C.elegans as a model system forC.neoformans pathogenesis

描述(由申请者提供)：本研究职业生涯奖申请书描述了候选人Mylonakis博士在真菌发病机制方面的指导研究计划。候选人接受过传染病方面的培训，并提出了一个研究新生隐球菌毒力的研究计划。候选人的赞助人卡尔德伍德博士长期以来一直对使用简单生物作为人类病原体研究的模型宿主感兴趣，而候选人的共同赞助人海特曼博士对新生葡萄球菌的遗传学有着长期的兴趣，并开发了许多相关的分子生物学方案。 这位候选人利用线虫模式生物秀丽线虫开发了一种新的系统，用于研究新生隐孢子虫发病的遗传和分子机制。他发现，新生弧菌可以杀死线虫，一些基因，如与信号转导途径、漆酶产生和阿尔法交配型相关的基因，以前被证明与哺乳动物的毒力有关，也在线虫的死亡中发挥作用。他使用这个系统筛选了一组随机插入突变体，这些突变体是在新生杆菌H99背景下开发的。在测试的350个突变体中，有7个在线虫中被鉴定为弱毒力，在突变进入干净的遗传背景后，低毒力仍然存在。对第一个突变体的遗传分析表明，该突变发生在与酿酒酵母KIN1同源的基因中。其具体目标如下：1)开发和使用线虫系统来筛选随机的新生线虫插入突变体文库，然后鉴定中断的基因，并利用致病的野生型菌株在每个已鉴定的基因中进行明确的缺失突变并开发相应的重组菌株，2)使用尾静脉和肺吸入小鼠模型来测试通过筛选在线虫中发现的基因是否与哺乳动物的毒力有关，3)对选定的数量有限的突变体进行深入分析，这些突变体在线虫和小鼠中都表现出最显著的低毒力。分析将包括对胶囊、黑色素产生和交配的评估。该研究计划提出的重点是使用线虫作为一种简单的模型来研究与隐球菌感染相关的基本的、进化上保守的途径。由于新生芽孢杆菌与其他致病酵母菌有相似之处，我们希望我们的发现将有助于从总体上研究真菌/宿主的相互作用。

DESCRIPTION (provided by applicant): This Research Career Award application describes a mentored research program for the candidate, Dr. Mylonakis, in fungal pathogenesis. The candidate is trained in Infectious Diseases and proposes a research program to study virulence of Cryptococcus neoformans. The candidate's sponsor, Dr. Calderwood, has a Iongtime interest in using simple organisms as model hosts for the study of human pathogens, while the candidate's cosponsor, Dr. Heitman, has a Iongtime interest in the genetics of C. neoformans and has developed a number of related molecular biology protocols. The candidate has developed a novel system for studying genetic and molecular mechanisms of C. neoformans pathogenesis using the nematode model organism Caenorhabditis elegans. He has found that C. neoformans kills C. elegans, and several genes, such as those associated with signal transduction pathways, laccase production and the alpha mating type, previously shown to be involved in mammalian virulence, also play a role in C. elegans killing. He used this system to screen a bank of randominsertional mutants that were developed in the C. neoformans H99 background. From 350 mutants tested, 7 were identified as attenuated in C. elegans, with the hypovirulence persisting after the mutation was crossed to a clean genetic background. Genetic analysis of the first mutant revealed that the mutation occurred in a gene homologous to KIN1 of Saccharomyces cerevisiae. The SPECIFIC AIMS are as follows: 1) to develop and use the C. elegans system to screen a library of random C. neoformans insertional mutants and then to identify the disrupted genes and utilize a pathogenic wild type strain to make defined deletion mutations in each of the genes identified and develop the corresponding reconstituted strains, 2) to use the tail vein and lung inhalation murine models to test if the genes identified through the screen in C. elegans are involved in mammalian virulence, 3) to perform in-depth analysis for a limited number of selected mutants that demonstrate the most dramatic hypovirulence in both C. elegans and mice. The analysis will include evaluation of capsule, melanin production and mating. The research plan proposed with focus on the use of C. elegans as a facile model to study basic, evolutionarily conserved pathways associated with cryptococcal infection. Because C. neoformanshas similarities with other pathogenic yeasts, we expect that our findings will facilitate the study of fungus/host interaction in general.