CNLAC 1 AND THE VIRULENCE OF CRYPTOCOCCUS

描述(改编自申请人的摘要)：新生链球菌属 艾滋病的一种主要机会性感染。黑色素的产生是独一无二的 与隐球菌的致病物种有关，并与 比较MEL和Mel致死率的三项单独研究中的毒力 梅尔--这种微生物在小鼠体内的菌株。然而，两者之间的联系 黑色素的形成和毒性还没有用真菌来确定 用分子生物学方法产生的菌株或以其为特征的菌株。 调查人员已经完成了初步工作，已经 结果提纯了二酚氧化酶 负责黑色素的形成和各自的克隆 新型弧菌结构基因CNLAC 1、CNLAC 1缺失突变株有 产生了一个MEL突变体，并与MEL进行了补充 CNLAC 1.本提案试图检验以下假设 CNLAC 1是负责黑色素之间联系的基因 在动物模型中的合成和毒力。第一个目标将是 构建并鉴定了C。 新生革兰氏菌及其与CNLAC 1互补突变株的研究 紧张。这些实验因此将提供两套MEL和MEL- 将是相似的突变体，除了在 能够制作CNLAC 1成绩单。第二个目标将是 构建等基因CNLAC-1和Cnlac-1新生链球菌菌株。 稳定的交配类型和以前的同源集合的产生 新生芽孢杆菌的菌株使研究人员减少了这种机会 一对突变体及其对照之间的无关突变 在转化后回交出未知的突变。在……里面 此外，第三组MEL和MEL突变体将由 将JEC 82(Mel-)回交到其同源Mel亲本。第三 目的将评估CNLAC 1基因对 用气管内小鼠模型研究新生葡萄球菌的毒力。老鼠 将被用来确定隐球菌的死亡时间 脑膜脑炎和定期器官培养评估 确定三个测试菌株之间的毒力差异和 它的控制力。

DESCRIPTION (Adapted from the applicant's abstract): C. neoformans is a major opportunistic infection in AIDS. Melanin production is unique to pathogenic species of Cyptococcus and has been associated with virulence in three separate studies comparing lethality of Mel+ and Mel- strains of this organism in mice. However, the association between melanin formation and virulence has not been established using fungal strains produced or characterized by molecular biological methods. Preliminary work has been done by the investigator which has resulted in the purification of the enzyme diphenol oxidase responsible for melanin formation and cloning of the respective structural gene, CNLAC 1, CNLAC 1 deleted mutants of C. neoformans have been produced and a Mel- mutant has been complemented to Mel+ with CNLAC 1. The present proposal seeks to test the hypothesis that CNLAC 1 is the gene responsible for the association between melanin synthesis and virulence in an animal model. The first aim will be to construct and fully characterize a gene deletant of CNLAC 1 in C. neoformans and a CNLAC 1 complemented mutant of Mel- C. neoformans strain. These experiments will thus provide two sets of Mel+ and Mel- mutants that will be similar except for a selective difference in the ability to produce CNLAC 1 transcripts. The second aim will be to construct isogenic CNLAC 1 and cnlac 1 sets of C. neoformans strains. Stable mating types and the previous production of congenic sets of C. neoformans strains allow the investigator to decrease the chance of irrelevant mutations between pairs of mutants and their controls by backcrossing out unidentified mutations after transformations. In addition, a third set of Mel+ and Mel- mutants will be produced by backcrossing JEC 82 (Mel-) to its congenic Mel+ parents. The third aim will be to assess the contribution of the CNLAC 1 gene to the virulence of C. neoformans using an intratracheal mouse model. Mice will be followed to determine time of death from Cryptococcal meningoencephalitis and assessed by periodic organ cultures to determine differences in virulence between each three test strains and its control.