TRANSPORT OF S-ADENOSYLMETHIONINE IN RICKETTSIA

立克次氏体属的成员是人类疾病的病原体，这些疾病对人类历史产生了重大影响，尽管有有效的治疗方法，但仍然对人类健康构成严重威胁。 流行性和地方性斑疹伤寒及落基山斑疹热。普劳瓦泽基河typhi和R.立克次氏体分别是最著名的立克次氏体疾病，尽管世界范围内已经认识到许多其他斑点热。 该属的所有成员都是专性细胞内寄生虫，只能在真核宿主细胞的细胞质（偶尔是细胞核）内生长，不受空泡膜的限制。 虽然局限于这种细胞内存在，但立克次氏体能够感染从节肢动物到人类的各种动物宿主，其中节肢动物充当将这些细菌传播到各种哺乳动物宿主的载体。 立克次体侵入真核细胞、在真核细胞内生长并最终裂解真核细胞的能力是立克次体致病性的基础，并依赖于利用这种新的细菌生态位的机制。本研究的目的是鉴定和表征立克次体S-腺苷甲硫氨酸（SAM）的转运系统，SAM是立克次体细胞内存活的重要组成部分。 这将是第一个在细菌中表征的SAM运输系统。 在具体目标1中，将完全表征该运输系统的摄取参数。 例如，将从动力学上定义立克次体SAM摄取，并确定底物特异性和抑制剂如SAM类似物sinefungin的作用。 然后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。 具体目标2将比较和对比SAM的运输和合成在R。prowazekii和与其关系密切的R.伤寒。 有证据表明，编码甲硫氨酸腺苷转移酶（MAT）的立克次体基因正在发生基因融合，这些物种合成和转运SAM的能力可能不同。 R.伤寒杆菌合成和/或转运SAM的能力将被确定并与R. prowazekii菌株为立克次体最小基因组的进化提供了独特的模型。 最后，在具体目标3中，我们将确定编码R的基因。prowazekii SAM转运蛋白，并建立在我们在立克次体转运蛋白的专业知识，在大肠杆菌中获得功能性表达。 这是确定这一运输系统的结构和功能的关键的第一步。 这些研究的完成将有助于我们了解这种病原体如何利用细胞内生态位，以及提供深入了解立克次体的演变。

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.