature peptide. The mature peptide has a predicted molecular weight of 84.9kDa, and a theoretical pI of 9.38. Phylogenetic analysis of Fi-Pen3 shows high resemblance with other Pen-3 from P. monodon, Litopenaeus stylirostris, Litopenaeus vannamei and Litopenaeus setiferus. Fi-Pen3 found to be expressed in haemocytes, heart, hepatopancreas, muscles, gills, intestine, and eyestalk with higher expression in haemocytes. Microbial challenge resulted in mRNA up-regulation, up to 6h post injection of Vibrio parahemolyticus. The Fi-Pen3 mRNA expression of F. indicus in the premolt stage (D(01) and D(02)) was significantly up-regulated than the postmolt (A and B) and intermolt stages (C). The findings of the present paper underline the involvement of Fi-Pen3 in innate immune system of F. indicus. Copyright © 2011 Elsevier GmbH. All rights reserved.

Characterization of on-target generated tryptic peptides from Giberella zeae conidia spore proteins by means of matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Dong, Hongjuan; Marchetti-Deschmann, Martina; Allmaier, Günter

2014-01-01

Traditionally characterization of microbial proteins is performed by a complex sequence of steps with the final step to be either Edman sequencing or mass spectrometry, which generally takes several weeks or months to be complete. In this work, we proposed a strategy for the characterization of tryptic peptides derived from Giberella zeae (anamorph: Fusarium graminearum) proteins in parallel to intact cell mass spectrometry (ICMS) in which no complicated and time-consuming steps were needed. Experimentally, after a simple washing treatment of the spores, the aliquots of the intact G. zeae macro conidia spores solution, were deposited two times onto one MALDI (matrix-assisted laser desorption ionization) mass spectrometry (MS) target (two spots). One spot was used for ICMS and the second spot was subject to a brief on-target digestion with bead-immobilized or non-immobilized trypsin. Subsequently, one spot was analyzed immediately by MALDI MS in the linear mode (ICMS) whereas the second spot containing the digested material was investigated by MALDI MS in the reflectron mode ("peptide mass fingerprint") followed by protonated peptide selection for MS/MS (post source decay (PSD) fragment ion) analysis. Based on the formed fragment ions of selected tryptic peptides a complete or partial amino acid sequence was generated by manual de novo sequencing. These sequence data were used for homology search for protein identification. Finally four different peptides of varying abundances have been identified successfully allowing the verification that our desorbed/ionized surface compounds were indeed derived from proteins. The presence of three different proteins could be found unambiguously. Interestingly, one of these proteins is belonging to the ribosomal superfamily which indicates that not only surface-associated proteins were digested. This strategy minimized the amount of time and labor required for obtaining deeper information on spore preparations within the nowadays widely used ICMS approach. Copyright © 2013 Elsevier Ltd. All rights reserved.

Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

PubMed Central

2011-01-01

Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK)2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli) when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs) under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might also provide hints for protein aggregation-related diseases. PMID:21320350

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

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

Booe, Jason M.; Walker, Christopher S.; Barwell, James

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE PAGES

Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

2015-05-14

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Peptide protected gold clusters: chemical synthesis and biomedical applications

NASA Astrophysics Data System (ADS)

Yuan, Qing; Wang, Yaling; Zhao, Lina; Liu, Ru; Gao, Fuping; Gao, Liang; Gao, Xueyun

2016-06-01

Bridging the gap between atoms and nanoparticles, noble metal clusters with atomic precision continue to attract considerable attention due to their important applications in catalysis, energy transformation, biosensing and biomedicine. Greatly different to common chemical synthesis, a one-step biomimetic synthesis of peptide-conjugated metal clusters has been developed to meet the demand of emerging bioapplications. Under mild conditions, multifunctional peptides containing metal capturing, reactive and targeting groups are rationally designed and elaborately synthesized to fabricate atomically precise peptide protected metal clusters. Among them, peptide-protected Au Cs (peptide-Au Cs) possess a great deal of exceptional advantages such as nanometer dimensions, high photostability, good biocompatibility, accurate chemical formula and specific protein targeting capacity. In this review article, we focus on the recent advances in potential theranostic fields by introducing the rising progress of peptide-Au Cs for biological imaging, biological analysis and therapeutic applications. The interactions between Au Cs and biological systems as well as potential mechanisms are also our concerned theme. We expect that the rapidly growing interest in Au Cs-based theranostic applications will attract broader concerns across various disciplines.

Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients

PubMed Central

Wang, Conan K.; Northfield, Susan E.; Colless, Barbara; Chaousis, Stephanie; Hamernig, Ingrid; Lohman, Rink-Jan; Nielsen, Daniel S.; Schroeder, Christina I.; Liras, Spiros; Price, David A.; Fairlie, David P.; Craik, David J.

2014-01-01

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog. PMID:25416591

Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients.

PubMed

Wang, Conan K; Northfield, Susan E; Colless, Barbara; Chaousis, Stephanie; Hamernig, Ingrid; Lohman, Rink-Jan; Nielsen, Daniel S; Schroeder, Christina I; Liras, Spiros; Price, David A; Fairlie, David P; Craik, David J

2014-12-09

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

PubMed

LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

2017-07-07

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Extensive characterization of peptides from Panax ginseng C. A. Meyer using mass spectrometric approach.

PubMed

Ye, Xueting; Zhao, Nan; Yu, Xi; Han, Xiaoli; Gao, Huiyuan; Zhang, Xiaozhe

2016-11-01

Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS-based data mining and sequencing. The screen on the charge states of precursor ions indicated that Ginseng is a peptide-rich herb in comparison of a number of commonly used herbs. The Ginseng peptides were then extracted and submitted to nano-LC-MS/MS analysis using different fragmentation modes, including CID, high-energy collisional dissociation, and electron transfer dissociation. Further database search and de novo sequencing allowed the identification of total 308 peptides, some of which might have important biological activities. This study illustrates the abundance and sequences of endogenous Ginseng peptides, thus providing the information of more candidates for the screening of active compounds for future biological research and drug discovery studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products.

