Toshihiro Aoyama

Transcription factors CgUPC2A and CgUPC2B regulate ergosterol biosynthetic genes in Candida glabrata

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 (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ‐mutant cells were as susceptible to the drugs as wild‐type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild‐type and upc2BΔ but not in upc2AΔ cells. Similarly, serum‐induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum‐induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ 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.

The Candida glabrata sterol scavenging mechanism, mediated by the ATP‐binding cassette transporter Aus1p, is regulated by iron limitation

During 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 wild‐type cells of C. glabrata but not in AUS1‐deleted mutant (aus1Δ) 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 aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ 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.

Growth defects resulting from inhibiting ERG20 and RAM2 in Candida glabrata

Farnesyl pyrophosphate (FPP) is utilized for many cellular processes, including the production of dolichols, ubiquinone (CoQ), sterols, farnesylated heme A and prenylated proteins. This lipid synthesized by FPP synthetase (ERG20) becomes attached to target proteins by the prenyltransferases, CDC43/RAM2 and RAM1/RAM2 complexes after the formation of the C15 and C20 units, respectively. Defects in protein prenylation as a result of inhibiting these enzyme complexes lead to pleiotropic effects in all eukaryotes. In this study, using Candida glabrata conditional mutants, the importance of the ERG20 and RAM2 genes for growth using both in vivo and in vitro assays was assessed by placing the RAM2 and ERG20 genes under the control of a regulatable promoter. Repression of RAM2 gene expression revealed growth defects under both conditions. However, repression of ERG20 gene expression did not impair fungal growth in a mouse host, but did result in growth defects on laboratory media. Thus, FPP synthase is not required for survival in an infected mouse, but the RAM2-encoded prenyltransferase was critical for growth under both conditions. This study strongly suggests that inhibitors of prenyltransferase may be promising antifungals.

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

Genome‐wide survey of transcriptional initiation in the pathogenic fungus, Candida glabrata

DNA sequencing of the 5′‐flanking region of the transcriptome effectively identifies transcription initiation sites and also aids in identifying unknown genes. This study describes a comprehensive polling of transcription start sites and an analysis of full‐length complementary DNAs derived from the genome of the pathogenic fungus Candida glabrata. A comparison of the sequence reads derived from a cDNA library prepared from cells grown under different culture conditions against the reference genomic sequence of the Candida Genome Database (CGD: http://www.candidagenome.org/) revealed the expression of 4316 genes and their acknowledged transcription start sites (TSSs). In addition this analysis also predicted 59 new genes including 22 that showed no homology to the genome of Saccharomyces cerevisiae, a genetically close relative of C. glabrata. Furthermore, comparison of the 5′‐untranslated regions (5′‐UTRs) and core promoters of C. glabrata to those of S. cerevisiae showed various global similarities and differences among orthologous genes. Thus, the C. glabrata transcriptome can complement the annotation of the genome database and should provide new insights into the organization, regulation, and function of genes of this important human pathogen.

The mannoprotein TIR3 (CAGL0C03872g) is required for sterol uptake in Candida glabrata.

Sterol uptake in the pathogenic fungus, Candida glabrata, occurs via the sterol transporter, CgAus1p. Azole inhibition of sterol biosynthesis can under certain circumstances be reversed by adding exogenously sterol. Here we demonstrate that the CgTIR3 (CAGL0C03872g) gene product is also required for sterol uptake, since Cgtir3Δ strains fail to take up sterol both aerobically and under hypoxic conditions. Western analysis using an HA-tagged TIR3 strain showed that CgTir3p localizes to the cell wall, and its expression is induced by serum. Semi-quantitative reverse transcriptase-PCR also showed that two transcription regulatory genes, CgUPC2A and CgUPC2B, control CgTIR3 as well as CgAUS1 gene expression. Interestingly, complementation studies using Cgtir3Δ showed that ScDAN1, a mannoprotein required for sterol uptake in Saccharomyces cerevisiae, could not complement the C. glabrata TIR3 function. Furthermore, sterol analyses, in which both the CgAUS1 and CgTIR3 genes were constitutively expressed, resulted in aerobic sterol uptake although the amount of uptake was considerably less than that of cells cultured aerobically with serum. These results suggest that additional factors other than CgAUS1 and CgTIR3 are required for sterol uptake in C. glabrata.

Quorum Sensing and Morphological Regulation in the Pathogenic Fungus Candida albicans

Quorum sensing is a cell-density effect and the regulation of gene expression in response to extracellular chemical signals (quorum-sensing molecules) produced by microorganisms. Hyphal induction in Candid albicans is inhibited by high cell density and also by farnesol, which is a quorum-sensing molecule of the organism. Here, we describe models of the regulation of dimorphism in Candida albicans by farnesol from the latest published data and gene regulation during the initial stages of quorum sensing, which was obtained through microarray analysis.