Molecular Basis of Francisella Virulence and Immunity

描述（申请人提供）：土拉菌弗朗西斯菌是土拉菌病的病原。这种革兰氏阴性细菌是一种潜在的生物恐怖主义病原体，因为它死亡率高，感染剂量极低，能够在动物宿主外长时间存活，并且有多种传播机制，包括吸入、摄入、皮肤接触、粘膜接触和昆虫媒介。目前对土拉菌的发病机制和宿主耐药性知之甚少。土拉菌可在巨噬细胞内存活，但被这些菌感染的巨噬细胞样细胞在接种后24小时内发生凋亡。本研究的第一个目的是研究土拉菌引起巨噬细胞中宿主细胞死亡的机制。无法诱导巨噬细胞凋亡的土拉菌转座子插入突变体将被分离出来，这些突变将被定义为促进宿主细胞死亡的细菌产物。这些突变株将在兔热病小鼠模型中进行潜在衰减测试。采用具有促凋亡和抗凋亡基因序列的小鼠基因芯片确定土拉菌诱导细胞凋亡的机制，比较对照组和土拉菌感染细胞的巨噬细胞mRNA水平。第二个目的是分离Francisella特异性CD4 T细胞杂种，并确定感染活菌或热杀菌的C57Bl/6小鼠识别的表位的差异。表达第一个目标中鉴定的凋亡诱导基因的大肠杆菌克隆将被创建并用于分离特异性CD4 T细胞杂交。最终目标将是创建特定的细胞因子分泌（Th1或Th2）的T细胞克隆（通过使用特定的肽表位），并确定克隆在发病机制和保护中的作用。这些目标旨在研究土拉菌病发病机制的分子基础，并结合一种阐明潜在免疫反应靶点的方法，从而改善对土拉菌病的预防。

DESCRIPTION (provided by applicant): Francisella tularensis is the etiologic agent of tularemia. This gram negative bacterium is a potential agent of bioterrorism because of its high mortality, extremely low infective dose, ability to survive for long periods of time outside of an animal host, and numerous transmission mechanisms including inhalation, ingestion, skin contact, mucous membrane contact, and insect vectors. Little is known about the mechanisms of F. tularensis pathogenesis and host resistance. F. tularensis will survive within macrophages, however macrophage-like cells infected with these organisms become apoptotic within 24 hours following inoculation. The first aim of this proposal is to examine the mechanism(s) by which F. tularensis provoke host cell death in macrophages. F. tularensis transposon insertion mutants that fail to induce macrophage apoptosis will be isolated and the mutations characterized to define bacterial products responsible for promoting host cell death. These mutant strains will be tested for potential attenuation in a mouse model of tularemia. The mechanism of apoptosis induction by F. tularensis will be determined using mouse gene chips possessing known pro- and anti- apoptotic gene sequences to compare macrophage mRNA levels from control and F. tularensis infected cells. The second aim is to isolate Francisella specific CD4 T cell hybrids and determine the differences in the epitopes recognized in C57Bl/6 mice infected with live or heat killed bacteria. E. coli clones expressing the apoptosis inducing genes identified in the first aim will be created and used to isolate specific CD4 T cell hybrids. The ultimate aim will be to create T cell clones of defined cytokine secretion (Th1 or Th2) (by using the defined peptide epitopes) and determine the effect of the clones on pathogenesis and protection. These aims are directed towards examining the molecular basis for F. tularensis pathogenesis, combined with an approach to elucidate potential immune response targets that could lead to improved prophylaxis against tularemia.