Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2 ⁄ ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AD) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BD-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AD cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BD but not in upc2AD cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AD cells but was unaffected in upc2BD cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AD and upc2BD cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.
CgAUS1 appears to function as a sterol transporter that may contribute to lower azole susceptibility in the presence of serum and to protect C. glabrata against azole toxicity in vivo.
SummaryDuring disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP-binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wildtype cells of C. glabrata but not in AUS1-deleted mutant (aus1D) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo-transferrin, growth of wild-type cells, but not aus1D cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1D strain caused a significantly decreased kidney fungal burden than the wild-type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.
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