PubMed

Liu, Rui; Xing, Lujuan; Fu, Qingquan; Zhou, Guang-Hong; Zhang, Wan-Gang

2016-09-20

Antioxidant peptides are gradually being accepted as food ingredients, supplemented in functional food and nutraceuticals, to positively regulate oxidative stress in the human body against lipid and protein oxidation. Meat muscle and meat by-products are rich sources of proteins and can be regarded as good materials for the production of bioactive peptides by use of enzymatic hydrolysis or direct solvent extraction. In recent years, there has been a growing number of studies conducted to characterize antioxidant peptides or hydrolysates derived from meat muscle and by-products as well as processed meat products, including dry-cured hams. Antioxidant peptides obtained from animal sources could exert not only nutritional value but also bioavailability to benefit human health. This paper reviews the antioxidant peptides or protein hydrolysates identified in muscle protein and by-products. We focus on the procedure for the generation of peptides with antioxidant capacity including the acquisition of crude peptides, the assessment of antioxidant activity, and the purification and identification of the active fraction. It remains critical to perform validation experiments with a cell model, animal model or clinical trial to eliminate safety concerns before final application in the food system. In addition, some of the common characteristics on structure-activity relationship are also reviewed based on the identified antioxidant peptides.

A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products

PubMed Central

Liu, Rui; Xing, Lujuan; Fu, Qingquan; Zhou, Guang-hong; Zhang, Wan-gang

2016-01-01

Antioxidant peptides are gradually being accepted as food ingredients, supplemented in functional food and nutraceuticals, to positively regulate oxidative stress in the human body against lipid and protein oxidation. Meat muscle and meat by-products are rich sources of proteins and can be regarded as good materials for the production of bioactive peptides by use of enzymatic hydrolysis or direct solvent extraction. In recent years, there has been a growing number of studies conducted to characterize antioxidant peptides or hydrolysates derived from meat muscle and by-products as well as processed meat products, including dry-cured hams. Antioxidant peptides obtained from animal sources could exert not only nutritional value but also bioavailability to benefit human health. This paper reviews the antioxidant peptides or protein hydrolysates identified in muscle protein and by-products. We focus on the procedure for the generation of peptides with antioxidant capacity including the acquisition of crude peptides, the assessment of antioxidant activity, and the purification and identification of the active fraction. It remains critical to perform validation experiments with a cell model, animal model or clinical trial to eliminate safety concerns before final application in the food system. In addition, some of the common characteristics on structure-activity relationship are also reviewed based on the identified antioxidant peptides. PMID:27657142

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

PubMed

Bergmann, Tobias; Lindvall, Mikaela; Moore, Erin; Moore, Eugene; Sidney, John; Miller, Donald; Tallmadge, Rebecca L; Myers, Paisley T; Malaker, Stacy A; Shabanowitz, Jeffrey; Osterrieder, Nikolaus; Peters, Bjoern; Hunt, Donald F; Antczak, Douglas F; Sette, Alessandro

2017-05-01

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Microbial sequencing methods for monitoring of anaerobic treatment of antibiotics to optimize performance and prevent system failure.

PubMed

Aydin, Sevcan

2016-06-01

As a result of developments in molecular technologies and the use of sequencing technologies, the analyses of the anaerobic microbial community in biological treatment process has become increasingly prevalent. This review examines the ways in which microbial sequencing methods can be applied to achieve an extensive understanding of the phylogenetic and functional characteristics of microbial assemblages in anaerobic reactor if the substrate is contaminated by antibiotics which is one of the most important toxic compounds. It will discuss some of the advantages and disadvantages associated with microbial sequencing techniques that are more commonly employed and will assess how a combination of the existing methods may be applied to develop a more comprehensive understanding of microbial communities and improve the validity and depth of the results for the enhancement of the stability of anaerobic reactors.

Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8 weeks.

PubMed

Hale, Vanessa L; Tan, Chia L; Knight, Rob; Amato, Katherine R

2015-06-01

Studies of the gut microbiome have become increasingly common with recent technological advances. Gut microbes play an important role in human and animal health, and gut microbiome analysis holds great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, many common fecal preservation protocols (e.g. freezing at -80 °C) are not suitable for field conditions, or have not been tested for long-term (greater than 2 weeks) storage. In this study, we collected fresh fecal samples from captive spider monkeys (Ateles geoffroyi) at the Columbian Park Zoo (Lafayette, IN, USA). The samples were pooled, homogenized, and preserved for up to 8 weeks prior to DNA extraction and sequencing. Preservation methods included: freezing at -20 °C, freezing at -80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater. At 0 (fresh), 1, 2, 4, and 8 weeks from fecal collection, DNA was extracted and microbial DNA was amplified and sequenced. DNA concentration, purity, microbial diversity, and microbial composition were compared across all methods and time points. DNA concentration and purity did not correlate with microbial diversity or composition. Microbial composition of frozen and ethanol samples were most similar to fresh samples. FTA card and RNAlater-preserved samples had the least similar microbial composition and abundance compared to fresh samples. Microbial composition and diversity were relatively stable over time within each preservation method. Based on these results, if freezers are not available, we recommend preserving fecal samples in ethanol (for up to 8weeks) prior to microbial extraction and analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Butelase-mediated cyclization and ligation of peptides and proteins.

PubMed

Nguyen, Giang K T; Qiu, Yibo; Cao, Yuan; Hemu, Xinya; Liu, Chuan-Fa; Tam, James P

2016-10-01

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Seasonal induced changes in spinach rhizosphere microbial community structure with varying salinity and drought

USDA-ARS?s Scientific Manuscript database

Salinity is a common problem under irrigated agriculture, especially in low rainfall and high evaporative demand areas of southwestern United States and other semi-arid regions around the world. However, studies on salinity effects on soil microbial communities are relatively few while the effects o...

Spatial and temporal variation of fecal indicator organisms in two creeks in Beltsville, Maryland

USDA-ARS?s Scientific Manuscript database

Evaluation of microbial water quality is commonly achieved by monitoring populations of indicator bacteria such as E. coli and enterococci. Monitoring data are utilized by water managers to predict potential fecal contaminations as well as a decision tool to improve microbial water quality. Both te...

Pilot-scale vadose zone microbial biobarriers removed nitrate leaching from a cattle corral

USDA-ARS?s Scientific Manuscript database

activities that involve animal wastes can result in the contamination of subsurface soils by nitrates. In saturated or nearly saturated soils microbial biobarriers are a common method used to remove contaminants from water. This field study was conducted beneath a cattle pen in northeast Colorado a...

