AIDS Related Oral Candidiasis: Drugs, Sterols, and Fungal Cells

描述(申请人提供)：致病酵母白色念珠菌(钙)是包括艾滋病患者在内的免疫受损人群中真菌感染的主要原因。它通常用抗真菌药物治疗，最常见的是在艾滋病患者中广泛使用的唑氟康唑。在这些患者中，很有可能会产生唑类耐药性。利用艾滋病患者的口服耐药菌株，已经确定了主要的耐药机制，包括ERG11(编码麦角固醇生物合成的一种酶的基因，也是唑类药物的靶标)的改变和外排泵表达的增加。麦角甾醇是真菌质膜中的主要甾醇，它的生物合成是唑类和许多其他抗真菌药物的靶标。真菌细胞与唑类化合物的相互作用是一个复杂的过程。新陈代谢和环境的特定方面会影响体外药物/细胞的相互作用，并有可能在临床上发挥重要作用。这项研究的总体目标是了解真菌细胞如何对唑类药物做出反应。这项建议调查了固醇的进口，随后由Upc2转录因子调节，以及环境因素对细胞对药物反应的影响。这项建议的具体目的是：1.表征进入真菌细胞的氟康唑。氮唑导入对细胞/药物的相互作用非常重要，它可以通过被动或主动转运来调节，以前还没有被研究过。2.分析Upc2的转录调控。一旦进入细胞，唑类化合物就会抑制ERG11p，改变固醇水平，并激活调控固醇生物合成和摄取的Upc2转录因子。将分析Upc2基因的调节，以了解包括摄取和生物合成在内的固醇代谢的变化如何与Upc2的表达相关。3.研究特定环境因素对药物敏感性和细胞表面的影响。环境因素，包括pH、氧气水平、氮源和外源甾醇，将利用微生物学、生化和基因表达方法分析它们对药物敏感性和细胞表面的影响。4.确定临床分离株的甾醇代谢变化特征。已知的耐药机制并不适用于许多耐药菌株。这一目标将测试导入、Upc2调控和对环境因素的反应，以确定这些分离株可能的新的耐药机制。了解氮唑类药物与真菌细胞之间的相互作用具有重要的临床意义，有助于改善真菌感染和耐药性的诊断、治疗和预防。

DESCRIPTION (provided by applicant): The pathogenic yeast Candida albicans (Ca) is a major cause of fungal infections in immune-compromised populations including AIDS patients. It is usually treated with antifungal drugs, most commonly the azole fluconazole which is used extensively in AIDS patients. In these patients, there is a significant probability that azole resistance will develop. Using oral resistant isolates from AIDS patients, major mechanisms of resistance have been identified including alterations in ERG11 (a gene encoding an enzyme in ergosterol biosynthesis and target of the azoles) and increased expression of efflux pumps. Ergosterol is the major sterol in the fungal plasma membrane; and its biosynthesis is the target for azoles and many other antifungals. The interaction of fungal cells with azoles is a complex process. Specific aspects of metabolism and the environment influence drug/cell interactions in vitro and have the potential to be important clinically. The Overall Goal of this research is to understand how a fungal cell responds to azoles. This proposal investigates import, subsequent regulation of sterols by the UPC2 transcription factor, and the influence of environmental factors on the cellular response to drugs. The Specific Aims of this proposal are: 1. To characterize fluconazole import into the fungal cell. Azole import is important to the cell/drug interaction; it may be mediated by passive or active transport; and it has not been studied previously. 2. To analyze the transcriptional regulation of UPC2. Once within the cell, azoles inhibit Erg 11 p, altering sterol levels, and activating the UPC2 transcription factor, which regulates sterol biosynthesis and uptake. Regulation of the UPC2 gene will be analyzed to understand how changes in sterol metabolism, including uptake and biosynthesis are correlated with expression of UPC2. 3. To characterize the effect of specific environmental factors on drug susceptibility and the cell surface. Environmental factors, including pH, oxygen levels, nitrogen sources, and exogenous sterols, will be analyzed for their effect on the drug susceptibility and the cell surface, using microbiological, biochemical and gene expression approaches. 4. To characterize clinical isolates for alterations in sterol metabolism. The known mechanisms of resistance do not apply to many resistant strains. This aim will assay import, UPC2 regulation, and response to environmental factors to identify possible new resistance mechanisms in these isolates. Understanding the interactions between azoles and fungal cells is a clinically significant issue, with the potential for improving diagnosis, treatment and prevention of fungal infections and resistance.