Fluconazole-Resistant Candida auris Is Susceptible to Salivary Histatin 5 Killing and to Intrinsic Host Defenses

Abstract

Candida auris is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic Candida species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against Candida albicans and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of C. auris to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of C. auris clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in C. auris cells; such translocation is required for the killing of C. albicans by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole. C. auris showed higher tolerance to oxidative stress than C. albicans, and higher survival within neutrophils, which correlated with resistance to oxidative stress in vitro Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of C. auris by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR C. auris strains effectively.

Keywords: Candida auris; histatin; innate host defense; neutrophils.

FIG 1

Basic morphology of the C. auris clinical isolates used in this study. (A to D) Micrographs of C. albicans SC5314, consisting of large ovoid cells (A), C. auris strains consisting of spherical to ovoid cells (CAU-05 shown) (B) or ellipsoidal cells (CAU-10 shown) (C), and some highly aggregative strains (CAU-02 shown) (D). (E) Colony morphology of C. auris (CAU-03 shown). C. auris forms smooth, unwrinkled colonies on Spider medium plates after 7 days of incubation at 37°C, while C. albicans SC5314 forms rough colonies. (F) C. auris strains form smooth, light- to dark-pink circular colonies on CHROMagar plates after incubation for 48 h at 37°C. Bars, 5 μm.

FIG 2

Many C. auris isolates are susceptible to Hst 5. C. auris cells in exponential phase were exposed to 7.5 μM Hst 5 in 10 mM NaPB for 60 min at 30°C; then aliquots containing 400 cells were plated to determine the percentage of killing. C. albicans showed 38% killing, while seven C. auris isolates (CAU-02, CAU-03, CAU-04, CAU-05, CAU-06, CAU-08, and CAU-10) showed significantly (P < 0.001) higher killing (55 to 90%), and three strains were insensitive (CAU-01, CAU-07, and CAU-09). Interestingly, C. glabrata and C. haemulonii, the closest phylogenetic relatives of C. auris, showed low susceptibility to Hst 5. Percentages of killing are shown as means and standard deviations for at least three independent replicates for each isolate, and significance was determined using one-way ANOVA with Dunnett's multiple-comparison test.

FIG 3

F-Hst 5 is taken up by C. auris cells. F-Hst 5 (30 μM) and PI (0.5 μM) were added to C. auris cells adherent to a glass coverslip, and intracellular uptake was imaged after 30 min at 23°C using DIC and fluorescence confocal microscopy. Regardless of the killing efficiency of Hst 5, both CAU-07 and CAU-08 transported F-Hst 5 to the cytosol (as visualized by green florescence), resulting in a change in cell topology followed by cell death (as visualized by red PI staining). Bar, 5 μm.

FIG 4

C. auris strains tolerate higher levels of oxidative stress than C. albicans. For spot assays, yeast cells in the mid-log-growth phase were serially diluted, inoculated onto YPD plates supplemented with 5 or 7.5 mM H₂O₂, and incubated at 30°C for 24 h. All C. auris isolates except CAU-01 showed higher tolerance of H₂O₂ stress at 7.5 mM than the reference strain, C. albicans SC5314.

FIG 5

C. auris strains have differential susceptibility to intracellular killing by human neutrophils. (A) C. auris cells are phagocytosed by human neutrophils. Shown are overlay images of green (neutrophils stained by phalloidin dye) and blue (nonphagocytosed yeast cells stained with calcofluor white) fluorescence channels. The bright-field view shows internalized C. auris cells. Bar, 5 μm. (B) C. auris strains were coincubated with human neutrophils for 3 h; phagocytosed yeast was released by lysing neutrophils; and intracellular survival was determined by plating. Results represent means ± standard deviations. Data were analyzed by one-way ANOVA with a post hoc Dunnett multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).