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

PubMed

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Synergy of antibacterial and antioxidant activities from crude extracts and peptides of selected plant mixture

PubMed Central

2013-01-01

Background A plant mixture containing indigenous Australian plants was examined for synergistic antimicrobial activity using selected test microorganisms. This study aims to investigate antibacterial activities, antioxidant potential and the content of phenolic compounds in aqueous, ethanolic and peptide extracts of plant mixture. Methods Well diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were used to test antibacterial activity against four pathogenic bacteria namely Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Pseudomonas aeruginosa. DPPH (2, 2-diphenyl-1- picrylhydrazyl) and superoxide dismutase (SOD) assays were used to evaluate antioxidant activity. HPLC and gel filtration were used for purification of the peptides. Scanning electron microscope was applied to investigate the mode of attachment of the peptides on target microbial membranes. Results Aqueous extraction of the mixture showed no inhibition zones against all the test bacteria. Mean diameter of inhibition zones for ethanol extraction of this mixture attained 8.33 mm, 7.33 mm, and 6.33 mm against S. aureus at corresponding concentrations of 500, 250 and 125 mg/ml while E .coli showed inhibition zones of 9.33 mm, 8.00 mm and 6.66 mm at the same concentrations. B. cereus exhibited inhibition zones of 11.33 mm, 10.33 mm and 10.00 mm at concentrations of 500, 250 and 125 mg/ml respectively. The peptide extract demonstrated antibacterial activity against S. aureus, E. coli and B. cereus. The MIC and MBC values for ethanol extracts were determined at 125 mg/ml concentration against S. aureus and E. coli and B. cereus value was 31.5 mg/ml. MIC and MBC values showed that the peptide extract was significantly effective at low concentration of the Australian plant mixture (APM). Phenolic compounds were detected in hot aqueous and ethanolic extracts of the plant mixture. Hot aqueous, ethanol and peptides extracts also exhibited antioxidant activities. Conclusions It was concluded that APM possessed good antibacterial and antioxidant activities following extraction with different solvents. The results suggest that APM provide a new source with antibacterial agents and antioxidant activity for nutraceutical or medical applications. PMID:24330547

Design of Embedded-Hybrid Antimicrobial Peptides with Enhanced Cell Selectivity and Anti-Biofilm Activity

PubMed Central

Xu, Wei; Zhu, Xin; Tan, Tingting; Li, Weizhong; Shan, Anshan

2014-01-01

Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents. PMID:24945359

Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

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

Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances themore » flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.« less

Pro-necrotic Activity of Cationic Mastoparan Peptides in Human Glioblastoma Multiforme Cells Via Membranolytic Action.

PubMed

da Silva, Annielle Mendes Brito; Silva-Gonçalves, Laíz Costa; Oliveira, Fernando Augusto; Arcisio-Miranda, Manoel

2018-07-01

Glioblastoma multiforme is the most common and lethal malignant brain tumor. Because of its complexity and heterogeneity, this tumor has become resistant to conventional therapies and the available treatment produces multiple side effects. Here, using multiple experimental approaches, we demonstrate that three mastoparan peptides-Polybia-MP1, Mastoparan X, and HR1-from solitary wasp venom exhibit potent anticancer activity toward human glioblastoma multiforme cells. Importantly, the antiglioblastoma action of mastoparan peptides occurs by membranolytic activity, leading to necrosis. Our data also suggest a direct relation between mastoparan membranolytic potency and the presence of negatively charged phospholipids like phosphatidylserine. Collectively, these data may warrant additional studies for mastoparan peptides as new agents for the treatment of glioblastoma multiforme brain tumor.

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

DOE PAGES

Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; ...

2016-01-24

Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac7 1–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac7 1–35,more » Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

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

Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.

Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac7 1–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac7 1–35,more » Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

A common structural motif in immunopotentiating peptides with sequences present in human autoantigens. Elicitation of a response mediated by monocytes and Th1 cells.

PubMed

López-Moratalla, N; Ruíz, E; López-Zabalza, M J; Santiago, E

1996-12-16

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

Remineralization of initial enamel caries in vitro using a novel peptide based on amelogenin

NASA Astrophysics Data System (ADS)

Li, Danxue; Lv, Xueping; Tu, Huanxin; Zhou, Xuedong; Yu, Haiyang; Zhang, Linglin

2015-09-01

Dental caries is the most common oral disease with high incidence, widely spread and can seriously affect the health of oral cavity and the whole body. Current caries prevention measures such as fluoride treatment, antimicrobial agents, and traditional Chinese herbal, have limitations to some extent. Here we design and synthesize a novel peptide based on the amelogenin, and assess its ability to promote the remineralization of initial enamel caries lesions. We used enamel blocks to form initial lesions, and then subjected to 12-day pH cycling in the presence of peptide, NaF and HEPES buffer. Enamel treated with peptide or NaF had shallower, narrower lesions, thicker remineralized surfaces and less mineral loss than enamel treated with HEPES. This peptide can promote the remineralization of initial enamel caries and inhibit the progress of caries. It is a promising anti-caries agent with various research prospects and practical application value.

Modification Site Localization in Peptides.

PubMed

Chalkley, Robert J

2016-01-01

There are a large number of search engines designed to take mass spectrometry fragmentation spectra and match them to peptides from proteins in a database. These peptides could be unmodified, but they could also bear modifications that were added biologically or during sample preparation. As a measure of reliability for the peptide identification, software normally calculates how likely a given quality of match could have been achieved at random, most commonly through the use of target-decoy database searching (Elias and Gygi, Nat Methods 4(3): 207-214, 2007). Matching the correct peptide but with the wrong modification localization is not a random match, so results with this error will normally still be assessed as reliable identifications by the search engine. Hence, an extra step is required to determine site localization reliability, and the software approaches to measure this are the subject of this part of the chapter.

Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress

PubMed Central

2015-01-01

Protein structure underlies essential biological processes and provides a blueprint for molecular mimicry that drives drug discovery. Although small molecules represent the lion’s share of agents that target proteins for therapeutic benefit, there remains no substitute for the natural properties of proteins and their peptide subunits in the majority of biological contexts. The peptide α-helix represents a common structural motif that mediates communication between signaling proteins. Because peptides can lose their shape when taken out of context, developing chemical interventions to stabilize their bioactive structure remains an active area of research. The all-hydrocarbon staple has emerged as one such solution, conferring α-helical structure, protease resistance, cellular penetrance, and biological activity upon successful incorporation of a series of design and application principles. Here, we describe our more than decade-long experience in developing stapled peptides as biomedical research tools and prototype therapeutics, highlighting lessons learned, pitfalls to avoid, and keys to success. PMID:24601557

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

PubMed Central

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-01

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

PubMed

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-02

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.

Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups.

PubMed

Haaf, Erik; Schlosser, Andreas

2012-01-03

We have developed a new method that applies acidic catalysis with hydrochloric acid for (18)O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95-97 atom % incorporation of (18)O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides. © 2011 American Chemical Society

Comparison of Urine and Plasma Peptidome Indicates Selectivity in Renal Peptide Handling.

PubMed

Magalhães, Pedro; Pontillo, Claudia; Pejchinovski, Martin; Siwy, Justyna; Krochmal, Magdalena; Makridakis, Manousos; Carrick, Emma; Klein, Julie; Mullen, William; Jankowski, Joachim; Vlahou, Antonia; Mischak, Harald; Schanstra, Joost P; Zürbig, Petra; Pape, Lars

2018-04-03

Urine is considered to be produced predominantly as a result of plasma filtration in the kidney. However, the origin of the native peptides present in urine has never been investigated in detail. Therefore, the authors aimed to obtain a first insight into the origin of urinary peptides based on a side-by-side comprehensive analysis of the plasma and urine peptidome. Twenty-two matched urine and plasma samples are analyzed for their peptidome using capillary electrophoresis coupled to mass spectrometry (CE-MS; for relative quantification) and CE or LC coupled to tandem mass spectrometry (CE- or LC-MS/MS; for peptide identification). The overlap and association of abundance of the different peptides present in these two body fluids are evaluated. The authors are able to identify 561 plasma and 1461 urinary endogenous peptides. Only 90 peptides are detectable in both urine and plasma. No significant correlation is found when comparing the abundance of these common peptides, with the exception of collagen fragments. This observation is also supported when comparing published peptidome data from both plasma and urine. Most of the plasma peptides are not detectable in urine, possibly due to tubular reabsorption. The majority of urinary peptides may in fact originate in the kidney. The notable exception is collagen fragments, which indicates potential selective exclusion of these peptides from tubular reabsorption. Experimental verification of this hypothesis is warranted. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anti-infective therapeutics from the Lepidopteran model host Galleria mellonella.

PubMed

Vilcinskas, Andreas

2011-01-01

The larvae of the greater wax moth Galleria mellonella prosper in use both as surrogate alternative model hosts for human pathogens and as a whole-animal-high-throughput-system for in vivo testing of antibiotics or mutant-libraries of pathogens. In addition, a broad spectrum of antimicrobial peptides and proteins has been identified in this insect during past decade among which some appear to be specific for Lepidoptera. Its arsenal of immunity-related effector molecules encompasses peptides and proteins exhibiting potent activity against bacteria, fungi or both, whose potential as new anti-infective therapeutics are presently being explored. Of particular interest is the insect metalloproteinase inhibitor (IMPI) which has been discovered in G. mellonella. The IMPI exhibits a specific and potent activity against thermolysin-like microbial metalloproteinases including a number of prominent virulence and/or pathogenic factors of human pathogens which are responsible for severe symptoms such as septicemia, hemorrhagic tissue bleeding, necrosis and enhancement of vascular permeability. The IMPI and antimicrobial peptides from G. mellonella may provide promising templates for the rational design of new drugs since evidence is available that the combination of antibiotics with inhibitors of pathogen-associated proteolytic enzymes yields synergistic therapeutic effects. The potential and limitations of insect-derived gene-encoded antimicrobial compounds as anti-infective therapeutics are discussed.

Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

PubMed Central

Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young

2014-01-01

Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension. PMID:25140307

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis.

PubMed

Bleich, Rachel; Watrous, Jeramie D; Dorrestein, Pieter C; Bowers, Albert A; Shank, Elizabeth A

2015-03-10

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi.

PubMed

Vieira Bard, Gabriela C; Nascimento, Viviane V; Ribeiro, Suzanna F F; Rodrigues, Rosana; Perales, Jonas; Teixeira-Ferreira, André; Carvalho, André O; Fernandes, Katia Valevski S; Gomes, Valdirene M

2015-04-01

Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation

PubMed Central

Yost, Christian C.; Schwertz, Hansjörg; Cody, Mark J.; Wallace, Jared A.; Campbell, Robert A.; Vieira-de-Abreu, Adriana; Araujo, Claudia V.; Schubert, Sebastian; Harris, Estelle S.; Rowley, Jesse W.; Rondina, Matthew T.; Koening, Curry L.; Weyrich, Andrew S.; Zimmerman, Guy A.

2016-01-01

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation. PMID:27599294

An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

PubMed

Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L

2016-12-01

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

Transglutaminases in Dysbiosis As Potential Environmental Drivers of Autoimmunity

PubMed Central

Lerner, Aaron; Aminov, Rustam; Matthias, Torsten

2017-01-01

Protein-glutamine γ-glutamyltransferases (transglutaminases, Tgs) belong to the class of transferases. They catalyze the formation of an isopeptide bond between the acyl group at the end of the side chain of protein- or peptide-bound glutamine residues and the first order 𝜀-amine groups of protein- or peptide-bound lysine. The Tgs are considered to be universal protein cross-linkers, and they play an essential role in a number of human diseases. In this review, we discuss mainly the bacterial Tgs in terms of the functionality of the enzymes and a potential role they may play in bacterial survival. Since microbial transglutaminases (mTgs) are functionally similar to the human homologs, they may be involved in the human disease provocation. We suggest here a potential involvement of Tgs in the pathologies such as autoimmune diseases. In this hypothesis, the endogenous mTgs that are secreted by the gut microbiota, especially in a dysbiotic configuration, are potential drivers of systemic autoimmunity, via the enzymatic posttranslational modification of peptides in the gut lumen. These mTg activities directed toward cross-linking of naïve proteins can potentially generate neo-epitopes that are not only immunogenic but may also activate some immune response cascades leading to the pathological autoimmune processes. PMID:28174571

Microbial host interactions and impaired wound healing in mice and humans: defining a role for BD14 and NOD2.

