Intracellular Proliferation of the fungal pathogen Histoplasma capsulatum

项目总结 摘要组织胞浆菌是一种感染免疫缺陷和免疫活性的真菌病原体。 个人。仅靠先天免疫系统无法有效控制组织胞浆酵母，因为 组织胞浆侵入并在宿主吞噬细胞内生长。巨噬细胞的组织胞浆寄生 需要从宿主细胞获取和代谢适当的营养资源，但宿主分子 作为营养物质的物质在很大程度上是未知的。初步研究表明，胞内酵母是 糖异生，可能会消耗氨基酸，特别是与谷氨酸有关的化合物。这些 这些发现为确定组织胞浆酵母所消耗的宿主分子提供了关键的立足点。 这项建议采取了多学科的方法，整合了来自分子生物学、遗传学和 代谢组学以确定吞噬小体内可供组织胞浆酵母菌利用的宿主分子及其作用方式 它们被代谢以满足宿主细胞内的碳和能量需求。细胞内生长 对谷氨酸脱氢酶活性的要求将代谢碳的潜在宿主分子缩小到 谷氨酸相关的氨基酸或产生谷氨酸的宿主分子。转录和代谢物 分析将与功能测试相结合，以确定组织胞浆所需的代谢途径 宿主巨噬细胞内的增殖。将使用宿主代谢物的同位素标记来跟踪它们的进口 并随后随着时间的推移掺入组织胞浆代谢中间体，以证明 从巨噬细胞到胞内酵母的碳，并确定宿主底物进入酵母中心的点 碳代谢。这些发现将通过组织胞浆体的氨基酸特征进行遗传测试 多肽转运体和基于RNAi的干扰候选宿主代谢物进入细胞内 组织胞浆酵母细胞。这些数据将提供多条证据来确定组织胞浆体是如何 在宿主感染期间，酵母菌利用吞噬小体作为复制允许的细胞内生态位。 本提案是为响应资助机会公告(FOA)PA-19-083《NORITY 了解、预防、治疗和诊断球孢子菌病(谷热)和其他选择的方法 地方性真菌感染。该提案回应了公告的目标，即解决霍奇金淋巴瘤的发病机制 地方性真菌，最终目标是推动该领域朝着地方性疾病治疗的解决方案迈进 真菌病。在二态地方性真菌病原体中，组织胞浆菌是分子特征最好的 它拥有最先进的分子遗传学，便于进行机械研究的功能测试。 我们的研究结果将通过强调运输和代谢来揭示新的疾病治疗途径 巨噬细胞内滞留的组织胞浆对组织胞浆的狭窄代谢所必需的反应 吞噬小体。我们的基因测试表明，这些关键新陈代谢反应的损害会减少 组织胞浆在哺乳动物肺中的增殖，显示了治疗的潜力。

PROJECT SUMMARY Histoplasma capsulatum is a fungal pathogen that infects both immunocompromised and immunocompetent individuals. The innate immune system alone is ineffective in controlling Histoplasma yeasts because Histoplasma invades and thrives within host phagocytic cells. Histoplasma’s parasitism of macrophages requires acquisition and metabolism of suitable nutritional resources from the host cell, yet the host molecules that serve as nutrients for are largely unknown. Preliminary investigations have shown intracellular yeasts are gluconeogenic and likely consume amino acids, particularly compounds that are related to glutamate. These findings provide a critical foothold for identifying the host molecules consumed by Histoplasma yeasts. This proposal takes a multidisciplinary approach, integrating evidence from molecular biology, genetics, and metabolomics to determine the host molecules available to Histoplasma yeasts within the phagosome and how they are metabolized to meet the carbon and energy needs within host cells. The intracellular growth requirement for glutamate dehydrogenase activity narrows the potential host molecules for metabolic carbon to glutamate-related amino acids or glutamate-generating host molecules. Transcriptional and metabolite profiling will be combined with functional tests to define the metabolic pathways necessary for Histoplasma proliferation within host macrophages. Isotopic labeling of host metabolites will be used to follow their import and subsequent incorporation into Histoplasma metabolism intermediates over time to demonstrate the flux of carbon from macrophage to intracellular yeasts and identify host substrate entry points into yeast central carbon metabolism. These findings will be genetically tested by characterization of Histoplasma’s amino acid and peptide transporters and RNAi-based interference with candidate host metabolite import into intracellular Histoplasma yeast cells. Together these data will provide multiple lines of evidence to define how Histoplasma yeasts exploit the phagosome as a replication-permissive intracellular niche during host infection. This proposal is submitted in response to Funding Opportunity Announcement (FOA) PA-19-083 “Novel approaches to understand, prevent, treat, and diagnose coccidioidomycosis (Valley Fever) and other select endemic fungal infections.” The proposal answers the announcement’s goal to address the pathogenesis of endemic fungi with the ultimate goal of advancing the field towards solutions for treatment of endemic mycoses. Of the dimorphic endemic fungal pathogens, Histoplasma is the best molecularly characterized fungus and it has the most advanced molecular genetics to facilitate functional testing for mechanistic studies. The results of our studies will reveal new disease treatment avenues by highlighting transport and metabolic reactions essential for the narrow metabolism imposed on Histoplasma by its residence within macrophage phagosomes. Our genetic tests have shown that impairment of these critical metabolic reactions reduces Histoplasma proliferation in the mammalian lung, demonstrating the therapeutic potential.