GENETIC ANALYSIS OF RICKETTSIAL INTRACELLULAR PARASITISM

流行性斑疹伤寒的病原体是普氏立克次体 一种专性的细胞内寄生细菌，直接生长 在其真核宿主细胞的细胞质内，不受 液泡膜。这项研究的总体目标是 增加我们对生物化学和遗传学的理解 细胞内寄生成功的机制 这种有机体。以前的工作，使用重组DNA 技术，已经导致了一些普罗瓦泽克乳杆菌的分离 基因。本申请中描述的工作的一个主要目标是 利用DNA对这些基因和基因产物进行表征 序列分析和随后的比较研究 在大肠杆菌中发现的相应基因和基因产物 和/或真核系统。额外的基因，编码酶 将会被分离出来，以便 研究立克次体基因的协同表达。这个 立克次体细胞表面蛋白编码基因的克隆 也将大力推行。金黄色葡萄球菌的分离与鉴定 这些基因和基因产物将构成 立克次体识别和渗透的后续研究 它的宿主细胞。这笔赠款的一个主要目标将是使用 从分离和表征中获得的知识 立克次体基因调控机制的研究 能存活的立克次体。克隆的基因或适当的片段将被 用作探针以检测特定的信使RNA。最后，这一点 将开始建立直接遗传系统的工作 PROWAZEKII的分析。这将涉及到低筛选 普罗瓦泽克乳杆菌传代菌株的自然产生质粒 可能作为立克次体病媒有用。此外，研究还包括 旨在评估R.prowazekii的吸收能力和 还将进行外源DNA的表达。这项工作应该是 提供对立克次体基因结构和 功能和定义直接遗传操作的限制 这种专属的细胞内寄生虫。

Rickettsiae prowazekii, the etiologic agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of its eucaryotic host cell, unbound by a vacuolar membrane. The overall goal of this research is to increase our understanding of the biochemical and genetic mechanisms involved in the successful intracellular parasitism by this organism. Previous work, employing recombinant DNA techniques, has led to the isolation of a number of R. prowazekii genes. A major goal of the work described in this application is the characterization of these genes and gene products using DNA sequence analysis and subsequent comparison studies with corresponding genes and gene products found in Escherichia coli and/or eucaryotic systems. Additional genes, coding for enzymes of the tricarboxylic acid cycle, will be isolated in order to investigate coordinate expression of rickettsial genes. The cloning of genes coding for proteins of the rickettsial cell surface will also be vigorously pursued. Isolation and characterization of these genes and gene products will form the foundation for subsequent studies on rickettsial recognition and penetration of its host cell. A major objective of this grant will be to use the knowledge gained from the isolation and characterization of rickettsial genes to probe the regulatory control mechanisms in viable rickettsiae. Cloned genes or appropriate fragments will be used as probes to detect specific messenger RNA. Finally, this work will begin to establish a system for the direct genetic analysis of R. prowazekii. This will involve screening low passaged R. prowazekii strains for naturally-occurring plasmids potentially useful as rickettsial vectors. In addition, studies designed to assess the ability or R. prowazekii to take-up and express exogenous DNA will also be performed. This work should provide an understanding of rickettsial gene structure and function and define the limits of direct genetic manipulation in this obligate intracellular parasite.