Dataset Information

Structure-function analysis of Avian ?-defensin-6 and ?-defensin-12: role of charge and disulfide bridges.

ABSTRACT: Avian beta-defensins (AvBD) are small, cationic, antimicrobial peptides. The potential application of AvBDs as alternatives to antibiotics has been the subject of interest. However, the mechanisms of action remain to be fully understood. The present study characterized the structure-function relationship of AvBD-6 and AvBD-12, two peptides with different net positive charges, similar hydrophobicity and distinct tissue expression profiles.AvBD-6 was more potent than AvBD-12 against E. coli, S. Typhimurium, and S. aureus as well as clinical isolates of extended spectrum beta lactamase (ESBL)-positive E. coli and K. pneumoniae. AvBD-6 was more effective than AvBD-12 in neutralizing LPS and interacting with bacterial genomic DNA. Increasing bacterial concentration from 10(5) CFU/ml to 10(9) CFU/ml abolished AvBDs' antimicrobial activity. Increasing NaCl concentration significantly inhibited AvBDs' antimicrobial activity, but not the LPS-neutralizing function. Both AvBDs were mildly chemotactic for chicken macrophages and strongly chemotactic for CHO-K1 cells expressing chicken chemokine receptor 2 (CCR2). AvBD-12 at higher concentrations also induced chemotactic migration of murine immature dendritic cells (DCs). Disruption of disulfide bridges abolished AvBDs' chemotactic activity. Neither AvBDs was toxic to CHO-K1, macrophages, or DCs.AvBDs are potent antimicrobial peptides under low-salt conditions, effective LPS-neutralizing agents, and broad-spectrum chemoattractant peptides. Their antimicrobial activity is positively correlated with the peptides' net positive charges, inversely correlated with NaCl concentration and bacterial concentration, and minimally dependent on intramolecular disulfide bridges. In contrast, their chemotactic property requires the presence of intramolecular disulfide bridges. Data from the present study provide a theoretical basis for the design of AvBD-based therapeutic and immunomodulatory agents.

SUBMITTER: Yang M

PROVIDER: S-EPMC5016922 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Structure-function analysis of Avian β-defensin-6 and β-defensin-12: role of charge and disulfide bridges.

Yang Ming M Zhang Chunye C Zhang Xuehan X Zhang Michael Z MZ Rottinghaus George E GE Zhang Shuping S

BMC microbiology 20160909

<h4>Background</h4>Avian beta-defensins (AvBD) are small, cationic, antimicrobial peptides. The potential application of AvBDs as alternatives to antibiotics has been the subject of interest. However, the mechanisms of action remain to be fully understood. The present study characterized the structure-function relationship of AvBD-6 and AvBD-12, two peptides with different net positive charges, similar hydrophobicity and distinct tissue expression profiles.<h4>Results</h4>AvBD-6 was more potent ...[more]

PMID: 27613063

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Project description:BackgroundDefensins 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 beta-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico 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 in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis.ResultsMD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico 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 Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein.ConclusionThe 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.

Project description:BACKGROUND:?-defensins and cathelicidins are two families of cationic antimicrobial peptides (AMPs) with a broad range of antimicrobial activities that are key components of the innate immune system. Due to their important roles in host defense against rapidly evolving pathogens, the two gene families provide an ideal system for studying adaptive gene evolution. In this study we performed phylogenetic and selection analyses on ?-defensins and cathelicidins from 53 avian species representing 32 orders to examine the evolutionary dynamics of these peptides in birds. RESULTS AND CONCLUSIONS:Avian ?-defensins are found in a gene cluster consisting of 13 subfamiles. Nine of these are conserved as one to one orthologs in all birds, while the others (AvBD1, AvBD3, AvBD7 and AvBD14) are more subject to gene duplication or pseudogenisation events in specific avian lineages. Avian cathelicidins are found in a gene cluster consisting of three subfamilies with species-specific duplications and gene loss. Evidence suggested that the propiece and mature peptide domains of avian cathelicidins are possibly co-evolving in such a way that the cationicity of the mature peptide is partially neutralised by the negative charge of the propiece prior to peptide secretion (further evidence obtained by repeating the analyses on primate cathelicidins). Negative selection (overall mean dN < dS) was detected in most of the gene domains examined, conserving certain amino acid residues that may be functionally crucial for the avian ?-defensins and cathelicidins, while episodic positive selection was also involved in driving the diversification of specific codon sites of certain AMPs in avian evolutionary history. These findings have greatly improved our understanding of the molecular evolution of avian AMPs and will be useful to understand their role in the avian innate immune response. Additionally, the large dataset of ?-defensin and cathelicidin peptides may also provide a valuable resource for translational research and development of novel antimicrobial agents in the future.

Dataset Information

Structure-function analysis of Avian ?-defensin-6 and ?-defensin-12: role of charge and disulfide bridges.

Publications

Structure-function analysis of Avian β-defensin-6 and β-defensin-12: role of charge and disulfide bridges.

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