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Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to upc2AΔ. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.ImportanceCandida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators of C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.s

Fungal allergic asthma is a subtype of asthma with high incidence. Although asthmatics are considered an ‘at risk’ group for severe influenza, these patients suffered less severe morbidity and mortality compared to non-asthmatics during the 2009 influenza pandemic. We developed a mouse model of fungal asthma and influenza comorbidities to recapitulate these epidemiologic findings and provide a means to explore mechanisms by which this protection may occur. We hypothesized that eosinophils guide antiviral responses against influenza virus infections in the allergic host. We characterized the immunophenotype of eosinophils in various niches including bone marrow, lymphoid organs, and lungs, in asthma and influenza comorbid conditions in comparison with individual disease conditions. Eosinophils from comorbid mice had high expression of MHCI, PIR-A/B, and CD62L in the lungs and spleens compared to influenza alone. The adoptive transfer of eosinophils led to a reduction in viral burden and increase in airway CD8+ T cells in virus-infected recipients. In addition, eosinophils were able to promote CD8+ T cell activation in vitro and ex vivo, and antigen-loaded eosinophils interacted with CD8+ T cells in vitro. Comorbid mice had more plasma cells in lymphoid organs although mature B cell numbers were similar to influenza alone. Antibody production in these cells was more prominent at early time points in comorbidity but was similar between the groups at late time points. Since eosinophils have been previously shown to regulate B cells and IgA production in the gut, we are now exploring the hypothesis that eosinophil-mediated B cell responses contribute to the increase in neutralizing antibodies noted in mice with asthma and influenza.