Molecular Basis of Francisella Virulence and Immunity

描述（由申请方提供）：土拉热弗朗西斯菌是土拉菌病的病原体。这种革兰氏阴性细菌是生物恐怖主义的潜在病原体，因为其死亡率高，感染剂量极低，能够在动物宿主外长时间存活，以及许多传播机制，包括吸入，摄入，皮肤接触，粘膜接触和昆虫媒介。关于F.土拉热病发病机制和宿主抗性。F.土拉热菌将在巨噬细胞内存活，然而，被这些生物体感染的巨噬细胞样细胞在接种后24小时内变得凋亡。本建议的第一个目的是研究F.土拉热引起巨噬细胞中宿主细胞死亡。 F.将分离不能诱导巨噬细胞凋亡的土拉热菌转座子插入突变体，并表征突变以确定负责促进宿主细胞死亡的细菌产物。将在兔热病小鼠模型中检测这些突变株的潜在减毒作用。探讨了F.将使用具有已知促细胞凋亡和抗细胞凋亡基因序列的小鼠基因芯片来比较对照和F的巨噬细胞mRNA水平来确定土拉菌病。土拉菌感染的细胞。第二个目的是分离弗朗西斯氏菌特异性CD 4 T细胞杂交体，并确定感染活菌或热灭活菌的C57 B1/6小鼠中识别的表位的差异。E.将产生表达在第一个目的中鉴定的凋亡诱导基因的大肠杆菌克隆，并用于分离特异性CD 4 T细胞杂交体。最终目的是产生确定的细胞因子分泌（Th 1或Th 2）的T细胞克隆（通过使用确定的肽表位），并确定克隆对发病机制和保护的作用。这些目标是针对检查F的分子基础。土拉菌病的发病机制，结合阐明潜在的免疫应答靶点的方法，可以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.