PubMed

Williams, Helen; Campbell, Laura; Crompton, Rachel A; Singh, Gurdeep; McHugh, Brian J; Davidson, Donald J; McBain, Andrew J; Cruickshank, Sheena M; Hardman, Matthew J

2018-04-30

Chronic wounds cause significant patient morbidity and mortality. A key factor in their etiology is microbial infection, yet skin host-microbiota interactions during wound repair remain poorly understood. Microbiome profiles of non-infected human chronic wounds are associated with subsequent healing outcome. Furthermore, poor clinical healing outcome was associated with increased local expression of the pattern recognition receptor NOD2. To investigate NOD2 function in the context of cutaneous healing, we treated mice with the NOD2 ligand muramyl dipeptide (MDP) and analyzed wound repair parameters and expression of anti-microbial peptides. MDP treatment of littermate controls significantly delayed wound repair associated with reduced re-epithelialization, heightened inflammation and upregulation of murine β-Defensins (mBD) 1, 3 and particularly 14. We postulated that although BD14 might impact on local skin microbial communities it may further impact other healing parameters. Indeed, exogenously administered mBD14 directly delayed mouse primary keratinocyte scratch wound closure in vitro. To further explore the role of mBD14 in wound repair, we employed Defb14 -/- mice, and showed they had a global delay in healing in vivo, associated with alterations in wound microbiota. Taken together these studies suggest a key role for NOD2-mediated regulation of local skin microbiota which in turn impacts on chronic wound etiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Stability of Microbiota Facilitated by Host Immune Regulation: Informing Probiotic Strategies to Manage Amphibian Disease

PubMed Central

Küng, Denise; Bigler, Laurent; Davis, Leyla R.; Gratwicke, Brian; Griffith, Edgardo; Woodhams, Douglas C.

2014-01-01

Microbial communities can augment host immune responses and probiotic therapies are under development to prevent or treat diseases of humans, crops, livestock, and wildlife including an emerging fungal disease of amphibians, chytridiomycosis. However, little is known about the stability of host-associated microbiota, or how the microbiota is structured by innate immune factors including antimicrobial peptides (AMPs) abundant in the skin secretions of many amphibians. Thus, conservation medicine including therapies targeting the skin will benefit from investigations of amphibian microbial ecology that provide a model for vertebrate host-symbiont interactions on mucosal surfaces. Here, we tested whether the cutaneous microbiota of Panamanian rocket frogs, Colostethus panamansis, was resistant to colonization or altered by treatment. Under semi-natural outdoor mesocosm conditions in Panama, we exposed frogs to one of three treatments including: (1) probiotic - the potentially beneficial bacterium Lysinibacillus fusiformis, (2) transplant – skin washes from the chytridiomycosis-resistant glass frog Espadarana prosoblepon, and (3) control – sterile water. Microbial assemblages were analyzed by a culture-independent T-RFLP analysis. We found that skin microbiota of C. panamansis was resistant to colonization and did not differ among treatments, but shifted through time in the mesocosms. We describe regulation of host AMPs that may function to maintain microbial community stability. Colonization resistance was metabolically costly and microbe-treated frogs lost 7–12% of body mass. The discovery of strong colonization resistance of skin microbiota suggests a well-regulated, rather than dynamic, host-symbiont relationship, and suggests that probiotic therapies aiming to enhance host immunity may require an approach that circumvents host mechanisms maintaining equilibrium in microbial communities. PMID:24489847

Remote Sensing between Liver and Intestine: Importance of Microbial Metabolites

PubMed Central

Fu, Zidong Donna; Cui, Julia Yue

2017-01-01

Recent technological advancements including metagenomics sequencing and metabolomics have allowed the discovery of critical functions of gut microbiota in obesity, malnutrition, neurological disorders, asthma, and xenobiotic metabolism. Classification of the human gut microbiome into distinct “enterotypes” has been proposed to serve as a new paradigm for understanding the interplay between microbial variation and human disease phenotypes, as many organs are affected by gut microbiota modifications during the pathogenesis of diseases. Gut microbiota remotely interacts with liver and other metabolic organs of the host through various microbial metabolites that are absorbed into the systemic circulation. Purpose of review The present review summarizes recent literature regarding the importance of gut microbiota in modulating the physiological and pathological responses of various host organs, and describes the functions of the known microbial metabolites that are involved in this remote sensing process, with a primary focus on the gut microbiota-liver axis. Recent findings Under physiological conditions, gut microbiota modulates the hepatic transcriptome, proteome, and metabolome, most notably down-regulating cytochrome P450 3a mediated xenobiotic metabolism. Gut microbiome also modulates the rhythmicity in liver gene expression, likely through microbial metabolites, such as butyrate and propionate that serve as epigenetic modifiers. Additionally, the production of host hormones such as primary bile acids and glucagon like peptide 1 is altered by gut microbiota to modify intermediary metabolism of the host. Summary Dysregulation of gut microbiota is implicated in various liver diseases such as alcoholic liver disease, non-alcoholic steatohepatitis, liver cirrhosis, cholangitis, and liver cancer. Gut microbiota modifiers such as probiotics and prebiotics are increasingly recognized as novel therapeutic modalities for liver and other types of human diseases. PMID:28983453

Pure and Oxidized Copper Materials as Potential Antimicrobial Surfaces for Spaceflight Activities

NASA Astrophysics Data System (ADS)

Hahn, C.; Hans, M.; Hein, C.; Mancinelli, R. L.; Mücklich, F.; Wirth, R.; Rettberg, P.; Hellweg, C. E.; Moeller, R.

2017-12-01

Microbial biofilms can lead to persistent infections and degrade a variety of materials, and they are notorious for their persistence and resistance to eradication. During long-duration space missions, microbial biofilms present a danger to crew health and spacecraft integrity. The use of antimicrobial surfaces provides an alternative strategy for inhibiting microbial growth and biofilm formation to conventional cleaning procedures and the use of disinfectants. Antimicrobial surfaces contain organic or inorganic compounds, such as antimicrobial peptides or copper and silver, that inhibit microbial growth. The efficacy of wetted oxidized copper layers and pure copper surfaces as antimicrobial agents was tested by applying cultures of Escherichia coli and Staphylococcus cohnii to these metallic surfaces. Stainless steel surfaces were used as non-inhibitory control surfaces. The production of reactive oxygen species and membrane damage increased rapidly within 1 h of exposure on pure copper surfaces, but the effect on cell survival was negligible even after 2 h of exposure. However, longer exposure times of up to 4 h led to a rapid decrease in cell survival, whereby the survival of cells was additionally dependent on the exposed cell density. Finally, the release of metal ions was determined to identify a possible correlation between copper ions in suspension and cell survival. These measurements indicated a steady increase of free copper ions, which were released indirectly by cells presumably through excreted complexing agents. These data indicate that the application of antimicrobial surfaces in spaceflight facilities could improve crew health and mitigate material damage caused by microbial contamination and biofilm formation. Furthermore, the results of this study indicate that cuprous oxide layers were superior to pure copper surfaces related to the antimicrobial effect and that cell density is a significant factor that influences the time dependence of antimicrobial activity.

