Genetic Analysis of Echinocandin Sensitivity

描述（由申请人提供）：真菌感染是免疫功能低下患者发病和死亡的主要原因。直到最近，可用的抗真菌药物受到全身毒性（多烯类和咪唑类）、缺乏杀真菌活性（三唑类和氟胞嘧啶类）或谱窄（烯丙胺类）的限制。棘白菌素（ECs），如最近fda批准的caspofungin，代表了一种新的抗真菌类，似乎解决了这些限制。ECs抑制真菌特异性酶-1,3-葡聚糖合成酶，该酶负责合成一种主要的细胞壁多糖。虽然常见的真菌病原体，如白色念珠菌和烟曲霉对ECs敏感，但其他真菌病原体，如新型隐球菌，由于尚不清楚的原因，具有内在抗性。此外，在通常敏感的真菌中选择抗ec或耐ec突变体的潜力尚未确定。以酿酒酵母菌为遗传模型，初步实验确定了一组独特的“ECH”基因，当这些基因过表达或缺失时，会产生EC抗性或超敏性。这些基因编码已知在细胞壁合成中起作用的高尔基体蛋白或质膜相关蛋白，以及蛋白激酶、转录因子和其他先前与该过程无关的蛋白质。但是，这些酿酒酵母的结果是否与临床重要真菌的EC活性有关？本R03提案将通过以下具体目标对此进行研究：(1)通过复制板筛选超敏缺失突变体完成酿酒酵母ECH基因的鉴定，并检测所有突变体的抗真菌敏感性和其他相关表型的改变。(2)构建具有代表性的ECH同源物缺失的白色念珠菌菌株，并检测对ECs和其他抗真菌药物的敏感性改变，以及其他相关表型，如酵母-菌丝形态发生的改变。(3)体外筛选白色念珠菌自发ec耐药或耐受突变体（初步研究显示后者频率较高），并通过RNA杂交和DNA测序检测基因表达的改变和基因产物的改变。在此基础上，未来的研究可能包括：(1)测试这些EC耐药或耐受突变体的体内敏感性和毒力，(2)确定与EC协同或拮抗作用的药物，并确定这些相互作用的基础，(3)确定临床分离株获得性和内在EC耐药的机制。

DESCRIPTION (provided by applicant): Fungal infections are a major cause of morbidity and mortality in the immunocompromised patient. Until recently, available antifungals were limited by systemic toxicity (polyenes and imidazoles), lack of fungicidal activity (triazoles and flucytosine), or narrow spectrum (allylamines). Echinocandins (ECs) such as the recently FDA-approved caspofungin represent a new antifungal class that appears to address these limitations. ECs inhibit the fungal specific enzyme beta-1,3-glucan synthase responsible for synthesizing a major cell wall polysaccharide. While common fungal pathogens such as Candida albicans and Aspergillus fumigatus are susceptible to ECs, others such as Cryptococcus neoformans are intrinsically resistant for reasons that are unclear. Also, there is an undefined potential for the selection of EC-resistant or tolerant mutants of normally susceptible fungi. Using Saccharomyces cerevisiae as genetic model, initial experiments identified unique sets of "ECH" genes which, when overexpressed or deleted, conferred EC resistance or hypersensitivity. These genes encode Golgi or plasma membrane-associated proteins known to play roles in cell wall synthesis, as well as protein kinases, transcription factors, and other proteins with no previous connection to this process. But do these S. cerevisiae results have relevance to EC activity in clinically important fungi? This R03 proposal will examine this through the following Specific Aims: (1) Complete the identification of S. cerevisiae ECH genes by replica plate screening for hypersensitive deletion mutants, and test all mutants for altered antifungal sensitivities and other relevant phenotypes. (2) Construct C. albicans strains with deletions in representative ECH homologs and test for altered susceptibilities to ECs and other antifungals, and for other relevant phenotypes such as altered yeast-hyphal morphogenesis. (3) Select in vitro for C. albicans spontaneous EC-resistant or tolerant mutants (initial studies revealed a high frequency of the latter) and examine for altered gene expression by RNA hybridization and altered gene products by DNA sequencing. Building on this foundation, future studies are likely to include: (1) testing the in vivo susceptibility and virulence of these EC-resistant or tolerant mutants, (2) identifying drugs which act synergistically or antagonistically with ECs and determining the basis for these interactions, (3) characterizing mechanisms for acquired and intrinsic EC resistance in clinical isolates.