SE REGIONAL CENTER FOR EXCELLENCE FOR EMERGING INFECTIONS & BIODEFENSE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 土拉热弗朗西丝菌是一种高度传染性的A类细菌病原体，可引起土拉菌血症，这是一种可能危及人类生命的疾病。 由于这种微生物易于气溶胶传播和引起严重疾病所需的最小接种量（d10细菌），F。土拉热菌已被武器化，用于生物战。 弗朗西斯菌的发病机制的关键是其在巨噬细胞内复制和破坏宿主免疫反应的能力。 我们最近在小鼠中采用了一种强有力的整体体内阴性选择筛选来鉴定F. novicida，弗朗西斯菌属的一个亚种，在小鼠中引起疾病，但不在人类中引起疾病。 该方法鉴定了164个毒力所需的基因，其中44个似乎编码新的毒力因子。我们发现164个基因中的65个是体外巨噬细胞复制所必需的。 巨噬细胞中复制所必需的具有最强表型的基因之一编码一种功能未知的新型蛋白质。 我们着手确定该基因编码的蛋白质如何在分子水平上促进弗朗西斯菌的发病机制和逃避巨噬细胞防御。 构建和测试缺乏该蛋白的干净缺失突变体证实了该基因（我们称之为fpeA -弗朗西斯菌吞噬体逃逸A）是F.噬菌体在巨噬细胞中的复制以及体内毒力。 此外，fpeA突变株与野生型基因拷贝的互补恢复了在巨噬细胞内复制的能力。 电子显微镜显示，突变株是有缺陷的逃避巨噬细胞吞噬体，解释其衰减的复制表型。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Francisella tularensis is a highly infectious Category A bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Due to the ease of aerosol dissemination of this organism and the minimal inoculum (d10 bacteria) necessary to cause severe disease, F. tularensis has been weaponized for use in biowarfare. Critical to Francisella's pathogenesis are its ability to replicate within macrophages and to subvert the host immune response. We recently employed a powerful global in vivo negative selection screen in mice to identify genes required for the pathogenesis of F. novicida, a subspecies of Francisella that causes disease in mice but not humans. This approach resulted in the identification of 164 genes that are required for virulence, 44 of which appear to encode novel virulence factors. We found that 65 of the 164 genes are required for replication in macrophages in vitro. One of the genes with the strongest phenotypes that was essential for replication in macrophages encodes a novel protein of unknown function. We set out to determine how the protein encoded by this gene contributes to Francisella pathogenesis and the evasion of macrophage defenses at the molecular level. Construction and testing of a clean deletion mutant lacking the protein confirmed that this gene (which we refer to as fpeA - Francisella phagosome escape A) is required for F. novicida replication in macrophages as well as virulence in vivo. Furthermore, complementation of the fpeA mutant strain with a wild-type copy of the gene restored the ability to replicate within macrophages. Electron microscopy revealed that the mutant strain is defective for escape from the macrophage phagosome, explaining its attenuated replication phenotype.