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Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide.

Wei GX, Campagna AN, Bobek LA - Ann. Clin. Microbiol. Antimicrob. (2007)

Bottom Line: To examine the effect of salts or EDTA, a checkerboard microdilution technique was used.The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity.MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, School of Dental Medicine, University at Buffalo, SUNY, 3435 Main Street, Buffalo, USA, NY 14214. gwei@buffalo.edu.

ABSTRACT

Background: MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity.

Methods: Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56-50 microM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry.

Results: The MICs of MUC7 12-mer against organisms tested ranged from 6.25-50 microM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1-2 mM). No antagonism but additivity or indifference (FICi 0.55-2.5) was observed for the combination of MUC7 12-mer and each K+, Na+, Mg2+, or Zn2+. MUC7 12-mer peptide (at 25 microM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity.

Conclusion: MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity. These results suggest that, especially in combination with EDTA, it could potentially be applied as an alternative therapeutic agent for the treatment of human oral candidiasis.

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Effect of sodium chloride on candidacidal activity of MUC7 12-mer and Hsn5 12-mer, determined by killing assay. MUC7 12-mer (blue column) or Hsn5 12-mer (red column) (at a final concentration of 25 μM) were incubated with C. albicans (105 cells/ml) in 10 mM Na-PB containing NaCl (6.25–150 mM). The killing activities were determined by viable counting on SDA, comparing the number of colonies at each salt concentration to those of the agent-free control. Data represents two independent experiments, each performed in duplicate.
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Figure 1: Effect of sodium chloride on candidacidal activity of MUC7 12-mer and Hsn5 12-mer, determined by killing assay. MUC7 12-mer (blue column) or Hsn5 12-mer (red column) (at a final concentration of 25 μM) were incubated with C. albicans (105 cells/ml) in 10 mM Na-PB containing NaCl (6.25–150 mM). The killing activities were determined by viable counting on SDA, comparing the number of colonies at each salt concentration to those of the agent-free control. Data represents two independent experiments, each performed in duplicate.

Mentions: The effect of peptides on cell viability of C. albicans in the presence of NaCl is shown in Figure 1. MUC7 12-mer exhibits 100% killing activity in the presence of NaCl up to 25 mM, a physiological concentration of Na+ in human saliva. Some decrease in the activity of the MUC7 12-mer was observed in the presence of 50 mM NaCl or higher. However, MUC7 12-mer still exhibited more resistance to NaCl than Hsn5 12-mer. In the presence of 50 mM NaCl, MUC7 retained 90% killing activity, while Hsn5 12-mer 40%. Additionally, MUC7 12-mer peptide was effective against E. coli in the presence of NaCl up to 150 mM (data not shown), a physiological concentration of Na+ in plasma.


Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide.

Wei GX, Campagna AN, Bobek LA - Ann. Clin. Microbiol. Antimicrob. (2007)

Effect of sodium chloride on candidacidal activity of MUC7 12-mer and Hsn5 12-mer, determined by killing assay. MUC7 12-mer (blue column) or Hsn5 12-mer (red column) (at a final concentration of 25 μM) were incubated with C. albicans (105 cells/ml) in 10 mM Na-PB containing NaCl (6.25–150 mM). The killing activities were determined by viable counting on SDA, comparing the number of colonies at each salt concentration to those of the agent-free control. Data represents two independent experiments, each performed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211505&req=5

Figure 1: Effect of sodium chloride on candidacidal activity of MUC7 12-mer and Hsn5 12-mer, determined by killing assay. MUC7 12-mer (blue column) or Hsn5 12-mer (red column) (at a final concentration of 25 μM) were incubated with C. albicans (105 cells/ml) in 10 mM Na-PB containing NaCl (6.25–150 mM). The killing activities were determined by viable counting on SDA, comparing the number of colonies at each salt concentration to those of the agent-free control. Data represents two independent experiments, each performed in duplicate.
Mentions: The effect of peptides on cell viability of C. albicans in the presence of NaCl is shown in Figure 1. MUC7 12-mer exhibits 100% killing activity in the presence of NaCl up to 25 mM, a physiological concentration of Na+ in human saliva. Some decrease in the activity of the MUC7 12-mer was observed in the presence of 50 mM NaCl or higher. However, MUC7 12-mer still exhibited more resistance to NaCl than Hsn5 12-mer. In the presence of 50 mM NaCl, MUC7 retained 90% killing activity, while Hsn5 12-mer 40%. Additionally, MUC7 12-mer peptide was effective against E. coli in the presence of NaCl up to 150 mM (data not shown), a physiological concentration of Na+ in plasma.

Bottom Line: To examine the effect of salts or EDTA, a checkerboard microdilution technique was used.The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity.MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, School of Dental Medicine, University at Buffalo, SUNY, 3435 Main Street, Buffalo, USA, NY 14214. gwei@buffalo.edu.

ABSTRACT

Background: MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity.

Methods: Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56-50 microM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry.

Results: The MICs of MUC7 12-mer against organisms tested ranged from 6.25-50 microM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1-2 mM). No antagonism but additivity or indifference (FICi 0.55-2.5) was observed for the combination of MUC7 12-mer and each K+, Na+, Mg2+, or Zn2+. MUC7 12-mer peptide (at 25 microM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity.

Conclusion: MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity. These results suggest that, especially in combination with EDTA, it could potentially be applied as an alternative therapeutic agent for the treatment of human oral candidiasis.

Show MeSH
Related in: MedlinePlus