Permeability of the Epidemic Typhus Rickettsia

描述（由申请人提供）：立克次体疾病，如流行性斑疹伤寒，一种人类的虱媒疾病，是一种世界性的祸害，需要对病原体、更好的诊断工具和疫苗有基本的了解。病原菌R.普氏立克次体是一种专性细胞内细菌寄生虫;立克次体仅直接在宿主和载体的细胞质中生长。立克次体是揭开专性胞内寄生生物学奥秘的奇妙生物，也是研究真核宿主细胞质的活体探针。R. prowazekii只有834个基因，因为许多代谢中间产物不需要合成，而是从宿主细胞的细胞质中运输-通常是通过不寻常的运输系统，而这些系统在自由生活的细菌中没有对应物。ATP/ADP转位酶（Tlc 1）是一种以ATP/ADP转位酶（Tlc 1）为底物的酶。prowazekii通过立克次体ADP与宿主细胞ATP的强制性交换来转运能量。我们现在有一个很好的二维拓扑结构的这种蛋白质和证据，跨膜结构域（TM）形成的水填充通道，ATP/ADP通过跨膜。我们目前的目标1是阐明其12个TM的结构，Tlc 1螺旋包装。在目标2中，Tlc 1的底物结合位点将通过DNA改组和嵌合蛋白的形成来确定。我们将通过对嗜核Caedibacter caryophilus和R. prowazekii。这些嵌合蛋白内的改组片段的序列分析将保守序列基序与底物特异性相关联。在目标3中，我们将确定和表征的运输系统的NAD，在R的主要代谢产物。目的4我们将研究R. prowazekii的磷脂酶样蛋白patatin和ExoU。prowazekii。我们已经证明立克次体与宿主细胞的相互作用导致磷脂酶活性的生化产物。这涉及立克次体生物学中最基本的问题之一：它的进入。假设激活和/或易位是酶活性和立克次体进入所必需的，我们将确定ExoU和马铃薯糖蛋白是否在此过程中发挥作用。

DESCRIPTION (provided by applicant): Rickettsial diseases, such as epidemic typhus, a louse-vectored disease of humans, are a world-wide scourge for which fundamental understanding of the etiologic agent, better diagnostic tools, and vaccines are needed. The etiologic agent, R. prowazekii, is an obligate intracellular bacterial parasite; rickettsiae only grow directly in the cytoplasm of hosts and vectors. Rickettsiae are marvelous organisms to unravel the biological mysteries of obligate intracellular parasitism and living probes for the investigation of the eukaryotic host cell cytoplasm. R. prowazekii has only 834 genes because many metabolic intermediates need not be synthesized, but are transported from the cytoplasm of the host cell - often by unusual transport systems that have no counterparts in free-living bacteria. The ATP/ADP translocase (Tlc1) of R. prowazekii transports energy via the obligatory exchange of rickettsial ADP for host cell ATP. We now have an excellent two-dimensional topology for this protein and evidence for which transmembrane domains (TMs) form the water-filled channel through which ATP/ADP crosses the membrane. Our current Aim 1 is to elucidate the structure of its 12 TMs, the Tlc1 helix packing. In Aim 2 the substrate-binding site of Tlc1 will be determined through DNA shuffling and formation of chimeric proteins. We will do 'evolution in the test-tube' by shuffling the tlc1 genes of Caedibacter caryophilus and R. prowazekii. Sequence analysis of shuffled fragments within these chimeric proteins will associate conserved sequence motifs with substrate specificity. In Aim 3 we will identify and characterize the transport system of NAD, a principle metabolite in R. prowazekii, and in Aim 4 we will investigate the phospholipase-like proteins, patatin and ExoU, in R. prowazekii. We have demonstrated that interaction of rickettsiae with host cells results in the biochemical products of phospholipase activity. This is involved in one of the most fundamental questions in rickettsial biology: its entry. The hypothesis is that activation and/or translocation is required for enzyme activity and rickettsial entry and we will determine if ExoU and patatin play a role in this process.