The Antimicrobial Peptides P-113Du and P-113Tri Function against Candida albicans

Abstract

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...

The interaction between Carbohydrates and the Antimicrobial Peptide P-113Tri is Involved in the Killing of Candida albicans

“…The killing assays were performed as previously described [19]. Briefly, C. albicans cells were grown overnight in YPD medium at 30 • C with shaking, subcultured into fresh YPD and further grown to the exponential phase (~5 h).…”

“…In our previous study, P-113Tri showed overall higher anti-candidacidal activity than its parental compound P-113 [19]. To further investigate the mechanisms of P-113Tri against C. albicans, the interaction between the cells and peptides conjugated with fluorescein isothiocyanate (FITC) was determined using confocal microscopy.…”

“…P-113Tri contained significant fractions of an α-helical conformation and was more resistant to high salt and low pH than P-113 [19] and Figure S1. Moreover, compared to P-113, P-113Tri exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of C. albicans and non-albicans Candida species [19]. However, the detailed mechanism by which P-113Tri functions differently from P-113 in its anti-C. albicans activity is still unknown.…”

“…However, the efficacy of P-113 is significantly reduced in the presence of high salt concentrations and at pH 4.5 [16][17][18]. In our previous study, novel P-113 derivatives, such as P-113Tri (a tandem arrangement of three P-113 repeats) were synthesized and characterized [19]. P-113Tri contained significant fractions of an α-helical conformation and was more resistant to high salt and low pH than P-113 [19] and Figure S1.…”

“…In our previous study, novel P-113 derivatives, such as P-113Tri (a tandem arrangement of three P-113 repeats) were synthesized and characterized [19]. P-113Tri contained significant fractions of an α-helical conformation and was more resistant to high salt and low pH than P-113 [19] and Figure S1. Moreover, compared to P-113, P-113Tri exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of C. albicans and non-albicans Candida species [19].…”