CNLAC 1 AND THE VIRULENCE OF CRYPTOCOCCUS

描述（改编自申请人的摘要）：C。新形式群岛 一种主要的艾滋病机会性感染 黑色素的产生是独一无二的 致病性的隐球菌属， 在三项单独的研究中比较了Mel+和 Mel-在小鼠中的这种生物体的菌株。 然而， 黑色素的形成和毒力还没有建立使用真菌 用分子生物学方法产生或表征的菌株。 调查人员已经做了初步工作， 导致酶二酚氧化酶的纯化 负责黑色素的形成和克隆 结构基因、CNLAC 1、CNLAC 1缺失突变体。新形式的人 已经产生了Mel-突变体，并且Mel-突变体已经补充到Mel+， CNLAC 1.本提案旨在检验以下假设： CNLAC 1是负责黑色素与 在动物模型中的合成和毒力。第一个目标是 构建并鉴定了C. neoformans和Mel- C的CNLAC 1互补突变体。 新生 株 因此，这些实验将提供两组Mel+和Mel-。 突变体将是相似的，除了选择性差异， 产生CNLAC 1转录本的能力。第二个目标是 构建C. neoformans菌株。 稳定的交配类型和以前产生的同类套 C. 新型菌株使研究人员能够减少 在突变体对和它们的对照之间的不相关的突变， 在转化后回交掉未识别的突变。 在 此外，第三组Mel+和Mel-突变体将通过 将JEC 82（Mel-）与其同系Mel+亲本回交。 第三 目的将是评估CNLAC 1基因对 C.毒力新生儿使用肠道内愈合的小鼠模型。 小鼠 将被跟踪以确定隐球菌感染的死亡时间 脑膜脑炎，并通过定期器官培养评估， 确定每三种试验菌株之间的毒力差异， 它的控制。

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.