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

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 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 reduced AvBD-6 and AvBD-12. Comparison of LPS-neutralizing activity of reduced (■) and wild-type (▲) AvBDs. a Reduced AvBD-6 neutralizing for E. coli O111:B4 LPS, b Reduced AvBD-6 neutralizing S. Typhimurium L6143 LPS, c Reduced AvBD-12 neutralizing for E. coli O111:B4 LPS, d Reduced AvBD-12 neutralizing S. Typhimurium L6143 LPS. The assay was repeated three times and data are presented as means ± SD (n = 3). Asterisks indicate significant difference of neutralizing LPS activity between wild-type and reduced AvBD-12 (**p < 0.01)
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Fig9: LPS-neutralizing activity of reduced AvBD-6 and AvBD-12. Comparison of LPS-neutralizing activity of reduced (■) and wild-type (▲) AvBDs. a Reduced AvBD-6 neutralizing for E. coli O111:B4 LPS, b Reduced AvBD-6 neutralizing S. Typhimurium L6143 LPS, c Reduced AvBD-12 neutralizing for E. coli O111:B4 LPS, d Reduced AvBD-12 neutralizing S. Typhimurium L6143 LPS. The assay was repeated three times and data are presented as means ± SD (n = 3). Asterisks indicate significant difference of neutralizing LPS activity between wild-type and reduced AvBD-12 (**p < 0.01)

Mentions: The reduced AvBD-6 and AvBD-12 showed antimicrobial activities similar to that of the wild-type AvBD-6 and AvBD-12, respectively (Fig. 8a). In contrast, reduced AvBDs lost their chemotactic effect on CCR2-CHO cells (p < 0.01, Fig. 8b). Reduction had significant negative impact on the LPS-neutralizing activity of AvBD-12 (Fig. 9c and d). For example, wild type AvBD-12 at 32 μg/ml neutralized 74.48 % of E. coli LPS (1EU/ml) whereas reduced AvBD-12 neutralized 28.78 % E. coli LPS at the same peptide and LPS concentrations. Similar pattern was observed with S. Typhimurium LPS.Fig. 8


Structure-function analysis of Avian β -defensin-6 and β -defensin-12: role of charge and disulfide bridges
LPS-neutralizing activity of reduced AvBD-6 and AvBD-12. Comparison of LPS-neutralizing activity of reduced (■) and wild-type (▲) AvBDs. a Reduced AvBD-6 neutralizing for E. coli O111:B4 LPS, b Reduced AvBD-6 neutralizing S. Typhimurium L6143 LPS, c Reduced AvBD-12 neutralizing for E. coli O111:B4 LPS, d Reduced AvBD-12 neutralizing S. Typhimurium L6143 LPS. The assay was repeated three times and data are presented as means ± SD (n = 3). Asterisks indicate significant difference of neutralizing LPS activity between wild-type and reduced AvBD-12 (**p < 0.01)
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Related In: Results  -  Collection

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Fig9: LPS-neutralizing activity of reduced AvBD-6 and AvBD-12. Comparison of LPS-neutralizing activity of reduced (■) and wild-type (▲) AvBDs. a Reduced AvBD-6 neutralizing for E. coli O111:B4 LPS, b Reduced AvBD-6 neutralizing S. Typhimurium L6143 LPS, c Reduced AvBD-12 neutralizing for E. coli O111:B4 LPS, d Reduced AvBD-12 neutralizing S. Typhimurium L6143 LPS. The assay was repeated three times and data are presented as means ± SD (n = 3). Asterisks indicate significant difference of neutralizing LPS activity between wild-type and reduced AvBD-12 (**p < 0.01)
Mentions: The reduced AvBD-6 and AvBD-12 showed antimicrobial activities similar to that of the wild-type AvBD-6 and AvBD-12, respectively (Fig. 8a). In contrast, reduced AvBDs lost their chemotactic effect on CCR2-CHO cells (p < 0.01, Fig. 8b). Reduction had significant negative impact on the LPS-neutralizing activity of AvBD-12 (Fig. 9c and d). For example, wild type AvBD-12 at 32 μg/ml neutralized 74.48 % of E. coli LPS (1EU/ml) whereas reduced AvBD-12 neutralized 28.78 % E. coli LPS at the same peptide and LPS concentrations. Similar pattern was observed with S. Typhimurium LPS.Fig. 8

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.