Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses

Abstract

The emergence of resistance in Candida albicans , an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans , here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress. Alternative splicing (AS)—a process by which a single gene gives rise to different protein isoforms in eukaryotes—has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans , has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans . Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3 , which contains a single intron. The sod3 Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans . IMPORTANCE The emergence of resistance in Candida albicans , an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans , here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.