Erg25 Controls Host-Cholesterol Uptake Mediated by Aus1p-Associated Sterol-Rich Membrane Domains in Candida glabrata.

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not the localization of the membrane proteins Pma1p and Hxt1p, which localize to sterol-poor domains. In the sterol-rich lipid domain, Aus1p-contining domain was mostly independent of MCC/eisosomes, and the nature of these domains was also different: Ausp1-contining domain was a dynamic network-like domain, whereas the MCC/eisosomes was a static dot-like domain. However, deletion of MCC/eisosomes was observed to influence the localization of Aus1p after Aus1p was transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. These findings suggest that ERG25 plays a key role in stabilizing sterol-rich lipid domains, constituting a promising candidate target for antifungal therapy.

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.

Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super Support Film) and other support films and grids in electron microscopy.

Hydrophilicity/hydrophobicity of the surfaces of plasma-polymerized naphthalene film (Super Support Film, Nisshin EM Co. Ltd., Tokyo), carbon and formvar support films, and copper and nickel grids were quantitatively estimated by contact angles measured from diameters and heights of water droplets placed on various specimens. With treatment of glow discharge, the surfaces of plasma-polymerized naphthalene and carbon support films became fully hydrophilic in 20 s. They remained hydrophilic for 6 h. The surfaces of copper and nickel grids became fully hydrophilic with 60 s of glow discharge treatment. They remained hydrophilic for only 1 h. This information is useful for negative staining, ultrathin sectioning and rapid freezing of biological specimens using the sandwich method.

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.

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 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.

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.

[Quorum sensing in fungal pathogenesis].

Quorum sensing is a function that allows microbes to monitor their own population densities by detecting their extracellular products collectively known as autoinducers. This function has been shown to be required for the expression of various virulence factors and drug resistance in a number of bacterial pathogens. In recent years, quorum sensing has been studied also in pathogenic fungi, particularly the dimorphic fungus Candida albicans. Here, we discuss (i) quorum sensing in C. albicans with emphasis on the role of farnesol as an autoinducer, (ii) quorum sensing in other fungi, (iii) comparison between fungi and bacteria regarding the quorum sensing mechanisms.

[Farnesol as a quorum-sensing molecule in Candida albicans].

Farnesol is one of the quorum sensing molecules of Candida albicans. In this report, we discuss the effects of farnesol on: 1. growth of Candida albicans in vitro and in vivo; 2. the incorporation of biomolecules into the cell wall of Candida albicans; and 3. cytokine expression by the immune system. Our results indicate genes of Candida albicans expressed at an early stage of quorum-sensing. Half of these genes are known and two-thirds of known genes are up-regulated by two types of transcription factors.

Transcriptional changes in Candida albicans Genes by both farnesol and high cell density at an early stage of morphogenesis in N-acetyl-D-glucosamine medium.

Quorum sensing through farnesol, a quorum sensing molecule, regulates virulence and morphogenesis in Candida albicans. Farnesol and high cell density of C. albicans repress hyphal formation in a minimal medium containing N-acetyl-D-glucosamine. Global transcription profiling at an early stage of quorum sensing by C. albicans in the N-acetyl-D-glucosamine medium was analyzed. Twenty-two of a total of 53 genes responded to both farnesol and high cell density. From in silico analysis and previous published data, nine of these genes including those encoding amino acid biosynthesis were controlled by the Gcn4p regulator. Nine other genes which included genes encoding central carbon metabolism were controlled by negative regulators including Nrg1p, Tup1p, Ssn6p, and/or Mig1p. Other genes not controlled by these regulators included genes related to oxidative stress, glucose metabolism, and agglutination. Expression of genes related to amino acid biosynthesis and central carbon metabolism in this study is similar to a previous report of transcription profiling in C. albicans following its internalization by phagocyte cells and adaptation to host challenges.

The Candida glabrata putative sterol transporter gene CgAUS1 protects cells against azoles in the presence of serum.

OBJECTIVES: The uptake of endogenous sterol from serum may allow Candida glabrata to survive azole treatment. This study aims to determine the contribution of a sterol transporter that alters fluconazole sensitivity in the presence of serum. METHODS: Bioinformatic analysis predicted CgAUS1 as the C. glabrata orthologue of the Saccharomyces cerevisiae transporters AUS1 and PDR11. To investigate whether the CgAUS1 gene has sterol transporter activity, we investigated the effects of an AUS1 deletion on the growth of a tetracycline-regulatable ERG9 strain (tet-ERG9aus1), wherein ERG9 expression is turned off giving rise to a sterol requirement. Tetracycline-dependent repression of CgAUS1 in the tet-AUS1 strain was used to determine the fluconazole susceptibility of CgAUS1 in the presence and absence of serum. RESULTS: The tetracycline-treated tet-ERG9aus1 strain failed to grow in the presence of serum, whereas the parental tet-ERG9AUS1 strain grew by incorporating sterol from exogenously supplied serum. Serum cholesterol protected cells against the antifungal effects of fluconazole and this protection was lost by repressing CgAUS1 gene expression. Furthermore, such protection was also observed during itraconazole treatment, but not observed in cells treated with non-azole antifungals. CONCLUSIONS: 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.

Concierge: personal database software for managing digital research resources.

This article introduces a desktop application, named Concierge, for managing personal digital research resources. Using simple operations, it enables storage of various types of files and indexes them based on content descriptions. A key feature of the software is a high level of extensibility. By installing optional plug-ins, users can customize and extend the usability of the software based on their needs. In this paper, we also introduce a few optional plug-ins: literature management, electronic laboratory notebook, and XooNlps client plug-ins. XooNIps is a content management system developed to share digital research resources among neuroscience communities. It has been adopted as the standard database system in Japanese neuroinformatics projects. Concierge, therefore, offers comprehensive support from management of personal digital research resources to their sharing in open-access neuroinformatics databases such as XooNIps. This interaction between personal and open-access neuroinformatics databases is expected to enhance the dissemination of digital research resources. Concierge is developed as an open source project; Mac OS X and Windows XP versions have been released at the official site (http://concierge.sourceforge.jp).

Sequence finishing and gene mapping for Candida albicans chromosome 7 and syntenic analysis against the Saccharomyces cerevisiae genome.

The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans.

Simulation analysis of receptive-field size of retinal horizontal cells by ionic current model.

The size of the receptive field of retinal horizontal cells changes with the state of dark/light adaptation. We have used a mathematical model to determine how changes in the membrane conductance affect the receptive-field properties of horizontal cells. We first modeled the nonlinear membrane properties of horizontal cells based on ionic current mechanisms. The dissociated horizontal cell model reproduced the voltage-current (V-I) relationships for various extracellular glutamate concentrations measured in electrophysiological studies. Second, a network horizontal cell model was also described, and it reproduced the V-I relationship observed in vivo. The network model showed a bell-shaped relationship between the receptive-field size and constant glutamate concentration. The simulated results suggest that the calcium current is a candidate for the bell-shaped length constant relationship.