C.elegans as a model system forC.neoformans pathogenesis

描述（由申请人提供）：本研究职业奖申请描述了候选人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，并提出了一项研究计划，以研究新型隐球菌的毒力。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的生物体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，长期以来一直对C. neoformans，并开发了一些相关的分子生物学协议。 该候选人开发了一种新的系统，用于研究C。使用线虫模式生物秀丽隐杆线虫（Caushabditis elegans）研究新型线虫的致病机理。他发现C。neoformans杀死C. elegans，和一些基因，如那些与信号转导途径，漆酶的生产和α交配型，以前被证明是参与哺乳动物的毒力，也发挥作用，在C。优雅的杀戮他使用这个系统来筛选C中开发的随机插入突变体库。neoformans H99背景。在350株突变株中，有7株为弱毒。elegans，在突变与干净的遗传背景杂交后，低毒力持续存在。对第一个突变体的遗传分析表明，突变发生在与酿酒酵母KIN 1同源的基因中。具体目标如下：1）开发和使用C。elegans系统筛选随机C.新形式的插入突变体，然后鉴定被破坏的基因，并利用致病性野生型菌株在每个鉴定的基因中产生确定的缺失突变，并开发相应的重建菌株，2）使用尾静脉和肺吸入小鼠模型来测试通过在C.线虫参与哺乳动物的毒力，3）对有限数量的选择突变体进行深入分析，这些突变体在两种线虫中表现出最显著的低毒力。线虫和老鼠分析将包括评价包膜、黑色素生成和交配。提出了利用C. elegans作为一个简单的模型来研究基本的，进化上保守的途径与隐球菌感染。因为C.由于neoformanshas与其他致病性酵母的相似性，我们希望我们的发现将促进真菌/宿主相互作用的研究。

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.