Candidiasis is the most common fungal infection in the US, with an estimated 63,000 episodes of invasive candidiasis occurring per year and an estimated cost of $2-4 billion. Candida albicans, the prevalent species, causes up to 50% of blood infections. Since 2001, C. albicans infections have been effectively treated first with caspofungin (CAS) and later with other echinocandins (ECNs). However, expanding use of ECNs has led to an increase in the number of breakthrough infections due to resistance. The only generally recognized mecha- nism of C. albicans ECN resistance are specific point mutations in the FKS1 gene encoding a catalytic subunit of 1,3-?-D-glucan synthase complex. Intriguingly, many clinical isolates that are free of FKS1 mutations exhibit increased minimum inhibitory concentrations (MICs) for ECNs in standard microdilution assays. Our own data show that direct exposure of C. albicans cells to CAS in vitro generates mutants that lack FKS1 resistance mu- tations but exhibit decreased CAS susceptibilities and remodeled cell walls. A half of these mutants remain as normal diploids, whereas the remainder either lose one copy of chromosome 5 (Ch5) or convert one copy of Ch5 into a chromosome with two right arms (iso-Ch5R). Importantly, expression of 54 genes on disomic Ch5 is downregulated twofold or more both in the mutants that are normal diploids, suggesting that some of these Ch5 genes affect CAS susceptibility. We are finding that the newly-discovered mechanisms control same genes in a similar fashion in vitro and can operate in clinical isolates with decreased ECN susceptibility. Fur- thermore, DNA-seq analysis of the above model mutants, revealed 2 independent mutational pathways with a limited number of mutational hot spots. A growing body of factors influencing ECN susceptibilities also in- cludes: a) multiple genes for either negative or positive regulators residing on Ch5; b) the orthologous gene FKS3, a negative regulator of FKS1 that is downregulated in all types of mutants and in an isolate with in- creased MICs; c) epigenetic regulation on aneuploid chromosomes that might also operate on disomic Ch5. In the absence of series of multiple consecutive isolates from ECN-treated patients including drug-nave isolate and clinically resistant isolate, characterization of in vitro mechanisms that can decrease ECN susceptibility in vivo is of a special importance. We propose that the genetic mechanisms discovered in vitro are adaptive initial changes that decrease ECN susceptibility in clinical isolates and allow survival until a resistance mutation in FKS1 occurs. To define mechanisms of evolution of ECN resistance we propose to 1) use the information pro- vided by the model mutants to determine metabolic and mutational pathways that are activated in clinical iso- lates with decreased ECN susceptibility; 2) determine whether epigenetic regulation governs gene regulation on the disomic Ch5, as well as 3) determine the relevance of mechanisms identified in vitro to evolution of true resistance in clinical isolates. Our findings will be of high significance to clinicians and researchers investigat- ing the phenomenon of drug resistance in C. albicans.
Candida infections are often fatal in immuno-compromised individuals, resulting in many thousands of deaths per year. Caspofungin has proven to be an excellent anti-Candida drug, and is now the frontline treatment for infections. However, as expected, the number of resistant cases is increasing, therefore new treatment modalities are needed. We are determining metabolic pathways leading to decreased drug susceptibility in order to identify mechanisms facilitating evolution of clinical resistance. Our study will also reveal genes for new drug targets.
