Novel Azole Resistance Mechanisms in Candida albicans

描述（由申请人提供）：在包括念珠菌在内的许多真菌物种引起的感染管理中，唑类抗真菌耐药性已成为一个重要问题。这一问题对免疫功能低下的患者群体，特别是艾滋病患者产生了重大影响。虽然高效抗逆转录病毒疗法（HAART）的使用降低了美国艾滋病患者OPC的频率，但在不发达国家，这种疗法的可及性有限，依从性差，以及与HAART相关的毒性可能会导致世界范围内艾滋病患者OPC问题的增加。了解唑类抗真菌药物的分子基础将有助于制定治疗策略来规避这一问题，并提高唑类抗真菌药物的效用。本提案的目的是鉴定和表征白色念珠菌对唑抗真菌耐药性的新分子机制。该建议背后的中心假设是，除了CDR1、CDR2、MDR1、ERG11和PDR16基因外，白色念珠菌还需要逐步获得唑类抗真菌耐药性。我们将使用综合功能基因组学和蛋白质组学方法来表征和比较多个匹配的白色念珠菌唑敏感和耐药分离株内部和之间的系列分离株的基因表达和蛋白质组学特征。这些研究将确定与唑抗性表型相关的基因和基因产物。他们还将识别与已知抗性机制协调调节的基因和基因产物，从而深入了解这些机制的转录调节。我们还将使用反义cDNA文库筛选抗唑菌株，以获得该表型所需的基因。靶向基因破坏和过表达将用于评估候选抗性基因在这一过程中的作用。然后检查这些突变体在体外对唑的敏感性变化。进一步的研究将检查可能在这一过程中起作用的点突变关键基因的核苷酸序列。这些研究将进一步阐明唑类抗真菌药物耐药性的分子基础，并为未来开发既能消除耐药性又能提高唑类抗真菌药物效用的化合物确定新的靶点。

DESCRIPTION (provided by the applicant): Azole antifungal resistance has emerged as a significant problem in the management of infections caused by a number of fungal species including Candida. This problem has had a significant impact in immune-compromised patient populations, particularly those suffering from AIDS. While the use of highly active antiretroviral therapy (HAART) has reduced the frequency of OPC among AIDS patients in the United States, limited access to such therapy in underdeveloped countries, poor compliance, and toxicity associated with HAART will likely contribute to an increase in this problem among AIDS patients world-wide. Understanding the molecular basis of azole resistance will facilitate the development of therapeutic strategies to circumvent this problem and improve the utility of the azole class of antifungal agents. The aim of this proposal is to identify and characterize novel molecular mechanisms of azole antifungal resistance in Candida albicans. The central hypothesis behind this proposal is that genes in addition to CDR1, CDR2, MDR1, ERG11 and PDR16 are required for the stepwise acquisition of azole antifungal resistance in C. albicans. We will use an integrated functional genomic and proteomic approach to characterize and compare the gene expression and proteomic profiles of serial isolates within and between multiple matched sets of azole-susceptible and -resistant isolates of C. albicans. These studies will identify genes and gene products that are associated with the azole resistance phenotype. They will also identify genes and gene products that are coordinately regulated with known resistance mechanisms and hence lend insight into the transcriptional regulation of these mechanisms. We will also screen azole resistant isolates for genes required for this phenotype using an antisense cDNA library. Targeted gene disruption and over-expression will be used to assess the role of candidate resistance genes in this process. These mutants will then be examined for changes in in vitro azole susceptibility. Additional studies will examine the nucleotide sequences of key genes for point mutations that may play a role in this process. These studies will further elucidate the molecular basis for azole antifungal resistance and will identify novel targets for future work towards the development of compounds that will both abrogate resistance and enhance the utility of the azole class of antifungal agents.