Molecular Basis of Francisella Virulence and Immunity

描述(申请人提供)：图拉氏方济氏菌是图拉热症的病原体。这种革兰氏阴性菌是潜在的生物恐怖主义病原体，因为它的死亡率高，感染量极低，能够在动物宿主外长时间存活，以及多种传播机制，包括吸入、摄取、皮肤接触、粘膜接触和昆虫媒介。关于图拉氏丝核菌的致病机制和寄主抗性，目前还知之甚少。图拉氏丝虫可以在巨噬细胞内存活，但感染了这些微生物的巨噬细胞样细胞在接种后24小时内就会发生凋亡。这项建议的第一个目的是研究图拉氏F菌引起巨噬细胞宿主细胞死亡的机制(S)。将分离出未能诱导巨噬细胞凋亡的图拉氏杆菌转座子插入突变体，并对突变进行定性，以确定负责促进宿主细胞死亡的细菌产物。这些突变菌株将在图拉热症的小鼠模型中进行潜在的衰减测试。利用具有已知的促和抗凋亡基因序列的小鼠基因芯片，比较对照细胞和感染图拉氏杆菌的细胞中巨噬细胞的mRNA水平，将确定图拉氏杆菌诱导细胞凋亡的机制。第二个目的是分离弗朗西斯杆菌特异性的CD4T细胞杂交种，并确定感染活细菌或热致死细菌的C57BL/6小鼠识别的表位的差异。表达第一个目的中确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定的CD4T细胞杂交体。最终目标将是创建具有特定细胞因子分泌(Th1或Th2)的T细胞克隆(通过使用已定义的多肽表位)，并确定这些克隆在发病机制和保护方面的作用。这些目的是为了研究图拉氏F菌致病的分子基础，并结合一种方法来阐明潜在的免疫反应靶点，从而改善对图拉热症的预防。

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.