Agents of Bioterrorism: Pathogenesis and Host Defense

肺鼠疫对人类是高度致命的，这种疾病的病原体，鼠疫杆菌，被认为是作为生物武器的主要威胁。目前预防肺炎鼠疫的策略，如使用疫苗或抗生素，都是无效的，或者可以通过对病原体的遗传操作来绕过。需要更好地了解鼠疫杆菌的致病机制，以指导新对策的开发。鼠疫杆菌是一种兼性胞内病原体，可在巨噬细胞内存活和复制。鼠疫杆菌在巨噬细胞中的存活在发病机制中起着重要作用，很可能是在感染的早期阶段。虽然关于鼠疫杆菌在巨噬细胞中存活的机制仍有许多有待了解的地方，但我们最近发现了几个与细胞内生存有关的细菌基因，确定了鼠疫杆菌在巨噬细胞中存在一个新的吞噬小体，称为含有耶尔森氏菌的空泡(YCV)，并显示了鼠疫杆菌在激活的巨噬细胞中生存的能力。YCV获得晚期内噬小体和自噬小体的标记，但不能酸化到吞噬小体的正常水平。我们假设1)鼠疫杆菌可能通过直接使空泡ATPase(VATPase)失活来有效地防止YCV的酸化；以及2)阻断酸化对于YCV的形成和巨噬细胞中鼠疫杆菌的生存是必不可少的。为了进一步阐明鼠疫杆菌在巨噬细胞中的存活机制，提出了以下目标。1)基因方法将被用来识别鼠疫杆菌细胞内增殖(RIP)和阻止巨噬细胞YCV酸化所需的基因。将使用分子、生化和免疫学技术研究由RIP基因编码的蛋白质，以揭示YCV形成的潜在细菌机制。2)在感染的巨噬细胞或纯化的YCV上使用免疫或化学探针研究YCV形成的细胞基础，以更好地描述YCV的环境，并确定vATPase的活性是如何调节的。宿主蛋白在YCV形成中的作用将通过利用基因缺陷的巨噬细胞来探索。

Pneumonic plague is highly lethal in humans, and the agent of this disease, Y. pestis, is considered a major threat as a biological weapon. Current strategies to prevent pneumonic plague, such as the use of vaccines or antibiotics, are non-effective or could be bypassed by genetic manipulation of the pathogen. A better understanding of Y. pestis pathogenesis is needed to guide the development of novel countermeasures. Y. pestis is a facultative intracellular pathogen that can survive and replicate in macrophages. Survival of Y. pestis in macrophages plays an important role in pathogenesis, most likely during the early stages of infection. Although much remains to be learned about the mechanism of Y. pestis survival in macrophages, we recently identified several bacterial genes important for intracellular survival, have determined that Y. pestis inhabits a novel phagosomal compartment in macrophages, called the Yersinia-containing vacuole (YCV), and shown the ability of Y. pestis to survive in activated macrophages. The YCV acquires markers of late endosomes and autophagosomes, but fails to acidify to the normal levels of a phagolysosome. We hypothesize 1) that Y. pestis actively prevents acidification of the YCV, possibly by direct inactivation of the vacuolar ATPase (vATPase); and 2) that blocking acidification is essential for YCV formation and survival of Y. pestis in macrophages. To further elucidate the mechanism of Y. pestis survival in macrophages, the following aims are proposed. 1) Genetic approaches will be used to identify Y. pestis genes that are required for intracellular proliferation (rip) and for blocking YCV acidification in macrophages. The proteins encoded by rip genes will be studied using molecular, biochemical and immunological techniques to reveal the underlying bacterial mechanism for YCV formation. 2) The cellular basis for YCV formation will be investigated using immunological or chemical probes on infected macrophages or purified YCVs, to better characterize the environment of YCV, and to determine how the activity of the vATPase is modulated. The contribution of host proteins to YCV formation will be explored by the use of gene deficient macrophages.