Virulence factor discovery in the secreted proteome of Histoplasma capsulatum

描述(申请人提供)：真菌疾病已经成为现代医学的主要威胁生命的并发症，以及在免疫受损和免疫能力正常的个人中的呼吸系统疾病。尤其是二相性真菌，即使在其他健康的人中也会导致疾病。尽管对人类健康有重大影响，但令人惊讶的是，几乎没有发现能够促进医学上重要真菌毒力的真菌基因产品。二相性真菌病原体，组织胞浆的酵母细胞入侵，在内部复制，并最终杀死宿主巨噬细胞。由于重大的技术限制，阻碍了在分子水平上理解组织胞浆致病机制的进展，组织胞浆是如何成功完成这些任务的尚不清楚。我们最近开发了强大的分子工具，能够抑制组织胞浆体的基因功能，从而提供了现在从功能上定义基因在毒力中的作用的手段。为了解决我们在理解上的不足，这项研究计划将在致病组织胞浆酵母分泌的因素中识别和定义更多的真菌毒力决定因素。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素。利用最近完成的基因组序列信息，将通过对组织胞浆酵母分泌的蛋白质进行基于蛋白质组学的分析来识别胞外因子。然后，编码这些因子的基因将成为通过RNA干扰或基因缺失方法来消除其产生的基因沉默的目标。这种创造缺乏单个蛋白质的组织胞浆菌株的能力使功能测试得以进行。菌株将被用来感染培养中的人和小鼠巨噬细胞，以评估细胞内的寄生性，并将被用于呼吸道组织胞浆菌病的动物感染模型。这些实验旨在评估个体因素在组织胞浆致病中的作用。与公共卫生相关：我们对致病真菌感染和导致人类宿主疾病的机制的了解有限，这损害了我们治疗威胁生命的真菌疾病的选择。这项提议将识别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。提高对病原真菌毒力机制的了解，特别是识别使它们能够致病的因素，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.