In Vivo Expression of Candida Drug Resistance Genes

描述(由申请人提供)： 致病酵母菌白色念珠菌会导致衰弱且经常反复发生的真菌感染，特别是在免疫功能低下的患者群体中，包括艾滋病患者。这些感染通常用唑类抗真菌药物治疗，耐药性是一个严重的问题，特别是在感染艾滋病毒的人中。唑类耐药的主要机制包括靶酶的点突变和过度表达以及外排泵的过度表达。然而，所有关于抗真菌耐药性的信息都是从临床分离株和培养中的实验室结构研究中获得的。这些耐药机制尚未在直接从患者身上采集的样本中确定或表征。本研究的总体目标是表征HIV患者口腔样本中白色念珠菌耐药基因的定量表达模式，以了解体内耐药机制。其具体目的是：1.采用定量RT-PCR方法检测患者唾液标本中耐药基因的体外、培养和体内表达水平。2.检测HIV+患者唾液中易感和耐药钙相关基因的表达水平。在可预见的未来，唑类药物与真菌细胞之间的相互作用以及对唑类药物的耐药性仍将是临床上重要的问题。这项建议将确定直接从患者样本中的人类样本中提取的敏感和耐药分离株中耐药基因的表达水平，扩展了在酵母培养中所做的观察。这些分析将在宿主的背景下进一步了解抗真菌药物与靶细胞的相互作用，并可能导致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 causes debilitating and often recurring fungal infections, particularly in immune-compromised patient populations including AIDS patients. These infections are usually treated with azole antifungal drugs, and resistance is significant problem, especially in HIV-infected individuals. The major mechanisms of azole resistance include point mutations and overexpression of the target enzyme and overexpression of efflux pumps. However, all of the information concerning antifungal drug resistance has been obtained from studies of clinical isolates and laboratory constructs in culture. These resistance mechanisms have not been identified or characterized in samples taken directly from the patient. The Overall Goal of this research is to characterize the quantitative expression patterns of C. albicans resistance genes in oral samples from HIV patients in order to understand the mechanisms of resistance in vivo. The Specific Aims are: 1. To adapt quantitative RT-PCR to determine levels of expression of resistance genes in vitro in culture and in vivo in patient saliva samples. 2. To determine the levels of expression for genes associated with resistance in saliva samples containing susceptible and resistant Ca from HIV + patients. The interactions between azoles and fungal cells, and azole drug resistance, will continue to be clinically significant issues for the foreseeable future. This proposal will determine the expression levels of resistance genes in susceptible and resistant isolates taken directly from human samples in patient samples, extending the observations made in yeast cultures. These analyses will further the understanding of the interactions of antifungal drugs and the target cells in the context of the host, and may lead to improvements in diagnosis, treatment and prevention of fungal infections and resistance.