A TALE OF TWO HAP1 OHNOLOGS, HAP1A AND HAP1B: ROLE IN ERGOSTEROL GENE REGULATION AND STEROL HOMEOSTASIS IN CANDIDA GLABRATA UNDER AZOLE AND HYPOXIC CONDITIONS
Candida glabrata is a member of the gut microbiota that can become an opportunistic pathogen under certain conditions. It is known for its inherent resistance to azole antifungal drugs and its ability to rapidly develop resistance during treatment. However, the regulatory mechanisms that enable this commensal organism to survive in low-oxygen environments, such as the gut, and to develop antifungal resistance when it becomes pathogenic, are not fully understood. In this study, we demonstrate for the first time the roles of two zinc cluster transcription factors in C. glabrata, Hap1A and Hap1B, in contributing to azole drug resistance in both laboratory strains and drug-resistant clinical isolates, adaptation to hypoxia, and resistance to other antifungal drugs like polyenes and echinocandins under specific conditions.
Azole drugs, which target the Erg11 protein, are widely used to treat Candida infections. The regulation of azole-induced ERG gene expression and activation of drug efflux pumps in C. glabrata has primarily been linked to the zinc cluster transcription factors Upc2A and Pdr1. Here, we investigated the roles of S. cerevisiae Hap1 orthologs, Hap1A and Hap1B, in C. glabrata as direct regulators of ERG genes upon azole exposure.
Our research shows that deleting HAP1 in the yeast model S. cerevisiae increases sensitivity to fluconazole due to the failure to induce ERG11 expression in the hap1Δ mutant compared to the wild-type strain. Although C. glabrata is closely related to S. cerevisiae, a whole genome duplication (WGD) event allowed C. glabrata to retain two HAP1 ohnologs, while S. cerevisiae lost one copy. Through phylogenetic and syntenic analyses, we identified Hap1A and Hap1B in C. glabrata as ohnologs of Hap1 in S. cerevisiae, which is known to regulate ERG gene expression under both aerobic and hypoxic conditions. Interestingly, deleting HAP1B in C. glabrata increased sensitivity to both triazole and imidazole drugs, similar to Hap1 in S. cerevisiae, while deleting HAP1A did not affect azole sensitivity.
Gene expression analysis revealed that the increased azole sensitivity in the hap1BΔ strain was due to reduced azole-induced ERG gene expression, leading to lower total endogenous ergosterol levels. Additionally, the loss of HAP1B in C. glabrata clinical isolates like SM1 and BG2, as well as in drug-resistant strains like SM3, also led to increased azole hypersusceptibility. While it was already known that losing UPC2A in C. glabrata increases azole sensitivity, our study is the first to demonstrate that the combined loss of both HAP1B and UPC2A makes C. glabrata strains even more sensitive to azoles than losing either gene alone. Additionally, we show that the loss of both HAP1B and the H3K4 histone methyltransferase SET1 increases azole hypersensitivity more than the loss of either gene alone.
Interestingly, the Hap1A protein is barely detectable under aerobic conditions but is specifically induced under hypoxia, where it plays a crucial role in repressing ERG genes. In the absence of Hap1A, Hap1B compensates by acting as a transcriptional repressor. Our RNA sequencing analysis further showed that losing both HAP1A and HAP1B not only affects genes in the ergosterol biosynthesis pathway but also upregulates iron transport-related genes FET3 and FTR1. Moreover, we found that the hypoxic growth defect caused by the loss of both HAP1A and HAP1B is exacerbated when treated with the echinocandin caspofungin and the cell wall-damaging agent calcofluor white, indicating that these Hap1 ohnologs contribute to maintaining cell wall integrity under hypoxic conditions. Since HAP1A transcript levels remain stable under aerobic conditions, we suspect that Hap1A expression is regulated post-transcriptionally.
Furthermore, we discovered that the simultaneous loss of both HAP1A and HAP1B leads to increased hypersensitivity to the polyene antifungal drug amphotericin B, though the exact mechanism behind this phenotype remains unclear. Altogether, our study is the first to show that Hap1A and Hap1B have evolved distinct roles, enabling C. glabrata to adapt to specific host and environmental conditions.
History
Degree Type
- Doctor of Philosophy
Department
- Biochemistry
Campus location
- West Lafayette