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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.


Antimicrobial activity of AvBD-6 and AvBD-12 against E. coli, S. Typhimurium and S. aureus. Bacteria (105 CFU/ml) were incubated with various concentrations of AvBD-6 or AvBD-12 in the presence of 5 mM NaCl (■), 50 mM NaCl (▲), or 150 mM () at 37 °C for 3 h. a Antimicrobial activities of AvBD-6. b Antimicrobial activities of AvBD-12. Antimicrobial activity was presented as percent of killing as compared to no-AvBD control. Data are means of three independent experiments ± SD (n = 3). Asterisks indicate statistically significant difference between 5 mM and 50 mM and 150 mM of NaCl (*p < 0.05, **p < 0.01)
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Fig1: Antimicrobial activity of AvBD-6 and AvBD-12 against E. coli, S. Typhimurium and S. aureus. Bacteria (105 CFU/ml) were incubated with various concentrations of AvBD-6 or AvBD-12 in the presence of 5 mM NaCl (■), 50 mM NaCl (▲), or 150 mM () at 37 °C for 3 h. a Antimicrobial activities of AvBD-6. b Antimicrobial activities of AvBD-12. Antimicrobial activity was presented as percent of killing as compared to no-AvBD control. Data are means of three independent experiments ± SD (n = 3). Asterisks indicate statistically significant difference between 5 mM and 50 mM and 150 mM of NaCl (*p < 0.05, **p < 0.01)

Mentions: Both AvBDs showed dose-dependent (1 to 128 μg/ml) bactericidal activities against three common bacterial pathogens, E. coli, S. Typhimurium, and S. aureus (Fig. 1). To detect antimicrobial activity of AvBDs over a wide range of concentrations, percentage killing of bacteria was evaluated. AvBD-6 was generally more potent than AvBD-12 in killing E. coli (2 to 64 μg/ml), S. Typhimurium and S. aureus (8 to 128 μg/ml). The susceptibility of three bacterial pathogens to AvBDs (AvBD-6 at lower concentrations and AvBD-12 at higher concentrations) could be classified as: E. coli > S. Typhimurium > S. aureus. The killing activities of AvBDs were impaired by increasing NaCl concentration from 5 mM to 50 mM or 150 mM (Fig. 1). The negative impact of increased NaCl concentration could be summarized as follows: AvBD-6/AvBD-12/E. coli > AvBD-6/S. Typhimurium > AvBD-6/S. aureus > AvBD-12/S. Typhimuirum > AvBD-12/S. aureus.Fig. 1


Structure-function analysis of Avian β -defensin-6 and β -defensin-12: role of charge and disulfide bridges
Antimicrobial activity of AvBD-6 and AvBD-12 against E. coli, S. Typhimurium and S. aureus. Bacteria (105 CFU/ml) were incubated with various concentrations of AvBD-6 or AvBD-12 in the presence of 5 mM NaCl (■), 50 mM NaCl (▲), or 150 mM () at 37 °C for 3 h. a Antimicrobial activities of AvBD-6. b Antimicrobial activities of AvBD-12. Antimicrobial activity was presented as percent of killing as compared to no-AvBD control. Data are means of three independent experiments ± SD (n = 3). Asterisks indicate statistically significant difference between 5 mM and 50 mM and 150 mM of NaCl (*p < 0.05, **p < 0.01)
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Fig1: Antimicrobial activity of AvBD-6 and AvBD-12 against E. coli, S. Typhimurium and S. aureus. Bacteria (105 CFU/ml) were incubated with various concentrations of AvBD-6 or AvBD-12 in the presence of 5 mM NaCl (■), 50 mM NaCl (▲), or 150 mM () at 37 °C for 3 h. a Antimicrobial activities of AvBD-6. b Antimicrobial activities of AvBD-12. Antimicrobial activity was presented as percent of killing as compared to no-AvBD control. Data are means of three independent experiments ± SD (n = 3). Asterisks indicate statistically significant difference between 5 mM and 50 mM and 150 mM of NaCl (*p < 0.05, **p < 0.01)
Mentions: Both AvBDs showed dose-dependent (1 to 128 μg/ml) bactericidal activities against three common bacterial pathogens, E. coli, S. Typhimurium, and S. aureus (Fig. 1). To detect antimicrobial activity of AvBDs over a wide range of concentrations, percentage killing of bacteria was evaluated. AvBD-6 was generally more potent than AvBD-12 in killing E. coli (2 to 64 μg/ml), S. Typhimurium and S. aureus (8 to 128 μg/ml). The susceptibility of three bacterial pathogens to AvBDs (AvBD-6 at lower concentrations and AvBD-12 at higher concentrations) could be classified as: E. coli > S. Typhimurium > S. aureus. The killing activities of AvBDs were impaired by increasing NaCl concentration from 5 mM to 50 mM or 150 mM (Fig. 1). The negative impact of increased NaCl concentration could be summarized as follows: AvBD-6/AvBD-12/E. coli > AvBD-6/S. Typhimurium > AvBD-6/S. aureus > AvBD-12/S. Typhimuirum > AvBD-12/S. aureus.Fig. 1

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.