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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.


AvBD interaction with S. Typhimurium genomic DNA. a Gel retardation assay revealed the binding of wild-type and reduced AvBDs to S. Typhimurium genomic DNA. M: DNA molecular marker; lanes 1–6: the mass ratios of DNA to AvBD were 1:0, 1:0.5, 1:1, 1:2, 1:4 and 1:8, respectively; lane 7: DNA: bovine serum albumin (BSA) at ratio of 1:8. b Densitomeric analysis of migrated DNA by ImageJ software. Density ratio between treatment and control (lane 1) groups were calculated. Displayed values are means ± SD (n = 3). Asterisks indicate significant difference (*p < 0.05, **p < 0.01) between wild-type and reduced AvBDs
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Fig11: AvBD interaction with S. Typhimurium genomic DNA. a Gel retardation assay revealed the binding of wild-type and reduced AvBDs to S. Typhimurium genomic DNA. M: DNA molecular marker; lanes 1–6: the mass ratios of DNA to AvBD were 1:0, 1:0.5, 1:1, 1:2, 1:4 and 1:8, respectively; lane 7: DNA: bovine serum albumin (BSA) at ratio of 1:8. b Densitomeric analysis of migrated DNA by ImageJ software. Density ratio between treatment and control (lane 1) groups were calculated. Displayed values are means ± SD (n = 3). Asterisks indicate significant difference (*p < 0.05, **p < 0.01) between wild-type and reduced AvBDs

Mentions: The ability of AvBDs to bind to bacterial genomic DNA was analyzed by a gel retardation assay [37]. Wild-type AvBDs at a mass ratio of 4:1 (AvBD:DNA) retarded more than 50 % of S. Typhimurium genomic DNA migration (Fig. 11). At a mass ration of 8:1 (AvBD:DNA), near complete retardation of genomic DNA migration by either AvBD was observed. BSA, as a negative control, had no effect at mass ratio of 8:1 (BSA:DNA). At a mass ratio of 4:1 (AvBD:DNA), AvBD-6 was significantly more effective than AvBD-12 in retarding genomic DNA migration (p < 0.01). Reduced AvBDs were less able than their respective wild-type peptides to retard Salmonella genomic DNA migration.Fig. 11


Structure-function analysis of Avian β -defensin-6 and β -defensin-12: role of charge and disulfide bridges
AvBD interaction with S. Typhimurium genomic DNA. a Gel retardation assay revealed the binding of wild-type and reduced AvBDs to S. Typhimurium genomic DNA. M: DNA molecular marker; lanes 1–6: the mass ratios of DNA to AvBD were 1:0, 1:0.5, 1:1, 1:2, 1:4 and 1:8, respectively; lane 7: DNA: bovine serum albumin (BSA) at ratio of 1:8. b Densitomeric analysis of migrated DNA by ImageJ software. Density ratio between treatment and control (lane 1) groups were calculated. Displayed values are means ± SD (n = 3). Asterisks indicate significant difference (*p < 0.05, **p < 0.01) between wild-type and reduced AvBDs
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Related In: Results  -  Collection

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Fig11: AvBD interaction with S. Typhimurium genomic DNA. a Gel retardation assay revealed the binding of wild-type and reduced AvBDs to S. Typhimurium genomic DNA. M: DNA molecular marker; lanes 1–6: the mass ratios of DNA to AvBD were 1:0, 1:0.5, 1:1, 1:2, 1:4 and 1:8, respectively; lane 7: DNA: bovine serum albumin (BSA) at ratio of 1:8. b Densitomeric analysis of migrated DNA by ImageJ software. Density ratio between treatment and control (lane 1) groups were calculated. Displayed values are means ± SD (n = 3). Asterisks indicate significant difference (*p < 0.05, **p < 0.01) between wild-type and reduced AvBDs
Mentions: The ability of AvBDs to bind to bacterial genomic DNA was analyzed by a gel retardation assay [37]. Wild-type AvBDs at a mass ratio of 4:1 (AvBD:DNA) retarded more than 50 % of S. Typhimurium genomic DNA migration (Fig. 11). At a mass ration of 8:1 (AvBD:DNA), near complete retardation of genomic DNA migration by either AvBD was observed. BSA, as a negative control, had no effect at mass ratio of 8:1 (BSA:DNA). At a mass ratio of 4:1 (AvBD:DNA), AvBD-6 was significantly more effective than AvBD-12 in retarding genomic DNA migration (p < 0.01). Reduced AvBDs were less able than their respective wild-type peptides to retard Salmonella genomic DNA migration.Fig. 11

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.