Genetic Analysis of Echinocandin Sensitivity

棘白菌素敏感性的遗传分析

• 批准号: 6835307
• 负责人: SANTOSH K KATIYAR
• 金额: $ 7.55万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6835307
• 关键词: Candida albicans; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; antifungal antibiotics; cell wall; drug resistance; drug tolerance; fungal genetics; gene expression; gene mutation; glucans; molecular cloning; mutant; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; polysaccharides

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.

描述（由申请方提供）：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（多烯和咪唑类）、缺乏杀真菌活性（三唑和氟胞嘧啶）或窄谱（烯丙胺）的限制。棘白菌素（ECs），如最近FDA批准的卡泊芬净，代表了一种新的抗真菌药物，似乎可以解决这些局限性。EC抑制负责合成主要细胞壁多糖的真菌特异性酶β-1，3-葡聚糖合酶。虽然常见的真菌病原体如白色念珠菌和烟曲霉对EC敏感，但其他病原体如新型隐球菌对EC具有内在抗性，原因尚不清楚。此外，有一个不确定的潜在的选择EC抗性或耐受突变体的正常敏感的真菌。使用酿酒酵母作为遗传模型，初始实验鉴定了独特的“ECH”基因组，当其过表达或缺失时，赋予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。但是这些S。酿酒酵母结果与临床重要真菌的EC活性相关？本R 03提案将通过以下具体目标对此进行研究：（1）完成S.通过复制板筛选超敏缺失突变体，检测所有突变体的抗真菌敏感性和其他相关表型。(2)构建体C.白色念珠菌菌株的代表性ECH同源物缺失，并测试对EC和其他抗真菌剂的敏感性改变，以及其他相关表型，如酵母菌丝形态发生改变。(3)对于C，选择体外。白念珠菌自发的EC抗性或耐受突变体（最初的研究显示后者的频率很高），并通过RNA杂交和DNA测序检查改变的基因表达和改变的基因产物。在此基础上，未来的研究可能包括：（1）测试这些EC耐药或耐受突变体的体内敏感性和毒力，（2）鉴定与EC协同或拮抗作用的药物，并确定这些相互作用的基础，（3）表征临床分离株中获得性和内在EC耐药的机制。