TRANSPORT OF S-ADENOSYLMETHIONINE IN RICKETTSIA

立克次体中 S-腺苷甲硫氨酸的转运

• 批准号: 6170876
• 负责人: DAVID WOOD
• 金额: $ 21.55万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170876
• 关键词: Escherichia coli; Rickettsia; Rickettsia prowazekii; Rickettsiales disease; S adenosylmethionine; bacterial cytopathogenic effect; bacterial genetics; computer assisted sequence analysis; enzyme activity; gene expression; host organism interaction; intracellular parasitism; intracellular transport; ion exchange chromatography; molecular biology information system; molecular cloning; polymerase chain reaction; protein biosynthesis; protein structure function; scintillation spectrometry; transport proteins

Members of the genus Rickettsia are the causative agents of human diseases that have had a significant impact on human history and that, despite the availability of effective treatment, continue to pose serious threats to human health. Epidemic and endemic typhus and rocky mountain spotted fever caused by R. prowazekii, R. typhi and R. rickettsii, respectively, are the most well known of the rickettsial diseases although numerous other spotted fevers have been recognized worldwide. All members of this genus are obligate intracellular parasites that can only grow within the cytoplasm (and occasionally the nucleus) of a eukaryotic host cell, unbounded by a vacuolar membrane. While restricted to this intracellular existence, the rickettsiae are capable of infecting an assortment of animal hosts ranging from arthropods to humans with arthropods serving as vectors for transmission of these bacteria to a variety of mammalian hosts. The ability to invade, grow within and eventually lyse eukaryotic cells is the basis for rickettsial pathogenicity and is dependent on mechanisms for exploiting this novel bacterial niche. The goal of this research is to identify and characterize the rickettsial transport system for S-adenosylmethionine (SAM), an essential component of rickettsial intracellular survival. This will be the first SAM transport system to be characterized in bacteria. In Specific Aim 1 the uptake parameters of this transport system will be completely characterized. For example, rickettsial SAM uptake will be defined kinetically and the substrate specificity and effect of inhibitors such as the SAM analog sinefungin determined. This bacterial transporter will then be compared to known SAM transporters found in mitochondria and yeast. Specific Aim 2 will compare and contrast SAM transport and synthesis in R. prowazekii and the very closely related R. typhi. Evidence suggests that gene meltdown is occurring in the rickettsial gene coding for methionine adenosyltransferase (MAT) and that these species may differ in their ability to synthesize and tranport SAM. The ability of R. typhi to synthesize and/or transport SAM will be determined and compared to R. prowazekii strains providing a unique model for evolution of the rickettsial minimal genome. Finally, in Specific Aim 3 we will identify the gene coding for the R. prowazekii SAM transporter and, building upon our expertise in rickettsial transporters, obtain functional expression in Escherichia coli. This is a critical first step in characterizing the structure and function of this transport system. Completion of these studies will contribute to our understanding of how this pathogen exploits the intracellular niche as well as provide insight into rickettsial evolution.

立克属的成员是对人类历史产生重大影响的人类疾病的原因，尽管有有效的治疗方法，但仍对人类健康构成了严重威胁。 流行病和流行的斑疹伤寒和落基山斑点是由R. prowazekii，R。Typhi和R. rickettsii引起的，是最著名的人力素性疾病中最著名的，尽管全球已认识到许多其他发现的发烧。 该属的所有成员都是只能在真核宿主细胞的细胞质（和偶尔是核）内生长的较强细胞内寄生虫，该寄生虫由液泡膜无限。 虽然仅限于这种细胞内的存在，但立克的人能够感染各种动物宿主，从节肢动物到人类，具有将这些细菌传播到各种哺乳动物宿主的载体的人类。 入侵，生长并最终裂解真核细胞的能力是立克疾病致病性的基础，并取决于利用这种新型细菌壁se的机制。这项研究的目的是识别和表征S-腺苷硫氨酸（SAM）的立克式转运系统，这是人力素内细胞内生存的重要组成部分。 这将是第一个在细菌中表征的SAM运输系统。 在特定的目标1中，该传输系统的吸收参数将完全表征。 例如，将通过动力学定义身心，并确定抑制剂的底物特异性和效果。 然后将将这种细菌转运蛋白与线粒体和酵母中发现的已知SAM转运蛋白进行比较。 特定的目标2将比较和对比Prowazekii和密切相关的Typhi中的SAM运输和合成。 有证据表明，基因崩溃发生在编码蛋氨酸腺苷转移酶（MAT）的立克基因基因中，并且这些物种在合成和Tranport SAM的能力上可能有所不同。 将鼠伤寒红杆菌合成和/或运输SAM的能力将被确定并与R. prowazekii菌株进行比较，该菌株为立克的最小基因组的进化提供了独特的模型。 最后，在特定目标3中，我们将确定R. prowazekii Sam Transporter的基因编码，并基于我们在立克转运蛋白的专业知识，在大肠杆菌中获得功能表达。 这是表征该传输系统的结构和功能的关键第一步。 这些研究的完成将有助于我们理解这种病原体如何利用细胞内生态裂市场，并提供对立克的进化的见解。