Two antimicrobial P-113 peptide derivatives, P-113Du and P-113Tri, were investigated in this study. Notably, P-113Du and P-113Tri contained significant fractions of ␣-helix conformation and were less sensitive to high salt and low pH than P-113. Moreover, compared to P-113, these peptides exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of Candida albicans and non-albicans Candida spp. These results suggest that P-113Du and P-113Tri are promising candidates for development as novel antifungal agents. Histatin 5 (Hst5) is a potent antimicrobial peptide (AMP) with activity against Candida albicans (1). A 12-mer amino acid fragment of Hst5, P-113, retains strong candidacidal activity compared to the parental Hst5 and has had no adverse effects in clinical trials (2-6). P-113 forms an amphipathic ␣-helix in trifluoroethanol, which mimics the hydrophobic environment of microbial membranes (7). However, the interaction between AMPs and microbial membranes is salt sensitive (8, 9). The efficacy of P-113 is significantly reduced in the presence of high salt concentrations, such as 150 mM sodium chloride or 100 mM sodium phosphate (4, 10).To improve the activity of P-113, our approach was based on the dimerization of histatin-derived peptides to increase bactericidal activity against Staphylococcus aureus (11). In this study, the duplicated and triplicated P-113 (designated P-113Du and P-113Tri, respectively) ( Table 1) were characterized. The secondary structures of the peptides in 85% trifluoroethanol (pH 6.0) were analyzed using an AVIV circular dichroism spectrometer at 25°C. Spectra were measured from 195 to 260 nm at 1-nm intervals, and mean residue molar ellipticity (MRE) was used to compare different peptides. Figure 1 shows an ␣-helical conformation characterized by a strong positive band at 195 nm and two negative bands at 208 and approximately 222 nm (12). The ␣-helical content of P-113 was estimated to be 2.9% using the ␤-structure selection (BeStSel) method (13). However, the ␣-helical contents of P-113Du and P-113Tri were 10.6% and 21.4%, respectively, suggesting the presence of a significant fraction of ␣-helix conformation.To examine the salt sensitivity of the peptides, spot assays were performed ( Fig. 2A). Cells (4.0 ϫ 10 5 cells) of the C. albicans strain SC5314 were incubated with different concentrations of sodium acetate (NaOAc) and peptides for 1 h. The mixtures were then spotted onto 1% yeast extract-2% peptone-2% glucose (YPD) agar. After 24 h of incubation, P-113 exhibited candidacidal activity in the presence of 12.5 mM sodium acetate but reduced activity in the presence of 62.25 and 93.75 mM salt. However, P-113Du and P-113Tri exhibited potent candidacidal activity even in the presence of 93.75 mM sodium acetate ( Fig. 2A). In addition, a previous study indicated that P-113 loses its bactericidal activity at pH 4.5 (14). Therefore, the pH sensitivities of the peptides were examined. P-113 exhibited candidacidal activity at pH 6 and 8, and...

Candida albicans is a commensal that inhabits the skin and mucous membranes of humans. Because of the increasing immunocompromised population and the limited classes of antifungal drugs available, C. albicans has emerged as an important opportunistic pathogen with high mortality rates. During infection and therapy, C. albicans frequently encounters immune cells and antifungal drugs, many of which exert their antimicrobial activity by inducing the production of reactive oxygen species (ROS). Therefore, antioxidative capacity is important for the survival and pathogenesis of C. albicans. In this study, we characterized the roles of the zinc finger transcription factor Sfp1 in the oxidative stress response against C. albicans. A sfp1-deleted mutant was more resistant to oxidants and macrophage killing than wild-type C. albicans and processed an active oxidative stress response with the phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1 and high CAP1 expression. Moreover, the sfp1-deleted mutant exhibited high expression levels of antioxidant genes in response to oxidative stress, resulting in a higher total antioxidant capacity, glutathione content, and glutathione peroxidase and superoxide dismutase enzyme activity than the wild-type C. albicans. Finally, the sfp1-deleted mutant was resistant to macrophage killing and ROS-generating antifungal drugs. Together, our findings provide a new understanding of the complex regulatory machinery in the C. albicans oxidative stress response.

The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.