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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.


Cytotoxicity of AvBD-6 and AvBD-12 on host cells. Effect of 256 μg/ml AvBD-6 and AvBD-12 on the viability of chicken macrophages MQ-NCSU and HD11 cells, mouse immature dendritic JAWSII, and hamster CHO-K1 cells at 4, 12, 24, 48 h of incubation. Results shown are percentages of viable cells in different treatment groups relative to the untreated control cells. The data are expressed as the mean ± SD (n = 3)
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Fig4: Cytotoxicity of AvBD-6 and AvBD-12 on host cells. Effect of 256 μg/ml AvBD-6 and AvBD-12 on the viability of chicken macrophages MQ-NCSU and HD11 cells, mouse immature dendritic JAWSII, and hamster CHO-K1 cells at 4, 12, 24, 48 h of incubation. Results shown are percentages of viable cells in different treatment groups relative to the untreated control cells. The data are expressed as the mean ± SD (n = 3)

Mentions: The cellular toxicity of AvBD-6 and AvBD-12 to chicken macrophage cell line HD11 and MQ-NCSU, mouse immature dendritic JAWSII cells, and hamster CHO-K1 cells were evaluated using a MTT cell proliferation assay (Thermo Fisher Scientific). Exposure of cells to AvBDs at concentrations of 4, 16, 64, 256 μg/ml for 4, 12, 24, and 48 h did not cause any change in cell variability. Data on the highest concentration (256 μg/ml) and longest exposure (48 h) were presented in Fig. 4.Fig. 4


Structure-function analysis of Avian β -defensin-6 and β -defensin-12: role of charge and disulfide bridges
Cytotoxicity of AvBD-6 and AvBD-12 on host cells. Effect of 256 μg/ml AvBD-6 and AvBD-12 on the viability of chicken macrophages MQ-NCSU and HD11 cells, mouse immature dendritic JAWSII, and hamster CHO-K1 cells at 4, 12, 24, 48 h of incubation. Results shown are percentages of viable cells in different treatment groups relative to the untreated control cells. The data are expressed as the mean ± SD (n = 3)
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Related In: Results  -  Collection

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Fig4: Cytotoxicity of AvBD-6 and AvBD-12 on host cells. Effect of 256 μg/ml AvBD-6 and AvBD-12 on the viability of chicken macrophages MQ-NCSU and HD11 cells, mouse immature dendritic JAWSII, and hamster CHO-K1 cells at 4, 12, 24, 48 h of incubation. Results shown are percentages of viable cells in different treatment groups relative to the untreated control cells. The data are expressed as the mean ± SD (n = 3)
Mentions: The cellular toxicity of AvBD-6 and AvBD-12 to chicken macrophage cell line HD11 and MQ-NCSU, mouse immature dendritic JAWSII cells, and hamster CHO-K1 cells were evaluated using a MTT cell proliferation assay (Thermo Fisher Scientific). Exposure of cells to AvBDs at concentrations of 4, 16, 64, 256 μg/ml for 4, 12, 24, and 48 h did not cause any change in cell variability. Data on the highest concentration (256 μg/ml) and longest exposure (48 h) were presented in Fig. 4.Fig. 4

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.