VIRULENCE AND GENETIC VARIATION IN C NEOFORMANS

新型隐球菌的毒力和遗传变异

• 批准号: 3456067
• 负责人: Eric D. Spitzer
• 金额: $ 9.75万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3456067
• 关键词: AIDS; Cryptococcus neoformans; carbon dioxide; cell capsule; cryptococcosis; fungal genetics; genetic library; genetic mapping; genetic strain; human immunodeficiency virus 1; molecular cloning; mutant; opportunistic infections; pulsed field gel electrophoresis; restriction fragment length polymorphism; southern blotting; subtraction hybridization; virulence

Cryptococcus neoformans, an encapsulated yeast, is a major cause of life-threatening opportunistic infections in AIDS patients and other immunocompromised patients. Little is known about the genetic structure of this pathogen. The long term goals of this project are to develop improved methods for genetic mapping in C neoformans; determine the genetic basis for differences between the two varieties of C neoformans (var. neoformans and var. gatti); determine whether C neoformans isolates from AIDS patients are different from other strains; and isolate genes affecting capsule synthesis and other virulence factors. These studies will contribute to a better understanding of the pathogenesis of cryptococcal infections and the development of new therapeutic agents. The experimental design of this project is as follows. Electrophoretic karyotypes of representative strains will be determined by pulsed field gel electrophoresis and Southern hybridization with chromosome-specific probes. Individual chromosomes will then be mapped by cloning large chromosomal fragments in yeast artificial chromosomes and aligning these clones with chromosomal restriction fragments generated by rare-cutting enzymes. These maps will be used to compare clinical isolates and to locate mutations affecting virulence factors. Genes specific to each variety or serotype mill be isolated by differential hybridization screening of cDNA subtraction libraries. These genes will be used to compare C. neoformans isolates from AIDS patients with other clinical isolates. Genes required for synthesis of the polysaccharide capsule (a major virulence determinant) will be cloned by complementation of a capsular mutants and sib selection using a genomic library. Genes required for growth in physiological concentrations of CO2 will be identified by differential hybridization screening and by isolation of CO2-sensitive mutants.

封装的酵母菌Neoformans是 艾滋病患者和其他艾滋病患者的机会性感染威胁生命 免疫功能低下的患者。 关于的遗传结构知之甚少 这种病原体。 该项目的长期目标是发展 C Neoformans遗传图的方法；确定遗传基础 对于两种c neoformans的差异（var。Neoformans） 和var。 gatti）;确定C Neoformans是否是从AIDS患者中分离出来的 与其他菌株不同；和影响胶囊的基因 合成和其他毒力因子。 这些研究将有助于 更好地了解隐球菌感染的发病机理和 开发新的治疗剂。 该项目的实验设计如下。 电泳 代表性菌株的核型将由脉冲场凝胶确定 电泳和南部与染色体特异性探针的杂交。 然后，将通过克隆大染色体来映射单个染色体 酵母人工染色体中的碎片，并将这些克隆对齐 稀有切割酶产生的染色体限制片段。 这些 地图将用于比较临床分离株并定位突变 影响毒力因素。 特定于每种品种或血清型的基因 通过cDNA的差异杂交筛选来隔离磨坊 减法库。 这些基因将用于比较梭菌C. neoformans 来自其他临床分离株的艾滋病患者的分离株。 需要基因 用于合成多糖胶囊（主要的毒力决定因素） 将通过囊膜突变体和SIB选择的互补来克隆 使用基因组库。 生理生长所需的基因 二氧化碳的浓度将通过差异杂交确定 筛选并通过分离二氧化碳敏感突变体。