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率和死亡率的主要原因。直到最近，可用的抗真菌药物还受到全身毒性(多烯和咪唑)、缺乏杀菌活性(三氮唑和氟胞嘧啶)或窄谱(烯丙胺)的限制。棘球菌素(ECs)，如最近FDA批准的Caspogenin，代表了一种新的抗真菌类别，似乎解决了这些限制。ECS抑制真菌特有的β-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，但其他病原体，如新生隐球菌，出于未知的原因而固有地耐药。此外，对正常敏感真菌的EC抗性或耐受性突变体的选择也存在未确定的潜力。以酿酒酵母为遗传模型，初步实验确定了一组独特的“ECH”基因，当这些基因过度表达或缺失时，会导致EC抗性或过敏性。这些基因编码高尔基体或质膜相关蛋白，已知在细胞壁合成中发挥作用，以及蛋白激酶、转录因子和其他与这一过程无关的蛋白质。但是，这些酿酒酵母的结果与临床重要真菌中的EC活性有相关性吗？这份R03提案将通过以下具体目标来检查这一点：(1)通过复制模板筛选超敏缺失突变体来完成对酿酒酵母ECH基因的鉴定，并测试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，检测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改变。(3)体外筛选白念珠菌自发耐EC或耐药突变株(初步研究发现后者频率较高)，并通过RNA杂交检测基因表达变化，通过DNA测序检测基因突变产物。在此基础上，未来的研究可能包括：(1)测试这些EC耐药或耐药突变株的体内敏感性和毒力；(2)识别与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.