The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene ( CgERG11 ), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata , of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2 , alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2 . Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11 , were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2 , were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1 -, CgCDR2 -, and CgSNQ2 -encoded efflux pumps might explain the azole resistance in our set of isolates.

Introduction: The culture-independent serum (1→3)-β-D-glucan (BG) detection test may allow early diagnosis of invasive fungal disease, but its clinical usefulness needs to be firmly established. A prospective single-center observational study was conducted to compare the diagnostic value of BG assay, Candida score (CS), and colonization index in intensive care unit (ICU) patients at risk for Candida sepsis. Methods: Of 377 patients, consecutively admitted to ICU for sepsis, 95 patients having an ICU stay of more than five days were studied. Blood specimens for fungal culture and BG measurement were obtained at the onset of clinical sepsis. For CS and colonization index calculations, surveillance cultures for Candida growth, and/or clinical data were recorded. Results: Sixteen (16.8%) patients were diagnosed with proven invasive fungal infection, 14 with candidiasis (13 candidemia and 1 mediastinitis) and 2 with pulmonary aspergillosis or fusariosis. Of 14 invasive Candida-infection patients, 13 had a serum sample positive for BG, 10 had a CS value ≥3, and 7 a colonization index ≥0.5. In the 12 candidemic patients, a positive BG result was obtained 24 to 72 hrs before a culture-documented diagnosis of invasive candidiasis. The positive and negative predictive values for the BG assay were higher than those of CS and colonization index (72.2% versus 57.1% and 27.3%; and 98.7% versus 97.2% and 91.7%, respectively). Conclusions: A single-point BG assay based on a blood sample drawn at the sepsis onset, alone or in combination withCS, may guide the decision to start antifungal therapy early in patients at risk for Candida infection.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata . Cells with mitochondrial DNA deficiency (so-called “petite mutants”) upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 ( CgCDR1 ), CgCDR2 , and CgSNQ2 . We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

A small molecule, inhibitor of a protein–protein interaction between the transcription factor Pdr1 and the Med15 subunit of Mediator in the fungal pathogen Candida glabrata, is identified and characterized here; the compound iKIX1 inhibits Pdr1-mediated gene activation and resensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models of disseminated and urinary tract infection. Joy Nishikawa et al. report the identification and characterization of a small-molecule inhibitor of a protein–protein interaction between the transcription factor Pdr1 and the MED15 subunit of Mediator in the fungal pathogen Candida glabrata. The lead compound, iKIX1, inhibits Pdr1-mediated gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models of disseminated and urinary tract infection. The NMR structure of the interaction between Mediator and Pdr1, has also improved our understanding of the mechanism of gene activation by Pdr1 and its inhibition by iKIX1. Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex1,2. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi3,4. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata5,6. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent7,8. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active9,10,11,12,13,14, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.