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

Abstract

BackgroundMUC7 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.MethodsMinimal 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 μM. 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.ResultsThe MICs of MUC7 12-mer against organisms tested ranged from 6.25–50 μM. 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 μM) 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°C did not affect the activity.ConclusionMUC7 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.

pH Dependence of Microbe Sterilization by Cationic Antimicrobial Peptides

“…The influence of pH on CAMP activity has also been examined for a small number of peptides (24,26,27,32). Impacts on CAMP activity due to a change in pH can occur directly, by affecting the charge-charge interactions involved in the initial binding step, or indirectly via a change in the ionic strength of the solution.…”

“…Due to histidine's pK a value near 6, the clavanins become much more cationic and more effective as the pH is decreased to below the pK a . More recently, several studies have been conducted over a wider range of salt concentrations and between pH 5 to 8 (27) or 5 to 9 (26,40). Such studies are often performed without an initial incubation step; the pH is simply adjusted in growth media without added buffers.…”

“…In the case of cystic fibrosis, lung infection by Pseudomonas aeruginosa has been linked to inactivity of CAMPs at the higher salt concentrations found in the lungs of cystic fibrosis patients (29). Other studies have observed that CAMPs work as well or better at lower pH than at neutral pH (26,31), particularly His-rich peptides such as the clavanins, which contain a number of His residues instead of the more typical Arg or Lys residues (32). In this work, we investigated the effect of a wide range of pH and ionic strength values, both separately and in combination, to gain a more comprehensive understanding of their effect on CAMP activity.…”

“…A number of studies have looked at the effects of pH or salt concentration on the ability of CAMPs to kill microorganisms (24,25). In most cases, only a narrow range of different pHs or salt concentrations were sampled, although a few studies have been more comprehensive (26,27). It is well known that higher salt concentrations often interfere with CAMP activity (25,28,29), but some CAMPs from marine organisms have been found to tolerate salt concentrations up to 450 mM (30).…”

Exploring the Mode of Action of Antimicrobial Peptide MUC7 12-Mer by Fitness Profiling of Saccharomyces cerevisiae Genomewide Mutant Collection

“…Haploid MATa deletion strains and parental strain S288C (Open Biosystems) were kindly provided by P. Cullen, University at Buffalo. Yeasts were grown at 30°C in full-strength Sabouraud dextrose broth (SDB), or in twofold-diluted SDB if used for the treatment with the MUC7 12-mer, since the peptide is ineffective against microorganisms in full-strength medium (32). The peptide (RKSYKCLHKRCR) was synthesized by Bio-Synthesis (Lewisville, TX).…”