Antifungal activity of a β-peptide in synthetic urine media: Toward materials-based approaches to reducing catheter-associated urinary tract fungal infections.

PubMed

Raman, Namrata; Lee, Myung-Ryul; Rodríguez López, Angélica de L; Palecek, Sean P; Lynn, David M

2016-10-01

Catheter-associated urinary tract infections (CAUTI) are the most common type of hospital-acquired infection, with more than 30 million catheters placed annually in the US and a 10-30% incidence of infection. Candida albicans forms fungal biofilms on the surfaces of urinary catheters and is the leading cause of fungal urinary tract infections. As a step toward new strategies that could prevent or reduce the occurrence of C. albicans-based CAUTI, we investigated the ability of antifungal β-peptide-based mimetics of antimicrobial peptides (AMPs) to kill C. albicans and prevent biofilm formation in synthetic urine. Many α-peptide-based AMPs exhibit antifungal activities, but are unstable in high ionic strength media and are easily degraded by proteases-features that limit their use in urinary catheter applications. Here, we demonstrate that β-peptides designed to mimic the amphiphilic helical structures of AMPs retain 100% of their structural stability and exhibit antifungal and anti-biofilm activity against C. albicans in a synthetic medium that mimics the composition of urine. We demonstrate further that these agents can be loaded into and released from polymer-based multilayer coatings applied to polyurethane, polyethylene, and silicone tubing commonly used as urinary catheters. Our results reveal catheters coated with β-peptide-loaded multilayers to kill planktonic fungal cells for up to 21days of intermittent challenges with C. albicans and prevent biofilm formation on catheter walls for at least 48h. These new materials and approaches could lead to advances that reduce the occurrence of fungal CAUTI. Catheter-associated urinary tract infections are the most common type of hospital-acquired infection. The human pathogen Candida albicans is the leading cause of fungal urinary tract infections, and forms difficult to remove 'biofilms' on the surfaces of urinary catheters. We investigated synthetic β-peptide mimics of natural antimicrobial peptides as an approach to kill C. albicans and prevent biofilm formation in media that mimics the composition of urine. Our results reveal these mimics to retain structural stability and activity against C. albicans in synthetic urine. We also report polymer-based approaches to the local release of these agents within urinary catheter tubes. With further development, these materials-based approaches could lead to advances that reduce the occurrence of fungal urinary tract infections. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Heat-induced alterations in cashew allergen solubility and IgE binding.

PubMed

Mattison, Christopher P; Bren-Mattison, Yvette; Vant-Hull, Barry; Vargas, Aurora M; Wasserman, Richard L; Grimm, Casey C

2016-01-01

Cashew nuts are an increasingly common cause of food allergy. We compare the soluble protein profile of cashew nuts following heating. SDS-PAGE indicate that heating can alter the solubility of cashew nut proteins. The 11S legumin, Ana o 2, dominates the soluble protein content in ready to eat and mildly heated cashew nuts. However, we found that in dark-roasted cashew nuts, the soluble protein profile shifts and the 2S albumin Ana o 3 composes up to 40% of the soluble protein. Analysis of trypsin-treated extracts by LC/MS/MS indicate changes in the relative number and intensity of peptides. The relative cumulative intensity of the 5 most commonly observed Ana o 1 and 2 peptides are altered by heating, while those of the 5 most commonly observed Ana o 3 peptides remaine relatively constant. ELISA experiments indicate that there is a decrease in rabbit IgG and human serum IgE binding to soluble cashew proteins following heating. Our findings indicate that heating can alter the solubility of cashew allergens, resulting in altered IgE binding. Our results support the use of both Ana o 2 and Ana o 3 as potential cashew allergen diagnostic targets.

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis.

PubMed

Castañeda-Delgado, J; Hernández-Pando, R; Serrano, C J; Aguilar-León, D; León-Contreras, J; Rivas-Santiago, C; Méndez, R; González-Curiel, I; Enciso-Moreno, A; Rivas-Santiago, B

2010-09-01

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti-microbial peptides in tuberculosis. The cathelin-related anti-microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well-characterized models of latent or chronic infection and progressive disease. The lung of non-infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post-infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelicidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis. © 2010 British Society for Immunology.

Innate immunity in the small intestine

PubMed Central

Santaolalla, Rebeca; Abreu, Maria T.

2012-01-01

Purpose of review This manuscript reviews the most recent publications on innate immunity in the small intestine. We will go over the innate immune receptors that act as sensors of microbial presence or cell injury, Paneth cells as the main epithelial cell type that secrete antimicrobial peptides, and mucosal production of IgA. In addition, we will give an update on examples of imbalance of the innate immune response resulting in clinical disease with the most relevant example being Crohn’s disease. Recent findings Toll-like receptors (TLRs) are involved in B-cell homing to the intestine, rejection of small intestinal allografts and recruitment of mast cells. The TLR adaptor TRIF is necessary to activate innate immunity after Yersinia enterocolitica infection. Moreover, MyD88 is required to keep the intestinal microbiota under control and physically separated from the epithelium and RegIIIγ is responsible for the bacterial segregation from the lining epithelial cells. In Crohn’s disease, ATG16L1 T300A variant promotes a pro-inflammatory response; and miR-196 downregulates a protective IRGM polymorphism leading to impaired clearance of adherent Escherichia coli in the intestine. Summary The intestine is continuously exposed to dietary and microbial antigens. The host has to maintain intestinal homeostasis to keep the commensal and pathogenic bacteria under control. Some of the mechanisms to do so are by expression of innate immune receptors, production of antimicrobial peptides, secretion of IgA or autophagy of intracellular bacteria. Unfortunately, in some cases the innate immune response fails to protect the host and chronic inflammation, transplant rejection, or other pathologies may occur. PMID:22241076

Identification and Characterization of the First Cathelicidin from Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and Special Mechanism*

PubMed Central

Wei, Lin; Gao, Jiuxiang; Zhang, Shumin; Wu, Sijin; Xie, Zeping; Ling, Guiying; Kuang, Yi-Qun; Yang, Yongliang; Yu, Haining; Wang, Yipeng

