Modelling study of dimerization in mammalian defensins

BACKGROUND: Defensins are antimicrobial peptides of innate immunity functioning by non-specific binding to anionic phospholipids in bacterial membranes. Their cationicity, amphipathicity and ability to oligomerize are considered key factors for their action. Based on structural information on human β-defensin 2, we examine homologous defensins from various mammalian species for conserved functional physico-chemical characteristics. RESULTS: Based on homology greater than 40%, structural models of 8 homologs of HBD-2 were constructed. A conserved pattern of electrostatics and dynamics was observed across 6 of the examined defensins; models backed by energetics suggest that the defensins in these 6 organisms are characterized by dimerization-linked enhanced functional potentials. In contrast, dimerization is not energetically favoured in the sheep, goat and mouse defensins, suggesting that they function efficiently as monomers. CONCLUSION: β-defensin 2 from some mammals may work as monomers while those in others, including humans, work as oligomers. This could potentially be used to design human defensins that may be effective at lower concentrations and hence have therapeutic benefits

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

<p>Abstract</p> <p>Background</p> <p>Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed <it>in silico </it>site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by <it>in vitro </it>site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis.</p> <p>Results</p> <p>MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic <it>in silico </it>mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the <it>Escherichia coli </it>expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein.</p> <p>Conclusion</p> <p>The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.</p

Gingival tissue human beta-defensin levels in relation to infection and inflammation.

Aim To profile gingival tissue levels of human beta-defensin (hBD)-2 and hBD-3 in relation to gingival inflammation, Th17-related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities. Materials and Methods Gingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real-time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation coefficients. Results Subgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p <.001), as well as interleukin (IL)-1 beta (p = .012), IL-10 (p = .024), IL-17A (p = .002), IL-17F (p = .006), and IL-23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD-2 and hBD-3 levels. Negative correlations were found between protease activity and hBD-2 (p = .033) and hBD-3(p = .003) levels. Conclusions Shift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.Peer reviewe

Optimization of expression, purification and handling anti bacteria feature protein of bovine neutrophil B-defensing BNBD2

زمینه و هدف: دیفنسین‎ها یکی از بزرگ‎ترین خانواده‎ی پپتید‎های ضد میکروب می‎باشند که به واسطه‎ی فعالیت بر علیه باکتری‎ها، قارچ‎ها و بسیاری از ویروس‎ها به عنوان آنتی‎بیوتیک‎های نسل جدید منفعت بسیاری دارند. هدف از این مطالعه بهینه سازی بیان، تخلیص و بررسی خاصیت ضد میکروبی پروتئین بتا دیفنسین 2 نوتروفیل‎های گاو (BNBD2) بوده است. روش بررسی: در این مطالعه‎ی تجربی-آزمایشگاهی باکتری اشرشیاکلی B‏L21(DE3) حامل وکتور pET-32a(+) که ژن BNBD2 در آن همسانه سازی شده بود جهت مطالعات مورد استفاده قرار گرفت. بیان پروتئین BNBD2 با تغییر در پارامترهای دانسیته‎ی سلولی، دمای رشد، مدت زمان القاء با استفاده از سیستم الکتروفورز عمودی (SDS-PAGE) و تست برادفورد به صورت کمی و کیفی بررسی گردید. مراحل تخلیص پروتئین نوترکیب با کمک روش شیمیایی شکافت در جایگاه فرمیک اسید و عبور از سانتریکون و اثر ضد باکتری پروتئین تخلیص شده بر چند نمونه‎ی باکتریایی گرم مثبت و گرم منفی مورد بررسی قرار گرفت. یافته ها: با استفاده از محیط کشت Luria–Bertani، شروع القاء در جذب نوری 8/0 در طول موج 600 نانومتر، غلظت یک میلی مولار ماده‎ی القاء کننده‎ی IPTG، دمای رشد 30 درجه و مدت زمان 4 ساعت پس از القاء بیشترین میزان بیان پروتئین به دست آمد. پروتئین نوترکیب با استفاده از شکافت در جایگاه فرمیک اسید و عبور از سانتریکون تخلیص گردید. نتایج آزمایش وسترن بلاتینگ نیز نشان داد که پروتئین نوترکیب به طور اختصاصی به آنتی‎بادی mouse anti-(His)6 peroxidase متصل می‎گردد. تشکیل هاله‎ی عدم رشد در محیط‎های کشت مولر هینتون آگار حاوی کشت سطحی باکتری های مورد آزمایش خاصیت ضد باکتری این پروتئین را نشان داد. نتیجه گیری: با توجه به خاصیت ضد میکروبی پروتئین BNBD2و امکان بیان پروتئین در باکتری E. coli می توان به تولید انبوه این پروتئین نوترکیب اقدام نمود

Computational Investigation of the Pore Formation Mechanism of Beta-Hairpin Antimicrobial Peptides

