Permeability of the Epidemic Typhus Rickettsia

描述(申请人提供)：立克次体疾病，如流行性斑疹伤寒，一种由虱子传播的人类疾病，是一种世界性的灾祸，需要对病原体的基本了解、更好的诊断工具和疫苗。病原为普氏立克次体，是一种专性的胞内细菌寄生虫；立克次体只直接生长在宿主和媒介的细胞质中。立克次体是揭开专性细胞内寄生的生物学奥秘的神奇生物，也是研究真核宿主细胞质的活探针。普罗瓦泽克杆菌只有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运输--通常是通过不寻常的运输系统，而这在自由生活的细菌中是没有的。普氏立克次体的ATP/ADP转位酶(Tlc1)是通过立克次体ADP与宿主细胞ATP的强制性交换来运输能量的。我们现在对这种蛋白质有了一个很好的二维拓扑结构，并有证据表明跨膜结构域(TM)形成了ATP/ADP跨膜的充满水的通道。我们目前的目标1是阐明其12个TM的结构，Tlc1螺旋填料。在目标2中，Tlc1的底物结合位置将通过DNA改组和嵌合蛋白的形成来确定。我们将通过重组嗜酸凯迪杆菌和普罗瓦泽克杆菌的tlc1基因在试管中进行进化。对这些嵌合蛋白中改组片段的序列分析将把保守的序列基序与底物专一性联系起来。在目标3中，我们将鉴定和表征普氏沼虾的主要代谢物NAD的转运系统，在目标4中，我们将研究普氏沼虾中的磷脂酶样蛋白Patatin和ExoU。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。这涉及到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Patatin是否在这一过程中发挥作用。

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.