Intracellular Biology of Francisella tularensis

土拉热弗朗西丝菌是一种兼性胞内细菌病原体， 通过在宿主细胞（主要是巨噬细胞）中存活和繁殖来威胁疾病。因为 细菌具有极高的传染性，导致严重的发病率和死亡率， 由于分散，它也被认为是生物恐怖主义的潜在制剂。虽然目前可用的抗生素 有效治疗兔热病，F.土拉热菌可以被改造为携带抗生素抗性基因。为这些 因此，需要新的方法来治疗和预防土拉菌病。然而，设计这样的 策略需要更好地理解F.土拉菌及其宿主细胞。我们 已经证明完全毒力的F.土拉热菌通过宽敞的伪足环进入巨噬细胞， 然后细菌进入一个吞噬体区室， 有限数量的溶酶体相关膜糖蛋白，不获得组织蛋白酶D，并且仅 在细菌逃逸到巨噬细胞细胞质中之前最低限度地酸化。我们建议定义 进一步研究了F.土拉菌，并确定毒力机制，使其能够 逃避吞噬体-溶酶体融合、吞噬体酸化，并逃逸到宿主细胞质中。 我们将鉴定出对抵抗吞噬体-溶酶体融合很重要的宿主和细菌蛋白质 通过基于质谱的纯化吞噬体的蛋白质组学比较和吞噬体逃逸 含有活的野生型F。tularensis，杀死F. tularensis和选择的F.土拉菌突变体不能 逃逸和交通在宿主细胞内是不同。我们将鉴定出细菌生长所需的基因， 巨噬细胞是改变细胞内运输和吞噬体逃逸所需的，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质 通过这些方法需要通过循环吞噬作用摄取细菌， 融合，或吞噬体逃逸，通过使用siRNA技术和通过检查表型， 通过免疫荧光显微镜和透射电子显微镜观察靶向细菌突变体。我们 确定F。在吞噬体-溶酶体融合中有缺陷的土拉菌突变体，或 吞噬体逃逸可以通过递送对应于吞噬体逃逸的蛋白质而恢复到野生型表型。 将突变基因导入宿主细胞质或F.土拉菌吞噬体这将提供信息 关于这些细菌蛋白的可能作用位点。

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.