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Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

Kiselar JG, Wang X, Dubyak GR, El Sanadi C, Ghosh SK, Lundberg K, Williams WM - PLoS ONE (2015)

Bottom Line: The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions.We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus

MGO-adducted rhBD-2 shows a concentration-dependent reduction of bactericidal activity, shown as a reduction in CFU vs unadducted peptide.CFU within a defined area were counted following radial diffusion assays performed with gram-negative, facultative anaerobes Escherichia coli (e.c.) and Pseudomonas aeruginosa (p.a.), and with the gram-positive, facultative anaerobe Staphylococcus aureus (s.a.) exposed to 0.5 μg/5 μl with or without MGO. Wild-type hBD-2 is highly bactericidal against most gram-negative bacterial strains, including E. coli, but this function is dramatically reduced following 72 h incubation of rhBD-2 (0.5 μg/5 μl) with 100 μM MGO at 37°C (inset). MGO (100 μM) also reduces rhBD-2 bactericidal function against the gram-positive S. aureus strain. Data is presented as the Mean ± S.D. for N = 5 (e.c.) or N = 6 experiments (p.a., s.a.). Graph line for e.c. is offset slightly for clarity. (♦, ◊: p = 0.01; *: p = 0.05).
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pone.0130533.g004: MGO-adducted rhBD-2 shows a concentration-dependent reduction of bactericidal activity, shown as a reduction in CFU vs unadducted peptide.CFU within a defined area were counted following radial diffusion assays performed with gram-negative, facultative anaerobes Escherichia coli (e.c.) and Pseudomonas aeruginosa (p.a.), and with the gram-positive, facultative anaerobe Staphylococcus aureus (s.a.) exposed to 0.5 μg/5 μl with or without MGO. Wild-type hBD-2 is highly bactericidal against most gram-negative bacterial strains, including E. coli, but this function is dramatically reduced following 72 h incubation of rhBD-2 (0.5 μg/5 μl) with 100 μM MGO at 37°C (inset). MGO (100 μM) also reduces rhBD-2 bactericidal function against the gram-positive S. aureus strain. Data is presented as the Mean ± S.D. for N = 5 (e.c.) or N = 6 experiments (p.a., s.a.). Graph line for e.c. is offset slightly for clarity. (♦, ◊: p = 0.01; *: p = 0.05).

Mentions: We show by radial diffusion assay [34] that bactericidal activity of rhBD-2 against gram-negative P. aeruginosa and E. coli is significantly reduced when either strain is grown in the presence of rhBD-2 exposed to the highest concentration of MGO (100 μM), and 25 μM MGO, the lowest shown (Fig 4). In several experiments we exposed the peptide to 5 uM MGO and still observed loss of bactericidal activity (data not shown). Bacteriostatic activity of rhBD-2 against gram-positive S. aureus was also adversely affected, but reduced activity reached statistical significance only when this strain was grown in the presence of rhBD-2 incubated with 100 μM MGO. Bacterial viability and growth rate were unaffected by the presence in the growth media of 100 μM MGO alone (Fig 4, inset).


Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

Kiselar JG, Wang X, Dubyak GR, El Sanadi C, Ghosh SK, Lundberg K, Williams WM - PLoS ONE (2015)

MGO-adducted rhBD-2 shows a concentration-dependent reduction of bactericidal activity, shown as a reduction in CFU vs unadducted peptide.CFU within a defined area were counted following radial diffusion assays performed with gram-negative, facultative anaerobes Escherichia coli (e.c.) and Pseudomonas aeruginosa (p.a.), and with the gram-positive, facultative anaerobe Staphylococcus aureus (s.a.) exposed to 0.5 μg/5 μl with or without MGO. Wild-type hBD-2 is highly bactericidal against most gram-negative bacterial strains, including E. coli, but this function is dramatically reduced following 72 h incubation of rhBD-2 (0.5 μg/5 μl) with 100 μM MGO at 37°C (inset). MGO (100 μM) also reduces rhBD-2 bactericidal function against the gram-positive S. aureus strain. Data is presented as the Mean ± S.D. for N = 5 (e.c.) or N = 6 experiments (p.a., s.a.). Graph line for e.c. is offset slightly for clarity. (♦, ◊: p = 0.01; *: p = 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526640&req=5

pone.0130533.g004: MGO-adducted rhBD-2 shows a concentration-dependent reduction of bactericidal activity, shown as a reduction in CFU vs unadducted peptide.CFU within a defined area were counted following radial diffusion assays performed with gram-negative, facultative anaerobes Escherichia coli (e.c.) and Pseudomonas aeruginosa (p.a.), and with the gram-positive, facultative anaerobe Staphylococcus aureus (s.a.) exposed to 0.5 μg/5 μl with or without MGO. Wild-type hBD-2 is highly bactericidal against most gram-negative bacterial strains, including E. coli, but this function is dramatically reduced following 72 h incubation of rhBD-2 (0.5 μg/5 μl) with 100 μM MGO at 37°C (inset). MGO (100 μM) also reduces rhBD-2 bactericidal function against the gram-positive S. aureus strain. Data is presented as the Mean ± S.D. for N = 5 (e.c.) or N = 6 experiments (p.a., s.a.). Graph line for e.c. is offset slightly for clarity. (♦, ◊: p = 0.01; *: p = 0.05).
Mentions: We show by radial diffusion assay [34] that bactericidal activity of rhBD-2 against gram-negative P. aeruginosa and E. coli is significantly reduced when either strain is grown in the presence of rhBD-2 exposed to the highest concentration of MGO (100 μM), and 25 μM MGO, the lowest shown (Fig 4). In several experiments we exposed the peptide to 5 uM MGO and still observed loss of bactericidal activity (data not shown). Bacteriostatic activity of rhBD-2 against gram-positive S. aureus was also adversely affected, but reduced activity reached statistical significance only when this strain was grown in the presence of rhBD-2 incubated with 100 μM MGO. Bacterial viability and growth rate were unaffected by the presence in the growth media of 100 μM MGO alone (Fig 4, inset).

Bottom Line: The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions.We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus