An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.

IMPORTANCE: The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach.

The World Health Organization recently published the first list of priority fungal pathogens highlighting multiple Candida species including C. glabrata, C. albicans, and C. auris. The use of CRISPR-Cas9 and auxotrophic C. glabrata and C. albicans strains have been instrumental in the study of these fungal pathogens. Dominant drug resistance cassettes are also critical for genetic manipulation and eliminate the concern of altered virulence when using auxotrophic strains. However, genetic manipulation has been mainly limited to the use of two drug resistance cassettes, NatMX and HphMX. Using an in vitro assembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system and 130-150 bp homology regions for directed repair, we expand the drug resistance cassettes for Candida to include KanMX and BleMX, commonly used in S. cerevisiae. As a proof of principle, we demonstrated efficient deletion of ERG genes using KanMX and BleMX. We also showed the utility of the CRISPR-Cas9 RNP system for generating double deletions of genes in the ergosterol pathway and endogenous epitope tagging of ERG genes using an existing KanMX cassette. This indicates that CRISPR-Cas9 RNP can be used to repurpose the S. cerevisiae toolkit. Furthermore, we demonstrated that this method is effective at deleting ERG3 in C. auris using a codon optimized BleMX cassette and effective at deleting the epigenetic factor, SET1, in C. albicans using a recyclable SAT1. Using this expanded toolkit, we discovered new insights into fungal biology and drug resistance.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

The Set1 Histone H3K4 Methyltransferase Contributes to Azole Susceptibility in a Species-Specific Manner by Differentially Altering the Expression of Drug Efflux Pumps and the Ergosterol Gene Pathway.

Fungal infections are a major health concern because of limited antifungal drugs and development of drug resistance. Candida can develop azole drug resistance by overexpression of drug efflux pumps or mutating ERG11, the target of azoles. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance is poorly understood in Candida glabrata. In this study, we show that Set1 mediates histone H3K4 methylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation increases azole susceptibility in both C. glabrata and S. cerevisiae. This increase in azole susceptibility in S. cerevisiae and C. glabrata strains lacking SET1 is due to distinct mechanisms. For S. cerevisiae, loss of SET1 decreased the expression and function of the efflux pump Pdr5, but not ERG11 expression under azole treatment. In contrast, loss of SET1 in C. glabrata does not alter expression or function of efflux pumps. However, RNA sequencing revealed that C. glabrata Set1 is necessary for azole-induced expression of all 12 genes in the late ergosterol biosynthesis pathway, including ERG11 and ERG3. Furthermore, chromatin immunoprecipitation analysis shows histone H3K4 trimethylation increases upon azole-induced ERG gene expression. In addition, high performance liquid chromatography analysis indicated Set1 is necessary for maintaining proper ergosterol levels under azole treatment. Clinical isolates lacking SET1 were also hypersusceptible to azoles which is attributed to reduced ERG11 expression but not defects in drug efflux. Overall, Set1 contributes to azole susceptibility in a species-specific manner by altering the expression and consequently disrupting pathways known for mediating drug resistance.

A Novel Sterol-Signaling Pathway Governs Azole Antifungal Drug Resistance and Hypoxic Gene Repression in Saccharomyces cerevisiae.

During antifungal drug treatment and hypoxia, genetic and epigenetic changes occur to maintain sterol homeostasis and cellular function. In this study, we show that SET domain-containing epigenetic factors govern drug efficacy to the medically relevant azole class of antifungal drugs. Upon this discovery, we determined that Set4 is induced when Saccharomyces cerevisiae are treated with azole drugs or grown under hypoxic conditions; two conditions that deplete cellular ergosterol and increase sterol precursors. Interestingly, Set4 induction is controlled by the sterol-sensing transcription factors, Upc2 and Ecm22 To determine the role of Set4 on gene expression under hypoxic conditions, we performed RNA-sequencing analysis and showed that Set4 is required for global changes in gene expression. Specifically, loss of Set4 led to an upregulation of nearly all ergosterol genes, including ERG11 and ERG3, suggesting that Set4 functions in gene repression. Furthermore, mass spectrometry analysis revealed that Set4 interacts with the hypoxic-specific transcriptional repressor, Hap1, where this interaction is necessary for Set4 recruitment to ergosterol gene promoters under hypoxia. Finally, an erg3Δ strain, which produces precursor sterols but lacks ergosterol, expresses Set4 under untreated aerobic conditions. Together, our data suggest that sterol precursors are needed for Set4 induction through an Upc2-mediated mechanism. Overall, this new sterol-signaling pathway governs azole antifungal drug resistance and mediates repression of sterol genes under hypoxic conditions.