No abstract

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 .

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.

Candida glabrata is an opportunistic pathogen that has developed the ability to adapt and thrive under azole treated conditions. The common mechanisms that can result in Candida drug resistance are due to mutations or overexpression of the drug efflux pump or the target of azole drugs, Cdr1 and Erg11, respectively. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance are poorly understood in C. glabrata. In this study, we show for the first time that Set1 mediates histone H3K4 mono-, di-, and trimethylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation results in increased susceptibility to azole drugs in both C. glabrata and S. cerevisiae. Intriguingly, this increase in susceptibility to azole drugs in strains lacking Set1-mediated histone H3K4 methylation is not due to altered transcript levels of CDR1, PDR1 or Cdr1’s ability to efflux drugs. Genome-wide transcript analysis revealed that Set1 is necessary for azole-induced expression of 12 genes involved in the late biosynthesis of ergosterol including ERG11 and ERG3. Importantly, chromatin immunoprecipitation analysis showed that histone H3K4 trimethylation was detected on chromatin of actively transcribed ERG genes. Furthermore, H3K4 trimethylation increased upon azole-induced gene expression which was also found to be dependent on the catalytic activity of Set1. Altogether, our findings show that Set1-mediated histone H3K4 methylation governs the intrinsic drug resistant status in C. glabrata via epigenetic control of azole-induced ERG gene expression.IMPORTANCEC. glabrata is the second most commonly isolated species from Candida infections, coming in second to C. albicans. Treatment of C. glabrata infections are difficult due to their natural resistance to antifungal azole drugs and their ability to adapt and become multidrug resistant. In this study, we investigated the contributing cellular factors for controlling drug resistance. We have determined that an epigenetic mechanism governs the expression of genes involved in the late ergosterol biosynthesis pathway, an essential pathway that antifungal drugs target. This epigenetic mechanism involves histone H3K4 methylation catalyzed by the Set1 methyltransferase complex (COMPASS). We also show that Set1-mediated histone H3K4 methylation is needed for expression of specific azole induced genes and azole drug resistance in C. glabrata. Identifying epigenetic mechanisms contributing to drug resistance and pathogenesis could provide alternative targets for treating patients with fungal infections.

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 a small fraction of normal Cdc14 activity. It remains unclear what fungal Cdc14 functions require high Cdc14 activity. 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 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 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 phenotypes previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reducing 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 promotes fungal cell wall integrity and, in C. albicans, is needed to orchestrate septation and hyphal differentiation, and for pathogenesis. Cdc14 may therefore be worth future exploration as an antifungal drug target.