Human Salivary Mucin MUC7 12-Mer-land 12-Mer-dPeptides: Antifungal Activity in Saliva, Enhancement of Activity with Protease Inhibitor Cocktail or EDTA, and Cytotoxicity to Human Cells

Abstract

MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 M concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 M peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.Candidiasis is the most common oral fungal infection diagnosed in humans. Due to the emergence of pathogens resistant to conventional antifungals and the toxicity of some antimycotics, intense efforts have been made to develop more effective antifungal agents for clinical use (1-3). Cationic antimicrobial peptides (CAMPs) have become attractive as novel candidates for this purpose. The native CAMPs contribute to innate host defense against a number of bacterial and fungal pathogens. They have been discovered from various natural sources, including humans, mammals, plants, insects, and bacteria (12, 13, 31). Among these, the well-characterized human salivary histatins and frog skin magainins show activity against a variety of microorganisms (18,31,32). MUC7, the low-molecular-mass human salivary mucin (comprised of 357 amino acid residues), exerts antimicrobial activity by binding to and clearing microorganisms from the oral cavity (25). While the full-length MUC7 has no appreciable candidacidal activity (11), we have shown that peptides derived from its N-terminal region have significant and broadspectrum fungicidal and...

A Novel Target-Specific, Salt-Resistant Antimicrobial Peptide against the Cariogenic Pathogen Streptococcus mutans

“…The major advantage of such an AMP is that the targeting moiety can guide the fusion peptide to bind selectively to the target organism, allowing peptide-guided killing (7,26). It is also desirable that such a fusion peptide should be relatively stable and retain its bactericidal activity under a range of physiological conditions, such as different salt concentrations, ionic strengths, pHs, and other factors in body fluids (8,15,34). By studying marine-derived pleurocidin variants (25,30) and an S. mutans quorum-sensing signaling peptide pheromone or CSP (17, 18, 31) we have The peptide bands were quantitated by densitometry to calculate percent degradation compared to the 0-min samples.…”

“…In addition, whole saliva contains a number of components, such as antimicrobial compounds, proteins, and glycoproteins from epithelial cells, host defense cells, and bacteria, which account for most of its proteolytic activity (29,34). The evidence from our study indicates that IMB-2 is capable of maintaining killing activity in physiological or even higher concentrations of salt, particularly for the short time (15 min) that is sufficient for the peptide to exhibit full killing activity.…”

Improvement of the Efficacy of Linear Undecapeptides against Plant-Pathogenic Bacteria by Incorporation of d -Amino Acids

“…In particular, incorporation of D-amino acids is an approach used to protect peptides against enzymatic hydrolysis, since only a few enzymes are known to digest amide bonds involving D-configuration (32). This strategy has been used to improve the biological activity profiles of synthetic antimicrobial peptides, not only increasing the resistance to proteolytic enzymes but also reducing the hemolytic activity while maintaining the antimicrobial activity (23,28,42,44,47,48,54).…”

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

“…The apparent lack of a single protein target is underscored by the fact that for at least some CAMPs, including those derived from the MUC7 protein, the all-D amino acid forms are as effective as the native all-L forms (31), precluding involvement of stereospecific interactions with cellular macromolecules in their modes of operation.…”