Candida glabrata maintains two Hap1 homologs, Zcf27 and Zcf4, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

Candida glabrata exhibits innate resistance to azole antifungal drugs but also has the propensity to rapidly develop clinical drug resistance. Azole drugs, which target Erg11, is one of the three major classes of antifungals used to treat Candida infections. Despite their widespread use, the mechanism controlling azole-induced ERG gene expression and drug resistance in C. glabrata has primarily revolved around Upc2 and/or Pdr1. In this study, we determined the function of two zinc cluster transcription factors, Zcf27 and Zcf4, as direct but distinct regulators of ERG genes. Our phylogenetic analysis revealed C. glabrata Zcf27 and Zcf4 as the closest homologs to Saccharomyces cerevisiae Hap1. Hap1 is a known zinc cluster transcription factor in S. cerevisiae in controlling ERG gene expression under aerobic and hypoxic conditions. Interestingly, when we deleted HAP1 or ZCF27 in either S. cerevisiae or C. glabrata, respectively, both deletion strains showed altered susceptibility to azole drugs, whereas the strain deleted for ZCF4 did not exhibit azole susceptibility. We also determined that the increased azole susceptibility in a zcf27Δ strain is attributed to decreased azole-induced expression of ERG genes, resulting in decreased levels of total ergosterol. Surprisingly, Zcf4 protein expression is barely detected under aerobic conditions but is specifically induced under hypoxic conditions. However, under hypoxic conditions, Zcf4 but not Zcf27 was directly required for the repression of ERG genes. This study provides the first demonstration that Zcf27 and Zcf4 have evolved to serve distinct roles allowing C. glabrata to adapt to specific host and environmental conditions. IMPORTANCE: Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata , a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Zcf27 and Zcf4, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance.

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.

Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by Candida species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in Candida albicans and Candida glabrata that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.

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.

Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata . We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.

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.