Virulence factor discovery in the secreted proteome of Histoplasma capsulatum

描述（由申请人提供）：真菌疾病已成为现代医学中危及生命的主要并发症，以及免疫功能低下和免疫功能正常个体的呼吸和全身疾病。尤其是二态真菌，即使在健康的人群中也能引起疾病。尽管这对人类健康有重大影响，但令人惊讶的是，很少有真菌基因产物被确定为促进医学上重要真菌的毒力。二态真菌病原体，荚膜组织浆酵母细胞侵入，复制，并最终杀死宿主巨噬细胞。由于重大的技术限制，组织浆体如何成功完成这些任务尚不清楚，这阻碍了在分子水平上理解组织浆体发病机制的进展。我们最近开发了强大的分子工具，能够抑制组织浆体基因功能，从而提供了现在功能上定义基因在毒力中的作用的手段。为了解决我们理解中的这一缺陷，本研究计划将在致病性酵母组织原体分泌的因子中确定和定义其他真菌毒力决定因素。这些细胞外因子是影响宿主巨噬细胞和免疫防御的主要候选因子。利用最近完成的基因组序列信息，细胞外因子将通过基于蛋白质组学的酵母菌组织分泌的蛋白质分析来鉴定。然后，编码这些因子的基因将作为RNA干扰或基因删除方法沉默基因的目标，以消除它们的产生。这种制造缺乏单个蛋白质的组织浆菌株的能力允许进行功能测试。菌株将用于感染培养的人和小鼠巨噬细胞以评估细胞内寄生，并将用于呼吸道组织浆虫病的动物感染模型。这些实验旨在评估个体因素在组织浆体发病中的作用。公共卫生相关性：我们对病原真菌感染和引起人类宿主疾病的机制的了解有限，这损害了我们治疗危及生命的真菌疾病的选择。这项建议将鉴定真菌细胞释放的分子，这些分子允许病原体克服宿主的免疫防御。进一步了解病原真菌的毒力机制，特别是确定使它们致病的因素，将有助于合理设计更好的抗生素和治疗临床真菌感染的方案。

DESCRIPTION (provided by applicant): Fungal diseases have emerged as major life-threatening complications of modern medicine as well as respiratory and systemic disease among both immunocompromised and immunocompetent individuals. The dimorphic fungi in particular can cause disease even among otherwise healthy people. Despite this significant impact on human health, surprisingly few fungal gene products have been identified that promote the virulence of medically important fungi. Yeast cells of the dimorphic fungal pathogen, Histoplasma capsulatum, invade, replicate within, and ultimately kill host macrophages. How Histoplasma succeeds in these tasks is unknown due to significant technical limitations which have hindered progress in understanding Histoplasma pathogenesis at a molecular level. We recently developed powerful molecular tools enabling suppression of Histoplasma gene function thereby providing the means to now functionally define the role of genes in virulence. To address this deficiency in our understanding, this research proposal will identify and define additional fungal virulence determinants among the factors secreted by pathogenic Histoplasma yeast. These extracellular factors are prime candidates for affecting the host macrophages and immune defenses. Taking advantage of recently completed genomic sequence information, extracellular factors will be identified through proteomic-based analyses of proteins secreted by Histoplasma yeast. The genes encoding these factors will then serve as targets for gene silencing by RNA interference or gene deletion methodologies to eliminate their production. This ability to create strains of Histoplasma lacking individual proteins permits functional tests to be performed. Strains will be used to infect human and mouse macrophages in culture to assess intracellular parasitism and will be employed in animal infection models of respiratory histoplasmosis. These experiments are designed to assess the contribution of individual factors in Histoplasma pathogenesis. PUBLIC HEALTH RELEVANCE: Our understanding of the mechanisms by which pathogenic fungi infect and cause disease in human hosts is limited which impairs our options to treat life-threatening fungal disease. This proposal will identify molecules released by fungal cells which allow the pathogen to overcome host immune defenses. Improved understanding of the virulence mechanisms employed by pathogenic fungi, specifically the identification of factors that enable them to cause disease, will facilitate the rational design of better antibiotics and regimens to treat clinical fungal infections.