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

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ABSTRACT

Background: 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.

Results: 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 105 CFU/ml to 109 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.

Conclusions: 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.

No MeSH data available.


LPS-neutralizing activity of AvBD-6 and AvBD-12. LPS-neutralizing activities of AvBD-6 and AvBD-12 were determined by the Limulus Amoebocyte Lysate (LAL) assay. a Neutralizing activities AvBD-6 (■) and AvBD-12 (▲) for E. coli O111:B4 LPS and S. Typhimurium L6143 LPS. b The effect of NaCl concentration on the ability of AvBDs to neutralize E. coli O111:B4 LPS (■) and S. Typhimurium L6143 LPS (▲). The data are presented as means ± SD (n = 3). Asterisks denote statistically significant difference in LPS-neutralizing activities between AvBD-6 and AvBD-12 (*p < 0.05, **p < 0.01)
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Fig3: LPS-neutralizing activity of AvBD-6 and AvBD-12. LPS-neutralizing activities of AvBD-6 and AvBD-12 were determined by the Limulus Amoebocyte Lysate (LAL) assay. a Neutralizing activities AvBD-6 (■) and AvBD-12 (▲) for E. coli O111:B4 LPS and S. Typhimurium L6143 LPS. b The effect of NaCl concentration on the ability of AvBDs to neutralize E. coli O111:B4 LPS (■) and S. Typhimurium L6143 LPS (▲). The data are presented as means ± SD (n = 3). Asterisks denote statistically significant difference in LPS-neutralizing activities between AvBD-6 and AvBD-12 (*p < 0.05, **p < 0.01)

Mentions: Both AvBD-6 and AvBD-12 neutralized LPS activity in a dose-dependent manner (Fig. 3a). At the concentration of 32 μg/ml, AvBDs were able to neutralize more than 70 % of equal volume of 1 EU/ml of LPS. The neutralizing capacity of AvBD-6 was significantly stronger (p < 0.05 or 0.01) than AvBD-12. While AvBD-6 (16 μg/ml) was more effective in neutralizing E. coli LPS than Salmonella LPS, AvBD-12 showed no difference in neutralizing E. coli LPS and Salmonella LPS. Interestingly, NaCl concentrations, ranging from 0.1 % (17.1 mM) to 0.8 % (137 mM), had no impact on AvBDs’ ability to neutralize LPS (Fig. 3b).Fig. 3


Structure-function analysis of Avian β -defensin-6 and β -defensin-12: role of charge and disulfide bridges
LPS-neutralizing activity of AvBD-6 and AvBD-12. LPS-neutralizing activities of AvBD-6 and AvBD-12 were determined by the Limulus Amoebocyte Lysate (LAL) assay. a Neutralizing activities AvBD-6 (■) and AvBD-12 (▲) for E. coli O111:B4 LPS and S. Typhimurium L6143 LPS. b The effect of NaCl concentration on the ability of AvBDs to neutralize E. coli O111:B4 LPS (■) and S. Typhimurium L6143 LPS (▲). The data are presented as means ± SD (n = 3). Asterisks denote statistically significant difference in LPS-neutralizing activities between AvBD-6 and AvBD-12 (*p < 0.05, **p < 0.01)
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Related In: Results  -  Collection

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Fig3: LPS-neutralizing activity of AvBD-6 and AvBD-12. LPS-neutralizing activities of AvBD-6 and AvBD-12 were determined by the Limulus Amoebocyte Lysate (LAL) assay. a Neutralizing activities AvBD-6 (■) and AvBD-12 (▲) for E. coli O111:B4 LPS and S. Typhimurium L6143 LPS. b The effect of NaCl concentration on the ability of AvBDs to neutralize E. coli O111:B4 LPS (■) and S. Typhimurium L6143 LPS (▲). The data are presented as means ± SD (n = 3). Asterisks denote statistically significant difference in LPS-neutralizing activities between AvBD-6 and AvBD-12 (*p < 0.05, **p < 0.01)
Mentions: Both AvBD-6 and AvBD-12 neutralized LPS activity in a dose-dependent manner (Fig. 3a). At the concentration of 32 μg/ml, AvBDs were able to neutralize more than 70 % of equal volume of 1 EU/ml of LPS. The neutralizing capacity of AvBD-6 was significantly stronger (p < 0.05 or 0.01) than AvBD-12. While AvBD-6 (16 μg/ml) was more effective in neutralizing E. coli LPS than Salmonella LPS, AvBD-12 showed no difference in neutralizing E. coli LPS and Salmonella LPS. Interestingly, NaCl concentrations, ranging from 0.1 % (17.1 mM) to 0.8 % (137 mM), had no impact on AvBDs’ ability to neutralize LPS (Fig. 3b).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Results: 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 105&nbsp;CFU/ml to 109&nbsp;CFU/ml abolished AvBDs&rsquo; antimicrobial activity. Increasing NaCl concentration significantly inhibited AvBDs&rsquo; 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&rsquo; chemotactic activity. Neither AvBDs was toxic to CHO-K1, macrophages, or DCs.

Conclusions: 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&rsquo; 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.

No MeSH data available.