• Human vaginal fluid
• Synergistic effect
• Human hepcidin 20
• Candida glabrata
• Antimicrobial peptides
• Antifungal activity
• Vaginal candidiasis

Vaginal infections due to Candida glabrata are difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which are considered novel therapeutic candidates for the treatment of fungal infections. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility.Interestingly, Hep-20 exerted higher fungicidal effect alone and in combination with common antifungal agents at acidic pH values. The enhancement of Hep-20 activity observed under acid conditions and the capacity of C. glabrata to cause vaginal infections suggested that this peptide could represent a promising candidate for the treatment of these infections. To verify this hypothesis, an artificial vaginal fluid and a human vaginal fluid were used to evaluate whether the activity of the peptide alone or in combination with common antifungals could be maintained in biological fluids. The activity of the peptide was maintained in the artificial and human vaginal fluid supplemented with EDTA. Despite the fact that Hep-20 is a human derived peptide,therapeutic concentrations are usually higher than physiological ones. For this reason,the final part of the study was aimed at evaluating the potential cytotoxic effect exerted by the peptide on human erythrocytes, peripheral blood mononuclear cells and on a human derived epithelial cell line. Cytotoxicity studies indicated that the peptide does not produce any cytotoxic effect at Hep-20 concentrations that exhibited fungicidal activity against C. glabrata in human vaginal fluid. An indirect evidence of Hep-20 stability was obtained from co-incubation experiments of the peptide with human vaginal fluid at 37°C for 90 minutes and 24 hours. SDS-PAGE gels indicated that the peptide is not naturally present in the human vaginal fluid, and when it was coincubated with vaginal fluid alone, Hep-20 was not degraded. However, in the presence
of EDTA, Hep-20 was partially degraded, and this finding was even more evident after 24 hours of co-incubation. Further experiments will be required to understand the exact mechanism leading to this partial peptide degradation following 90 minutes and 24 hours of incubation. Nevertheless, killing experiments performed in human vaginal fluid clearly demonstrated that, even if partially degraded, the peptide is still able to exert fungicidal activity against C. glabrata at both 90 minutes and within 24 hours. Collectively, these results indicate that this peptide could be a potential candidate for the development of alternative strategies in the topical treatment of vaginal C. glabrata infections due to fluconazole resistant strains.