2015-01-01

Cathelicidins are a family of gene-encoded peptide effectors of innate immunity found exclusively in vertebrates. They play pivotal roles in host immune defense against microbial invasions. Dozens of cathelicidins have been identified from several vertebrate species. However, no cathelicidin from marine reptiles has been characterized previously. Here we report the identification and characterization of a novel cathelicidin (Hc-CATH) from the sea snake Hydrophis cyanocinctus. Hc-CATH is composed of 30 amino acids, and the sequence is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL. Circular dichroism spectroscopy and structure modeling analysis indicated that Hc-CATH mainly assumes an amphipathic α-helical conformation in bacterial membrane-mimetic solutions. It possesses potent broad-spectrum and rapid antimicrobial activity. Meanwhile, it is highly stable and shows low cytotoxicity toward mammalian cells. The microbial killing activity of Hc-CATH is executed through the disruption of cell membrane and lysis of bacterial cells. In addition, Hc-CATH exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Hc-CATH directly binds with LPS to neutralize its toxicity, and it also binds to Toll-like receptor 4 (TLR4/MD2 complex), which therefore inhibits the binding of LPS to TLR4/MD2 complex and the subsequent activation of LPS-induced inflammatory response pathways. Taken together, our study demonstrates that Hc-CATH, the first cathelicidin from sea snake discovered to have both antimicrobial and anti-inflammatory activity, is a potent candidate for the development of peptide antibiotics. PMID:26013823

Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression.

PubMed Central

Jonas, V; Lin, C R; Kawashima, E; Semon, D; Swanson, L W; Mermod, J J; Evans, R M; Rosenfeld, M G

1985-01-01

Two mRNAs generated as a consequence of alternative RNA processing events in expression of the human calcitonin gene encode the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Both calcitonin and CGRP RNAs and their encoded peptide products are expressed in the human pituitary and in medullary thyroid tumors. On the basis of sequence comparison, it is suggested that both the calcitonin and CGRP exons arose from a common primordial sequence, suggesting that duplication and rearrangement events are responsible for the generation of this complex transcription unit. Images PMID:3872459

Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

PubMed

Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

1988-12-01

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4.

PubMed

Liu, Peng; Chen, Yong; Wang, Dan; Tang, Yanqiong; Tang, Hongqian; Song, Haichao; Sun, Qun; Zhang, Yueling; Liu, Zhu

2016-01-01

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii.

Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4

PubMed Central

Liu, Peng; Chen, Yong; Wang, Dan; Tang, Yanqiong; Tang, Hongqian; Song, Haichao; Sun, Qun; Zhang, Yueling; Liu, Zhu

2016-01-01

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii. PMID:27588015

Recent approaches in design of peptidomimetics for antimicrobial drug discovery research.

PubMed

Lohan, Sandeep; Bisht, Gopal Singh

2013-06-01

Resistant pathogenic microbial strains are emerging at a rate that far exceeds the pace of new anti-infective drug development. In order to combat resistance development, there is pressing need to develop novel class of antibiotics having different mechanism of action in comparison to existing antibiotics. Antimicrobial peptides (AMPs) have been identified as ubiquitous components of innate immune system and widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from broad spectrum of activity to low propensity of resistance development. However, AMPs present several drawbacks that strongly limit their clinical applicability as ideal drug candidates such as; poor bioavailability, potential immunogenicity and high production cost. Thus, to overcome the limitations of native peptides, peptidomimetic becomes an important and promising approach. The different research groups worldwide engaged in antimicrobial drug discovery over the past decade have paid tremendous effort to design peptidomimetics. This review will focus on recent approaches in design of antimicrobial peptidomimetics their structure-activity relationship studies, mode of action, selectivity & toxicity.

A two-step enzymatic modification method to reduce immuno-reactivity of milk proteins.

PubMed

Damodaran, Srinivasan; Li, Yan

2017-12-15

A two-step enzymatic approach to reduce immuno-reactivity of whey protein isolate and casein has been studied. The method involves partial hydrolysis of proteins with proteases, followed by repolymerization with microbial transglutaminase. Whey protein isolate partially hydrolyzed with chymotrypsin, trypsin, or thermolysin retained about 80%, 30%, and 20% of the original immuno-reactivity, respectively. Upon repolymerization the immuno-reactivity decreased to 45%, 35%, and 5%, respectively. The immuno-reactivity of hydrolyzed and repolymerized casein was negligible compared to native casein. The repolymerized products were partially resistant to in vitro digestion. Peptides released during digestion of repolymerized thermolysin-whey protein hydrolysate had less than 5% immuno-reactivity, whereas those of whey protein control exhibited a sinusoidal immuno-reactivity ranging from 5 to 20%. Peptides released during digestion of repolymerized thermolysin-casein hydrolysates had no immuno-reactivity. These results indicated that it is possible to produce hypoallergenic milk protein products using the two-step enzymatic modification method involving thermolysin and transglutaminase. Copyright © 2017. Published by Elsevier Ltd.

Phase-assisted synthesis and DNA unpacking evaluation of biologically inspired metallo nanocomplexes using peptide as unique building block.

PubMed

Raman, N; Sudharsan, S

2011-12-01

The goal of nanomaterials' surface modification using a biomaterial is to preserve the materials' bulk properties while modifying only their surface to possess desired recognition and specificity. Here, we have developed a phase-assisted, modified Brust-Schiffrin methodological synthesis of metallo nanocomplexes anchored by a peptide, N,N'-(1,3-propylene)-bis-hippuricamide. The spectral, thermal and morphological characterizations assure the formation of nanocomplexes. Therapeutic behavior of all the nanocomplexes has been well sighted by evaluating their DNA unpacking skills. In addition, we demonstrate their biological inspiration by targeting few bacterial and fungal strains. The in vitro antimicrobial investigation reports that all the nanocomplexes disrupt microbial cell walls/membranes efficiently and inhibit the growth of microbes. These sorts of nanocomplexes synthesized in large quantities and at low cost, deliver versatile biomedical applications, and can be used to treat various diseases which may often cause high mortality. Copyright © 2011 Elsevier Inc. All rights reserved.

Metagenomics and Bioinformatics in Microbial Ecology: Current Status and Beyond.

PubMed

Hiraoka, Satoshi; Yang, Ching-Chia; Iwasaki, Wataru

2016-09-29

Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems. However, the findings of typical metagenomic and bioinformatic analyses still do not completely describe the ecology and evolution of microbes in their environments. Most analyses still depend on straightforward sequence similarity searches against reference databases. We herein review the current state of metagenomics and bioinformatics in microbial ecology and discuss future directions for the field. New techniques will allow us to go beyond routine analyses and broaden our knowledge of microbial ecosystems. We need to enrich reference databases, promote platforms that enable meta- or comprehensive analyses of diverse metagenomic datasets, devise methods that utilize long-read sequence information, and develop more powerful bioinformatic methods to analyze data from diverse perspectives.

