Structure-function analysis of Avian β-defensin-6 and β-defensin-12: role of charge and disulfide bridges.
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.
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA.
Ming
M
Yang
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA.
Chunye
C
Zhang
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, People's Republic of China.
Xuehan
X
Zhang
Department of Biomedical Science, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.|Department of Veterinary Pathobiology, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.
Michael Z
MZ
Zhang
Department of Biomedical Science, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.|Department of Veterinary Pathobiology, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.
George E
GE
Rottinghaus
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA. zhangshup@missouri.edu.|Department of Veterinary Pathobiology, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA. zhangshup@missouri.edu.
Shuping
S
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000890:Anti-Infective Agents / Q000494:pharmacology; D058491:Bacterial Load; D001426:Bacterial Proteins; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival; D002633:Chemotaxis / Q000187:drug effects; D002645:Chickens; D006224:Cricetinae; D004269:DNA, Bacterial; D003713:Dendritic Cells / Q000276:immunology; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000187:drug effects; D016680:Genome, Bacterial; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D008826:Microbial Sensitivity Tests; D046529:Microscopy, Electron, Transmission; D009500:Neutralization Tests; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology; D019707:Receptors, Chemokine; D012486:Salmonella typhimurium / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D012965:Sodium Chloride / Q000737:chemistry; D013211:Staphylococcus aureus / Q000187:drug effects; D023083:beta-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology*; D001618:beta-Lactamases / Q000378:metabolism
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004220:Disulfides; D008070:Lipopolysaccharides; D010455:Peptides; D019707:Receptors, Chemokine; D023083:beta-Defensins; D012965:Sodium Chloride; D001618:beta-Lactamases
Antimicrobial activity; Avian β-defensins; Chemotactic activity; Disulfide bridges; LPS-neutralizing activity; Net positive charge