Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance

Abstract

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

Figure 1. Deletion of MSH2 in C. glabrata leads to significantly more resistant colonies upon selection on multiple antifungal drugs.

Wild type, msh2Δ and rad50Δ strains were selected on media containing caspofungin (an echinocandin), fluconazole (a triazole) and amphotericin B (a polyene) at concentrations from 16- to 32-fold greater than wild type MICs as described in Methods section. The plots show means of resistant colony frequencies from ≥3 independent experiments±s.d. See Supplementary Fig. 1 for resistant frequencies to voriconazole (triazole) and micafungin (echinocandin). **P<0.01 (student’s t-test; two-tailed). Representative images of selection plates are shown. Scale bars=1 cm.

Figure 2. Msh2 alterations identified in diverse clinical isolates cause a mutator phenotype and increased emergence of antifungal resistance.

(a) The 357 clinical isolates obtained were classified according to their susceptibilities to fluconazole (FLC) and the echinocandins (ECH), and the percentage of isolates within each group demonstrating a nonsynonymous msh2 mutation were determined. P value was determined through χ² analysis (compared with susceptible group). (b) All isolates were categorized by institution. Isolates demonstrating an msh2 mutation/total isolates are shown for each susceptibility group. See Supplementary Data 1 for a list of all individual isolates analyzed. (c) Echinocandin- (caspofungin) resistant colony frequencies of various clinical isolates were measured. (d) Wild type or msh2Δ cells expressing an empty or MSH2-containing plasmid were selected on caspofungin and 5-fluoroanthranilic acid. See Supplementary Table 4 for strains. Frequency data in c,d are mean±s.d. from three independent experiments; representative images are shown. *P<0.05, **P<0.01 (student’s t-test; two-tailed). Scale bars=1 cm.

Figure 3. Colonization with msh2Δ leads to increased echinocandin-resistance in vivo.

(a) GI colonization of CF-1 mice represented through fecal C. glabrata burdens. Daily administration (i.p.) of caspofungin (CSF; 0.5 mg ml⁻¹) or saline occurred from days 6 to 26 post-inoculation. (CSF-treated groups: n=8 wild type inoculated mice, 8 msh2Δ inoculated mice; Saline-treated groups: n=2 wild type, 2 msh2Δ). Means±s.d. are calculated. (b) Fecal samples from each mouse at days 3, 9, 12, 15 and 18 (−3, 3, 6, 9 and 12 days post-treatment initiation, respectively) were selected on caspofungin (2 μg ml⁻¹) containing YPD media (see Methods section for additional details). Five of eight CSF-treated mice that were inoculated with msh2Δ demonstrated increase in resistant colony frequencies between days 12 and 18. (c) fks1 mutants identified within fecal isolates. Colonies on antifungal-free plates (produced in part a) were replica plated onto caspofungin-containing media. Hotspot regions of FKS1 and FKS2 (echinocandin drug targets) were amplified and sequenced in resistant colonies.

Figure 4. Wild type cells partially outcompete msh2Δ in mixed inoculation mouse models.

DNA was isolated from kidney tissue of systemically infected, immunosuppressed mice (a) and from fecal samples of GI-colonized, immune competent mice (b) and then subjected to SYBR green quantitative PCR with strain specific primers. Wild type (WT) to mutant ratios were calculated from qPCR DNA copy numbers. Means±s.d. are calculated.