Genetic Analysis of Echinocandin Sensitivity

棘白菌素敏感性的遗传分析

• 批准号: 6590937
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6590937
• 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，可用的抗真菌药物仍受到全身毒性（多烯和咪唑）、缺乏杀菌活性（三唑和氟胞嘧啶）或窄谱（烯丙胺）的限制。最近 FDA 批准的卡泊芬净等棘白菌素 (EC) 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。虽然白色念珠菌和烟曲霉等常见真菌病原体对 EC 敏感，但新型隐球菌等其他真菌病原体具有本质上的耐药性，原因尚不清楚。此外，选择通常敏感真菌的 EC 抗性或耐受突变体的潜力也不确定。使用酿酒酵母作为遗传模型，最初的实验鉴定了独特的“ECH”基因组，当这些基因过度表达或删除时，会赋予 EC 抗性或超敏性。这些基因编码已知在细胞壁合成中发挥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没有联系的蛋白。但这些酿酒酵母结果与临床重要真菌中的 EC 活性相关吗？该 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来完成酿酒酵母 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 ECH 同源物缺失的白色念珠菌菌株，并测试对 EC 和其他抗真菌药物的敏感性改变，以及其他相关表型，例如改变的酵母菌丝形态发生。 (3) 在体外选择白色念珠菌自发的 EC 抗性或耐受突变体（初步研究显示后者的频率很高），并通过 RNA 杂交检查改变的基因表达和通过 DNA 测序检查改变的基因产物。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。