Dialysis Unmasks the Fungicidal Properties of Glandular Salivary Secretions

Isevi

2011

J. Basic Microbiol.

Two Enterococcus faecium and one E. faecalis strains isolated and identified from wheat silage were characterized based on plasmid content, hemolytic activity, antibiotic resistance patterns, bacteriocin production potential, and presence of enterocin structural genes (entA, entB, entP, entL50B). Among the isolates, only the E. faecium U7 strain exhibited bacteriocin activity against Listeria monocytogenes ATCC 7644, and vancomycin resistant Enterococcus spp. (VRE). A combination of three structural genes (entA, entB, and entP) was detected in E. faecium U7. A relationship between the presence of enterocin structural genes, and bacteriocin activity was detected in E. faecium U7; therefore partially purified enterocin (PPE) was further investigated from the isolate. Several bands of different molecular weights were expressed from PPE extracts following tricine SDS-PAGE analysis. However, the only band showing bacteriocin activity was in an approximate 4-kDa region. PPE treatment with proteinase K, lysozyme, and α -amylase caused complete loss of bacteriocin activity. PPE heat treatment at various temperatures resulted in a notable reduction in bacteriocin expression. Enterocin U7 was relatively heat stable, and presumably exhibits a glucoprotein nature with distinct inhibitory properties. Specific bacterial inhibitory activity of enterocin U7, and the producer strain absence of β -hemolysis and vancomycin susceptibility features deserves further investigation to evaluate its potential application in silage inoculation and food preservation.

Section: Discussionsupporting

confidence: 85%

Characterization of an anti‐listerial enterocin from wheat silage based Enterococcus faecium

Isevi

2011

J. Basic Microbiol.

Antimicrob Agents Chemother

“…Ionic strength has also been demonstrated to be one of the important factors that affect the efficiency of antimicrobial peptides in the medium (17) and in saliva (14). Both monoand divalent ions exert inhibitory effects on some antimicrobial peptides.…”

Section: Discussionmentioning

confidence: 99%

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

Wei

Bobek

2005

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...

“…Perplexingly, purified Hst 5 exhibits only 10 to 15% of its in vitro fungicidal activity when added into whole saliva (62,63). Two major processes are likely to account for these differences: (i) the dynamic turnover of salivary proteins balancing secretion with proteolytic degradation (64,65) and (ii) the phenomenon first described as "masking" (62) that is likely due to Hsts binding with salts and possibly with various metals, including Ca (66), Cu, and Zn in saliva.…”

Section: Hst 5 Metal Binding Abilities As a Potential Candidacidal Mementioning

confidence: 99%

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

2014

Histatins are salivary cationic peptides that provide the first line of defense against oral candidiasis caused by Candida albicans. This minireview presents a critical evaluation of our knowledge of the candidacidal mechanism of histatin 5 (Hst 5). Hst 5 is the most potent among all histatin family members with regard to its antifungal activity. The mode of action of Hst 5 has been a subject of intense debate. Unlike other classical host innate immune proteins, pore formation or membrane lysis by Hst 5 has largely been disproven, and it is now known that all targets of Hst 5 are intracellular. Hst 5 binds C. albicans cell wall proteins (Ssa1/2) and glycans and is taken up by the cells through fungal polyamine transporters in an energy-dependent manner. Once inside the fungal cells, Hst 5 may affect mitochondrial functions and cause oxidative stress; however, the ultimate cause of cell death is by volume dysregulation and ion imbalance triggered by osmotic stress. Besides these diverse targets, a novel mechanism based on the metal binding abilities of Hst 5 is discussed. Finally, translational approaches for Hst 5, based on peptide design and synergy with other known drugs, are considered a step forward for bench-to-bed application of Hst 5.