β-hairpin antimicrobial peptides (AMPs) are small, usually cationic peptides that provide innate biological defenses against multiple agents. They have been proposed as the basis for novel antibiotics, but their pore formation has not been directly observed on a molecular level. We review previous computational studies of peptide-induced membrane pore formation and report several new molecular dynamics simulations of β-hairpin AMPs to elucidate their pore formation mechanism. We simulated β-barrels of various AMPs in anionic implicit membranes, finding that most of the AMPs’ β-barrels were not as stable as those of protegrin. We also performed an optimization study of protegrin β-barrels in implicit membranes, finding that nonamers were the most stable, but that multiplicities 7–13 were almost equally favorable. This indicated the possibility of a diversity of pore states consisting of various numbers of protegrin peptides. Finally, we used the Anton 2 supercomputer to perform multimicrosecond, all-atom molecular dynamics simulations of various protegrin-1 oligomers on the membrane surface and in transmembrane topologies. We also considered an octamer of the β-hairpin AMP tachyplesin. The simulations on the membrane surface indicated that protegrin dimers are stable, while trimers and tetramers break down because they assume a bent, twisted β-sheet shape. Tetrameric arcs remained stably inserted, but the pore water was displaced by lipid molecules. Unsheared protegrin β-barrels opened into long, twisted β-sheets that surrounded stable aqueous pores, whereas tilted barrels with sheared hydrogen bonding patterns were stable in most topologies. A third type of observed pore consisted of multiple small oligomers surrounding a small, partially lipidic pore. The octameric tachyplesin bundle resulted in small pores surrounded by 6 peptides as monomers and dimers. The results imply that multiple protegrin configurations may produce aqueous pores and illustrate the relationship between topology and pore formation steps. However, these structures’ long-term stability requires further investigation

Establishing bioinformatics research in the Asia Pacific

In 1998, the Asia Pacific Bioinformatics Network (APBioNet), Asia's oldest bioinformatics organisation was set up to champion the advancement of bioinformatics in the Asia Pacific. By 2002, APBioNet was able to gain sufficient critical mass to initiate the first International Conference on Bioinformatics (InCoB) bringing together scientists working in the field of bioinformatics in the region. This year, the InCoB2006 Conference was organized as the 5(th )annual conference of the Asia-Pacific Bioinformatics Network, on Dec. 18–20, 2006 in New Delhi, India, following a series of successful events in Bangkok (Thailand), Penang (Malaysia), Auckland (New Zealand) and Busan (South Korea). This Introduction provides a brief overview of the peer-reviewed manuscripts accepted for publication in this Supplement. It exemplifies a typical snapshot of the growing research excellence in bioinformatics of the region as we embark on a trajectory of establishing a solid bioinformatics research culture in the Asia Pacific that is able to contribute fully to the global bioinformatics community

STRUCTURAL, FUNCTIONAL AND EVOLUTIONARY STUDIES OF ANTIMICROBIAL PEPTIDES

Antimicrobial peptides represent a heterogeneous group that displays multiple modes of action such as bacteriostatic, microbicidal and cytolytic properties that are sequence and concentration dependent. Life threatening infectious disease is now a worldwide crisis and treating them effectively is becoming difficult day by day, due to the emergence of antibiotic resistant strains at alarming rates. Hence, there is an urgent need for new class of antibiotics and, antimicrobial peptides (AMPs) are an ideal candidate for this job. AMPs are gene encoded short (<100 amino acids), amphipathic molecules with hydrophobic and cationic amino acids arranged spatially which exhibit broad-spectrum antimicrobial activity. AMPs form an ancient non-specific type of innate immunity found universally in all living organisms and used as the principal first line of defense against the invading pathogen. AMPs have been in the process of evolution, as have the microbes, for hundreds of years. Despite the long history of co-evolution, AMPs have not lost their ability to kill the microbes totally nor have the microbes learnt to avoid the lethal punch of AMPs. Based upon accumulating positive data, we are encouraged to believe that antimicrobial peptides have a great potential to be the next breakthrough and first novel, truly biological in nature, class of antibiotics. The purpose of this study was twofold; primarily to elucidate the factors involved in governing the peptide activity and toxicity against membranes, and secondly to design a simple approach where we can boost and spread the spectrum of antimicrobial activity against pathogens such as S. aureus and P. aeruginosa for a peptide that is otherwise non-lethal to the bacteria. Results presented in this thesis show that antimicrobial domains of the anaphylatoxin C3a are structurally and evolutionary conserved. Moreover antimicrobial activity is not governed by a single factor, but instead by a combination of net charge, amphipathicity and helicity. By utilizing a low number of amino acid substitutions at strategic positions in an antimicrobial peptide derived from C3a, CNY20, we were able to develop peptides, which exert a significant activity on both S. aureus and C. albicans in contrast to the parent peptide. Although, antimicrobial activity is not governed by single parameter, the activity can still be boosted by end-tagging of a peptide with hydrophobic oligopeptide stretches. This modification promotes peptide binding to bacteria and subsequent cell wall rupture, but does not increase the toxicity or the protease susceptibility of the peptide. It is noteworthy that end tagging of ultra short peptides spanning 5-7 amino acids with hydrophobic amino acids enhances bactericidal activity, while preserving low toxicity and protease resistance

Molecular mechanism of action of tyrocidine antimicrobial peptides using NMR spectroscopy and computational techniques

Includes abstract.Includes bibliographical references.The need to come up with new and novel antibiotics that utilize unique mechanisms, to which bacteria cannot generate resistance, was the main motivation of this study. Tyrocidine peptides are non-selective antibiotics that have such properties. However, very limited information is available about their mechanism of action. The aim of this study was to determine the mechanism of action of tyrocidine peptides, tyrocidine A, tyrocidine B and tyrocidine C

Gingival Tissue Human Beta-Defensin Levels in Relation to Infection and Inflammation

AimTo profile gingival tissue levels of human beta‐defensin (hBD)‐2 and hBD‐3 in relation to gingival inflammation, Th17‐related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities.Materials and MethodsGingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real‐time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal–Wallis and Mann–Whitney U tests and Spearman correlation coefficients.ResultsSubgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p p = .012), IL‐10 (p = .024), IL‐17A (p = .002), IL‐17F (p = .006), and IL‐23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD‐2 and hBD‐3 levels. Negative correlations were found between protease activity and hBD‐2 (p = .033) and hBD‐3(p = .003) levels.ConclusionsShift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.</p