The Roles of Conserved outer-membrane proteins in SFG rickettsia pathogenesis

描述（由申请方提供）：康氏立克次体是斑点热组（SFG）立克次体的成员，是一种专性细胞内蜱传播病原体，是地中海斑点热的病原体。立克次体的气溶胶传播可能发生，因此对美国构成生物恐怖主义威胁。对该菌的致病机理进行了研究。在传播到宿主中时，病原体对宿主细胞的影响取决于病原体结合和侵入靶宿主细胞的能力。几个测序的立克次体基因组的注释已经确定了一个大家族的基因称为表面细胞抗原（SCA），类似于革兰氏阴性菌中的自转运蛋白和毒力因子。虽然该家族中的许多基因是简并的或分裂的，但至少有4个基因，sca 0（rompA），sca 1，sca 2和sca 5（rompB）在R中以完整的开放阅读框架存在。Conorii和许多其他SFG立克次体。以前的结果已经证明，Sca 0（rOmpA）和Sca 5（rOmpB）的粘附和随后的入侵的非吞噬性哺乳动物细胞和保护性体液免疫反应的产生是重要的。然而，很少有人知道其他外膜蛋白在SFG立克次体发病机制的功能。我们已经确定了第一个立克次体蛋白-宿主细胞受体对（rOmpB-Ku 70），它在靶人类细胞内成功感染的建立中起着重要作用。有趣的是，我们的研究结果还表明，虽然rOmpB-Ku 70相互作用在入侵过程中很重要，但其他保守的SFG立克次体蛋白，即rOmpA，Sca 1和Sca 2，可能在发病过程中发挥重要作用。本提案中概述的实验将涉及以下研究兴趣：利用重组大肠杆菌分析rOmpB和Ku 70对定殖的贡献。大肠杆菌，纯化的蛋白质和蛋白质偶联的乳胶珠。还将使用细胞培养模型分析摄取过程中涉及的宿主信号传导途径的诱导。二.我们将使用野生型和Ku 70基因敲除小鼠在鼠感染模型中确定rOmpB和Ku 70对SFG立克次体病的起始和进展的贡献。三.我们还将确定保守的表面抗原，即rOmpA，Sca 1和Sca 2介导的非吞噬性哺乳动物细胞的入侵的作用。这些蛋白质在产生保护性体液免疫应答中的作用将在感染的鼠模型中进行检查。此外，我们将确定与这些保守的表面蛋白相互作用的新的配体，希望确定新的受体在SFG立克次体的发病机制中发挥作用。

DESCRIPTION (provided by applicant): Rickettsia conorii, a member of the spotted fever group (SFG) rickettsia is an obligate intracellular tick borne pathogen and is the causative agent of Mediterranean spotted fever. Aerosol transmission of rickettsia can occur and therefore, represents a bioterrorist threat for the United States. The pathogenesis of R. conorii upon transmission into the host depends on the pathogen's ability to bind to and invade target host cells. Annotation of several sequenced rickettsial genomes has identified a large family of genes termed surface cell antigens (sca) that resemble autotransporter proteins and virulence factors in Gram-negative bacteria. While many genes in this family are degenerate or split, at least 4 genes, sca0 (rompA), sca1, sca2, and sca5 (rompB) are present as complete open reading frames in R. conorii and many other SFG rickettsia. Previous results have demonstrated that Sca0 (rOmpA) and Sca5 (rOmpB) are important for the adhesion to and subsequent invasion of non-phagocytic mammalian cells and in the generation of protective humoral immune responses. However, very little is known about the function of other outermembrane proteins in SFG rickettsia pathogenesis. We have identified the first rickettsial protein-host cell receptor pair (rOmpB-Ku70) that plays an important role in the establishment of a successful infection within target human cells. Interestingly, our results have also suggested that while rOmpB-Ku70 interactions are important during the invasion process, other conserved SFG rickettsia proteins, namely rOmpA, Sca1 and Sca2, likely play important roles during pathogenesis. The experiments outlined in this proposal will address the following research interests: i. The contributions of rOmpB and Ku70 to colonization will be analyzed by using recombinant E. coli, purified proteins and protein coupled latex beads. The induction of host signaling pathways involved in the uptake process will also be analyzed using a cell culture model. ii. We will determine the contribution of rOmpB and Ku70 to the initiation and progression of SFG rickettsial disease in a murine model of infection using wild-type and Ku70 knockout mice. iii. We will also determine the roles of conserved surface antigens, namely rOmpA, Sca1 and Sca2 in mediating invasion of non-phagocytic mammalian cells. The roles of these proteins in generating protective humoral immune responses will be examined in a murine model of infection. In addition, we will identify novel ligands that interact with these conserved surface proteins in the hopes of identifying new receptors that play roles in the pathogenesis of SFG rickettsia.