Intracellular Biology of Francisella tularensis

图拉氏方济氏菌是一种兼性的细胞内细菌病原体，可导致严重和潜在的生命 通过在宿主细胞，主要是巨噬细胞内存活和繁殖来威胁疾病。因为 细菌具有极高的传染性，会导致严重的发病率和死亡率，而且很容易 由于分散，它也被认为是潜在的生物恐怖主义因素。虽然目前可用的抗生素是 在治疗图拉热症方面，图拉氏F菌可以被改造成携带抗生素耐药性基因。为了这些 因此，需要有新的治疗和预防图拉热症的方法。然而，设计出这样的 应对策略需要更好地理解图拉氏丝孢子菌与其宿主细胞之间的相互作用。我们 已经证明了完全毒力的图拉氏杆菌通过宽敞的伪足环路进入巨噬细胞，并且 然后细菌进入吞噬小体隔间，该吞噬小体在获得 有限数量的溶酶体相关的膜糖蛋白，不获得组织蛋白酶D，并且只是 在细菌逃逸到巨噬细胞细胞质之前进行最低限度的酸化。我们建议定义 进一步研究图拉氏丝孢子菌的细胞内生物学，并鉴定其毒力机制 躲避吞噬小体-溶酶体融合、吞噬小体酸化，并逃逸进入宿主细胞胞浆。 我们将鉴定宿主和细菌蛋白，它们对抵抗吞噬小体-溶酶体融合很重要。 基于质谱学的纯化吞噬小体蛋白质组学比较 含有活的野生型图拉氏丝虫，杀死图拉氏丝虫，并选择未能 则在宿主小区内以不同方式逃逸和该业务。我们将确定生长所需的细菌基因。 改变细胞内转运和吞噬小体逃逸所需的巨噬细胞通过筛选 转座子突变体文库。我们将确定我们鉴定的特定宿主和细菌蛋白 通过这些方法需要通过环状吞噬来摄取细菌，抵抗吞噬小体 融合，或吞噬小体逃逸通过使用siRNA技术和通过检查表型 免疫荧光显微镜和透射电子显微镜定位细菌突变体。我们 应确定在吞噬小体-溶酶体融合方面有缺陷的图拉氏杆菌突变体或 吞噬小体逃逸可以通过传递相应的蛋白而恢复为野生型 突变基因进入宿主细胞胞质或进入图拉氏丝虫吞噬小体。这将提供信息 关于这些细菌蛋白可能的作用部位。

Francisella tularensis is a facultative intracellular bacterial pathogen that causes serious and potentially life threatening illness by surviving and multiplying within host cells, primarily macrophages. Because the bacterium has extraordinarily high infectivity, causes serious morbidity and mortality, and is easily dispersed, it is also considered a potential agent of bioterrorism. While currently available antibiotics are effective in treating tularemia, F. tularensis can be engineered to carry antibiotic resistance genes. For these reasons, new approaches to treatment and prevention of tularemia are needed. However, devising such strategies requires an improved understanding of the interaction between F. tularensis and its host cells. We have demonstrated that fully virulent F. tularensis enter macrophages via spacious pseudopod loops and that the bacterium then enters a phagosomal compartment that exhibits arrested maturation in that it acquires limited amounts of lysosome associated membrane glycoproteins, does not acquire cathepsin D, and is only minimally acidified prior to escape of the bacterium into the macrophage cytoplasm. We propose to define further the intracellular biology of F. tularensis and to identify the virulence mechanisms that allow it to evade phagosome-lysososome fusion, phagosome acidification, and to escape into the host cell cytoplasm. We shall identify host and bacterial proteins that are important to resistance to phagosome-lysosome fusion and phagosome escape by a mass spectrometry based proteomic comparison of purified phagosomes containing live wild type F. tularensis, killed F. tularensis, and selected F. tularensis mutants that fail to escape and that traffic differently within the host cell. We shall identify bacterial genes required for growth in macrophages that are required for altering the intracellular trafficking and phagosome escape by screening a transposon mutant library. We shall determine whether specific host and bacterial proteins that we identify by these methods are required for uptake of the bacteria by looping phagocytosis, resistance to phagosomelysosome fusion, or phagosome escape by using siRNA techniques and by examining the phenotype of targeted bacterial mutants by immunofluorescence microscopy and transmission electron microscopy. We shall determine whether F. tularensis mutants that are defective in phagosome-lysosome fusion or phagosome escape can be restored to wild-type phenotype by delivering the protein corresponding to the mutated gene into the host cell cytoplasm or into the F. tularensis phagosome. This will provide information regarding the possible site of action of these bacterial proteins.