Isogenic strain construction and gene mapping in Candida albicans.

Virulence factors of Candida albicans

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.

Cell Wall Assembly in Saccharomyces cerevisiae

It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.

Engineered Control of Cell Morphology In Vivo Reveals Distinct Roles for Yeast and Filamentous Forms of Candida albicans during Infection

Candida albicans is an opportunistic fungal pathogen that is found in the normal gastrointestinal flora of most healthy humans. However, in immunocompromised patients, blood-stream infections often cause death, despite the use of anti-fungal therapies. The recent completion of the C. albicans genome sequence, the availability of whole-genome microarrays and the development of tools for rapid molecular-genetic manipulations of the C. albicans genome are generating an explosion of information about the intriguing biology of this pathogen and about its mechanisms of virulence. They also reveal the extent of similarities and differences between C. albicans and its benign relative, Saccharomyces cerevisiae.

Candida albicans : A molecular revolution built on lessons from budding yeast

Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus Candida albicans

Purpose: The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. Experimental Design: Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. Results: CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. Conclusions: CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings. Clin Cancer Res; 17(10); 3272–81. ©2011 AACR .

https://clincancerres.aacrjournals.org/content/clincanres/early/2011/05/06/1078-0432.CCR-10-2882.full.pdf

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

Cyclothialidine is a new DNA gyrase inhibitor isolated from Streptomyces filipinensis NR0484. Structurally, it belongs to a new class of natural products containing a unique 12-membered lactone ring that is partly integrated into a pentapeptide chain. Cyclothialidine was found to be one of the most active of all the DNA gyrase inhibitors tested in the DNA supercoiling reaction of Escherichia coli DNA gyrase; 50% inhibitory concentrations (in micrograms per milliliter) of 0.03 (cyclothialidine), 0.06 (novobiocin), 0.06 (coumermycin A1), 0.66 (norfloxacin), 0.88 (ciprofloxacin), and 26 (nalidixic acid) were found. In addition, DNA gyrases from gram-positive species were inhibited equally as well as DNA gyrase from E. coli. Cyclothialidine also inhibited the in vitro DNA replication directed from oriC of E. coli. Among the bacterial species tested, only Eubacterium spp. were inhibited by cyclothialidine, suggesting that it can enter the cells of Eubacterium and exert antibacterial activity through interference with the DNA gyrase within the cells, although its penetration into most bacterial cells appears to be poor. These results provide a basis for cyclothialidine to be a lead structure for novel antibacterial agents with DNA gyrase inhibitory activities. Images

Biological characterization of cyclothialidine, a new DNA gyrase inhibitor.

Squalene synthase (farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is the first committed enzyme of the sterol biosynthesis pathway. Inhibitors of this enzyme have been intensively studied as potential antifungal agents. To assess the effect of deactivating squalene synthase on the growth of fungi in mice, we isolated the squalene synthase (ERG9) gene from the pathogenic fungus Candida glabrata and generated strains in which the CgERG9 gene was under the control of the tetracycline-regulatable promoter. Depletion of the ERG9 gene by doxycycline (DOX), a derivative of tetracycline, decreased the cell viability in laboratory media, whereas it did not affect cell growth in mice at all. The growth defect caused by DOX in laboratory media was suppressed by the addition of serum. Analyses of the sterol composition of the restored cells in serum-containing media suggest that the defect of ergosterol biosynthesis can be complemented by the incorporation of exogenous cholesterol into the cells. Thus, deactivation of squalene synthase did not affect fungal growth in mice, presumably because the cells were able to incorporate cholesterol from the serum. These results showed that squalene synthase could not be a suitable target of antifungals for the treatment of C. glabrata infection.

Depletion of the Squalene Synthase (ERG9) Gene Does Not Impair Growth of Candida glabrata in Mice

Azoxybacilin, produced by Bacillus cereus, has a broad spectrum of antifungal activity in methionine-free medium and has been suggested to inhibit sulfite fixation. We have further investigated the mode of action by which azoxybacilin kills fungi. The compound inhibited the incorporation of [35S] sulfate into acid-insoluble fractions of Saccharomyces cerevisiae under conditions in which virtually no inhibition was observed for DNA, RNA, or protein synthesis. It did not interfere with the activity of the enzymes for sulfate assimilation but clearly inhibited the induction of those enzymes when S. cerevisiae cells were transferred from rich medium to a synthetic methionine-free medium. Particularly strong inhibition was observed in the induction of sulfite reductase. Northern (RNA) analysis revealed that azoxybacilin decreased the level of mRNA of genes for sulfate assimilation, including MET10 for sulfite reductase and MET4, the transactivator of MET10 and other sulfate assimilation genes. When activities of azoxybacilin were compared for mRNA and enzyme syntheses from MET10, the concentration required for inhibition of transcription of the gene was about 10 times higher (50% inhibitory concentration = 30 micrograms/ml) than that required for inhibition of induction of enzyme synthesis (50% inhibitory concentration = 3 micrograms/ml). The data suggest that azoxybacilin acts on at least two steps in the expression of sulfite reductase; the transcriptional activation of MET4 and a posttranscriptional regulation in MET10 expression. We conclude that azoxybacilin exhibits antifungal activity by interfering with the regulation of expression of sulfite reductase activity.

Yuko Aoki

Papers

Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus Candida albicans

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

Biological characterization of cyclothialidine, a new DNA gyrase inhibitor.

Depletion of the Squalene Synthase (ERG9) Gene Does Not Impair Growth of Candida glabrata in Mice

Antifungal azoxybacilin exhibits activity by inhibiting gene expression of sulfite reductase.