Connecting the Dots: Could Microbial Translocation Explain Commonly Reported Symptoms in HIV Disease?

PubMed Central

Wilson, Natalie L.; Vance, David E.; Moneyham, Linda D.; Raper, James L.; Mugavero, Michael J.; Heath, Sonya L.; Kempf, Mirjam-Colette

2017-01-01

Microbial translocation within the context of HIV disease has been described as one of the contributing causes of inflammation and disease progression in HIV infection. HIV-associated symptoms have been related to inflammatory markers and sCD14, a surrogate marker for microbial translocation, suggesting a plausible link between microbial translocation and symptom burden in HIV disease. Similar pathophysiological responses and symptoms have been reported in inflammatory bowel disease. We provide a comprehensive review of microbial translocation, HIV-associated symptoms, and symptoms connected with inflammation. We identify studies showing a relationship among inflammatory markers, sCD14, and symptoms reported in HIV disease. A conceptual framework and rationale to investigate the link between microbial translocation and symptoms is presented. The impact of inflammation on symptoms supports recommendations to reduce inflammation as part of HIV symptom management. Research in reducing microbial translocation-induced inflammation is limited, but needed, to further promote positive health outcomes among HIV-infected patients. PMID:25305025

Connecting the dots: could microbial translocation explain commonly reported symptoms in HIV disease?

PubMed

Wilson, Natalie L; Vance, David E; Moneyham, Linda D; Raper, James L; Mugavero, Michael J; Heath, Sonya L; Kempf, Mirjam-Colette

2014-01-01

Microbial translocation within the context of HIV disease has been described as one of the contributing causes of inflammation and disease progression in HIV infection. HIV-associated symptoms have been related to inflammatory markers and sCD14, a surrogate marker for microbial translocation, suggesting a plausible link between microbial translocation and symptom burden in HIV disease. Similar pathophysiological responses and symptoms have been reported in inflammatory bowel disease. We provide a comprehensive review of microbial translocation, HIV-associated symptoms, and symptoms connected with inflammation. We identify studies showing a relationship among inflammatory markers, sCD14, and symptoms reported in HIV disease. A conceptual framework and rationale to investigate the link between microbial translocation and symptoms is presented. The impact of inflammation on symptoms supports recommendations to reduce inflammation as part of HIV symptom management. Research in reducing microbial translocation-induced inflammation is limited, but needed, to further promote positive health outcomes among HIV-infected patients. Published by Elsevier Inc.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

PubMed

Schirmer, Melanie; Smeekens, Sanne P; Vlamakis, Hera; Jaeger, Martin; Oosting, Marije; Franzosa, Eric A; Ter Horst, Rob; Jansen, Trees; Jacobs, Liesbeth; Bonder, Marc Jan; Kurilshikov, Alexander; Fu, Jingyuan; Joosten, Leo A B; Zhernakova, Alexandra; Huttenhower, Curtis; Wijmenga, Cisca; Netea, Mihai G; Xavier, Ramnik J

2016-11-03

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP. Copyright © 2016 Elsevier Inc. All rights reserved.

Predominant MIC Mechanisms in the Oil and Gas Industry

DTIC Science & Technology

literature, terms such as microbial corrosion, biocorrosion, microbially influenced/induced corrosion, and biodegradation are often applied. All descriptions...express that microorganisms (bacteria, archaea, and fungi) influence the corrosion process of a given material. In this chapter, an overview of the common MIC mechanisms encountered in the oil and gas industry is presented.

Estimating removal rates of bacteria from poultry carcasses using two whole-carcass rinse volumes

USDA-ARS?s Scientific Manuscript database

Rinse sampling is a common method for determining the level of microbial contamination on poultry carcasses. One of the advantages of rinse sampling, over other carcass sampling methods, is that the results can be used for both process control applications and to estimate the total microbial level o...

Canopy gaps decrease microbial densities and disease risk for a shade-intolerant tree species

Treesearch

Kurt O. Reinhart; Alejandro A. Royo; Stacie A. Kageyama; Keith. Clay

2010-01-01

Canopy disturbances such as windthrowevents have obvious impacts on forest structure and composition aboveground, but changes in soil microbial communities and the consequences of these changes are less understood.We characterized the densities of a soil-borne pathogenic oomycete (Pythium) and a common saprotrophic zygomycete (Mortierella...

Productivity and salinity structuring of the microplankton revealed by comparative freshwater metagenomics

PubMed Central

Eiler, Alexander; Zaremba-Niedzwiedzka, Katarzyna; Martínez-García, Manuel; McMahon, Katherine D; Stepanauskas, Ramunas; Andersson, Siv G E; Bertilsson, Stefan

2014-01-01

Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes, and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared with freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities. PMID:24118837

Microbial exudate promoted dissolution and transformation of chromium containing minerals

NASA Astrophysics Data System (ADS)

Saad, E. M.; Sun, J.; Tang, Y.

2015-12-01

Because of its utility in many industrial processes, chromium has become the second most common metal contaminant in the United States. The two most common oxidation states of chromium in nature are Cr(III), which is highly immobile, and Cr(VI), which is highly mobile and toxic. In both natural and engineered environments, the most common remediation of Cr(VI) is through reduction, which results in chromium sequestration in the low solubility mixed Cr(III)-Fe(III) (oxy)hydroxide phases. Consequently, the stability of these minerals must be examined to assess the fate of chromium in the subsurface. We examined the dissolution of mixed Cr(III)-Fe(III) (oxy)hydroxides in the presence of common microbial exudates, including the siderophore desferrioxamine B (DFOB; a common organic ligand secreted by most microbes with high affinity for ferric iron and other trivalent metal ions) and oxalate (a common organic acid produced by microbes). The solids exhibited incongruent dissolution with preferential leaching of Fe from the solid phase. Over time, this leads to a more Cr rich mineral, which is known to be more soluble than the corresponding mixed mineral phase. We are currently investigating the structure of the reacted mineral phases and soluble Cr(III) species, as well as the potential oxidation and remobilization of the soluble Cr species. Results from this study will provide insights regarding the long term transport and fate of chromium in the natural environment in the presence of microbial activities.