Permeability of the Epidemic Typhus Rickettsia

流行性斑疹伤寒立克次体的渗透性

• 批准号: 7570077
• 负责人: JONATHON PETER AUDIA
• 金额: $ 57.48万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570077
• 关键词: Actins; Adenine Nucleotide Translocase; Bacteria; Binding Sites; Biochemical; Biological; Biology; Cells; Characteristics; Chimeric Proteins; Collection; Complex; Conserved Sequence; Cosmids; Cysteine; Cytoplasm; Cytosol; DNA Shuffling; Diagnostic; Environment; Epidemic; Erythrocytes; Escherichia coli; Evaluation; Evolution; Family; Fever; Genes; Genome; Hydrolysis; Insect Vectors; Investigation; Label; Laboratories; Lice; Life; Mammals; Mass Spectrum Analysis; Membrane; Metabolic; Methodology; Methods; Modeling; Nature; Nicotinamide adenine dinucleotide; Nonesterified Fatty Acids; Nucleotides; Open Reading Frames; Organism; Parasites; Pathway interactions; Permeability; Phospholipase; Plastids; Play; Process; Proteins; Rickettsia; Rickettsia Infections; Role; Scourge; Screening procedure; Sequence Alignment; Sequence Analysis; Sequence Homologs; Site; Spottings; Structure; Substrate Specificity; Sulfhydryl Reagents; System; Tail; Testing; Transmembrane Domain; Travel; Tube; Typhus; Vaccines; Vacuole; Walkers; Water; base; crosslink; enzyme activity; human disease; intracellular parasitism; member; mutant; parasitism; positional cloning; success; tool; two-dimensional; vector

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.

描述（由申请人提供）：立克次体疾病，例如流行性斑疹伤寒，一种由虱子传播的人类疾病，是一种世界性的祸害，需要对病原有基本的了解，需要更好的诊断工具和疫苗。病原体普罗瓦泽基罗氏菌 (R. prowazekii) 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。立克次体是解开专性细胞内寄生生物学奥秘的奇妙生物体，也是研究真核宿主细胞质的活探针。 R. prowazekii 只有 834 个基因，因为许多代谢中间体不需要合成，而是从宿主细胞的细胞质中转运 - 通常通过在自由生活的细菌中没有对应物的不寻常的转运系统。普氏立克次体的 ATP/ADP 转位酶 (Tlc1) 通过立克次体 ADP 与宿主细胞 ATP 的强制性交换来运输能量。现在，我们对该蛋白质有了出色的二维拓扑结构，并有证据表明跨膜结构域 (TM) 形成了 ATP/ADP 穿过膜的充满水的通道。我们当前的目标 1 是阐明其 12 个 TM（Tlc1 螺旋堆积）的结构。在目标 2 中，将通过 DNA 改组和嵌合蛋白的形成来确定 Tlc1 的底物结合位点。我们将通过改组 Caedibacter caryophilus 和 R. prowazekii 的 tlc1 基因来进行“试管进化”。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，我们将鉴定和表征 R. prowazekii 中的主要代谢物 NAD 的转运系统，在目标 4 中，我们将研究 R. prowazekii 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会产生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。假设酶活性和立克次体进入需要激活和/或易位，我们将确定 ExoU 和 patatin 是否在此过程中发挥作用。