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Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor.

ABSTRACT: Resistance to azole drugs, the major clinical antifungal compounds, is most commonly due to gain-of-function (GOF) substitution mutations in a gene called PDR1 in the fungal pathogen Candida glabrata. PDR1 encodes a zinc cluster-containing transcription factor. GOF forms of Pdr1 drive high level expression of downstream target gene expression with accompanying azole resistance. PDR1 has two homologous genes in Saccharomyces cerevisiae, called ScPDR1 and ScPDR3. This study provides evidence that the PDR1 gene in C. glabrata represents a blend of the properties found in the two S. cerevisiae genes. We demonstrated that GOF Pdr1 derivatives are overproduced at the protein level and less stable than the wild-type protein. Overproduction of wild-type Pdr1 increased target gene expression but to a lesser extent than GOF derivatives. Site-directed mutagenesis of Pdr1 binding sites in the PDR1 promoter provided clear demonstration that autoregulation of PDR1 is required for its normal function. An internal deletion mutant of Pdr1 lacking its central regulatory domain behaved as a hyperactive transcription factor that was lethal unless conditionally expressed. A full understanding of the regulation of Pdr1 will provide a new avenue of interfering with azole resistance in C. glabrata.

SUBMITTER: Khakhina S

PROVIDER: S-EPMC5842128 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor.

Khakhina Svetlana S Simonicova Lucia L Moye-Rowley W Scott WS

Molecular microbiology 20180212 6

Resistance to azole drugs, the major clinical antifungal compounds, is most commonly due to gain-of-function (GOF) substitution mutations in a gene called PDR1 in the fungal pathogen Candida glabrata. PDR1 encodes a zinc cluster-containing transcription factor. GOF forms of Pdr1 drive high level expression of downstream target gene expression with accompanying azole resistance. PDR1 has two homologous genes in Saccharomyces cerevisiae, called ScPDR1 and ScPDR3. This study provides evidence that ...[more]

PMID: 29363861

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Project description:The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCECandida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C. glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen.

Project description:Candida glabrata is the second most common Candida species causing disseminated infection, after C. albicans. C. glabrata is intrinsically less susceptible to the widely used azole antifungal drugs and quickly develops secondary resistance. Resistance typically relies on drug efflux with transporters regulated by the transcription factor Pdr1. Gain-of-function (GOF) mutations in PDR1 lead to a hyperactive state and thus efflux transporter upregulation. Our laboratory has characterized a collection of C. glabrata clinical isolates in which azole resistance was found to correlate with increased virulence in vivo. Contributing phenotypes were the evasion of adhesion and phagocytosis by macrophages and an increased adhesion to epithelial cells. These phenotypes were found to be dependent on PDR1 GOF mutation and/or C. glabrata strain background. In the search for the molecular effectors, we found that PDR1 hyperactivity leads to overexpression of specific cell wall adhesins of C. glabrata. Further study revealed that EPA1 regulation, in particular, explained the increase in adherence to epithelial cells. Deleting EPA1 eliminates the increase in adherence in an in vitro model of interaction with epithelial cells. In a murine model of urinary tract infection, PDR1 hyperactivity conferred increased ability to colonize the bladder and kidneys in an EPA1-dependent way. In conclusion, this study establishes a relationship between PDR1 and the regulation of cell wall adhesins, an important virulence attribute of C. glabrata. Furthermore, our data show that PDR1 hyperactivity mediates increased adherence to host epithelial tissues both in vitro and in vivo through upregulation of the adhesin gene EPA1. IMPORTANCE Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients.

Dataset Information

Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor.

Publications

Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor.

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