pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

Abstract

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis.

Keywords: Candida albicans; antifungal; vacuole; valproic acid; vulvovaginal candidiasis.

Figure 1

In vitro antifungal activity of valproic acid is pH-dependant. (A) Effect of different pHs on antifungal activity of VPA. The C. albicans SC5314 strain was grown in SC medium with different pH (4.5–8) supplemented with different concentration of VPA. SC5314 strain was grown at 30°C and OD_595nm reading was taken after 24 h of incubation. ODs measurement for each VPA concentration is the mean of triplicate. (B) VPA inhibit the growth of non-albicans Candida species. C. glabrata, C. parapsilosis, C. tropicalis, C. krusei in addition to S. cerevisiae were grown in SC medium pH 4.5 with different concentration of VPA. OD_595nm reading was taken after 24 h of incubation at 30°C under agitation. (C) Time-kill curve demonstrating the fungistatic activity of VPA. C. albicans SC5314 strain was exposed to two different concentrations (1,000 and 3,000 μg/ml) at different times (6, 24, and 48). CFUs were calculated as described in the method section.

Figure 2

Valproic acid attenuate damage of vaginal epithelial cells caused by C. albicans. Damage of human epithelial vaginal cell line VK2/E6E7 infected by C. albicans SC5314 strain was assessed using LDH release assay. For each pH, cell damage was calculated as percentage of LDH activity of VPA-treated experiment relatively to that of the control experiment (C. albicans invading VK2/E6E7 cells in the absence of VPA). Results are the mean of three independent replicates.

Figure 3

Anti-biofilm activity of valproic acid. The effect of VPA on biofilm formation was evaluated using the metabolic colorimetric assay based on the reduction of XTT at pH4. Sensitive (SC5314) and azole- (S2 and F5), and echinocandin-resistant (DPL-1008 and DPL-1010) C. albicans strains were tested. Results represent growth inhibition (%) and are the mean of at least three independent replicates.

Figure 4

Valproic acid alters vacuolar morphology. C. albicans SC5314 strains was grown in RPMI pH 4.5 in the absence (A) or presence of 50 μg/ml of VPA (B) and stained for 3 min with the vacuole membrane marker, MDY-64. Cells were visualized using confocal microscopy. The white arrows indicate representatively intact vacuole lumens. Fluorescence PMT gain were increased five times for VPA-treated cells due to low incorporation of MDY-64. Bars, 8 μm.