Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

Abstract

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Anticandidal effect of endophytic bacteria isolated from Equisetum arvense L. against Candida albicans and Candida glabrata

“…Despite modern management options, mortality rates due to fungal infection are in the front-line for C. glabrata infection. It is believed that C. glabrata emerged as a human pathogen from additional Candida species 7 . Leading cause of disseminated candidiasis is due to C. glabrata.…”

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology

“…The most widely applied include (i) multilocus sequence typing (MLST), which analyzes 6 relatively conserved housekeeping loci for single nucleotide polymorphisms (SNPs) (29,30), (ii) pulsed-field gel electrophoresis (PFGE), which compares total DNA banding patterns with or without restriction enzyme digestion (14,23,31), (iii) multilocus variable-number tandem-repeat analysis (MLVA, also known as microsatellite analysis), which examines length variation in 6 to 9 PCR-amplified loci that contain polymorphic tandem repeats (32)(33)(34)(35), and (iv) random amplification of polymorphic DNA (RAPD), which compares banding patterns following PCR with a nonspecific primer (26,36). In general, these methods are comparable in their strain resolution, achieving diversity indexes of ca.…”

Mechanisms of Antifungal Drug Resistance

“…Selection of these mutants in the clinical context is much less frequent (Bouchara et al 2000;Ferrari et al 2011a). Although some C. glabrata clinical isolates with mitochondrial dysfunction do not show fitness defects compared with wild-type parents in a mouse infection model (Ferrari et al 2011a), other C. glabrata petite mutants are avirulent Singh-Babak et al 2012). C. albicans cannot undergo loss of mitochon-drial DNA and therefore is considered a "petitenegative" species.…”

“…Genetic or pharmacological compromise of Hsp90 function reduces basal echinocandin tolerance and resistance of C. albicans, C. glabrata, and A. fumigatus (Cowen and Lindquist 2005;Cowen 2009;Cowen et al 2009;Singh et al 2009;Singh-Babak et al 2012;Lamoth et al 2014a). Inhibition of Hsp90 reduces echinocandin resistance acquired by mutation in the drug target, and resistance that evolved in the human host Singh-Babak et al 2012). Hsp90 affects resistance to echinocandins through its client proteins calcineurin and Mkc1 LaFayette et al 2010).…”