TRANSPORT OF S-ADENOSYLMETHIONINE IN RICKETTSIA

立克次体属成员是人类疾病的病原体，这些疾病在人类历史上产生了重大影响，尽管有有效的治疗方法，但仍对人类健康构成严重威胁。由普氏立克次体、伤寒杆菌和立克次体分别引起的流行性斑疹伤寒和地方性斑疹伤寒是最广为人知的立克次体疾病，尽管全世界已认识到许多其他斑点热病。该属的所有成员都是专性的细胞内寄生虫，只能生长在真核宿主细胞的细胞质(有时也包括细胞核)内，不受液泡膜的限制。虽然局限于这种细胞内的存在，但立克次体能够感染从节肢动物到人类的各种动物宿主，节肢动物作为媒介将这些细菌传播给各种哺乳动物宿主。侵入、生长并最终溶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的转运系统，S-腺苷蛋氨酸是立克次体细胞内生存的重要组成部分。这将是第一个以细菌为特征的SAM运输系统。在具体目标1中，将全面描述这一运输系统的吸收参数。例如，立克次体对SAM的摄取将被动力学定义，底物特异性和抑制剂，如SAM类似的新诺明，将被确定。然后将这种细菌转运蛋白与在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将比较和对比沙门氏菌和非常接近的伤寒沙门氏菌的SAM运输和合成。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熔解，这些物种合成和运输SAM的能力可能不同。伤寒杆菌合成和/或运输SAM的能力将被确定，并与普氏立克次体菌株进行比较，为立克次体最小基因组的进化提供一个独特的模式。最后，在具体目标3中，我们将确定普氏立克次体SAM转运蛋白的编码基因，并在我们在立克次体转运蛋白方面的专业知识的基础上，获得在大肠杆菌中的功能表达。这是确定这一运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的生态位，并为立克次体的进化提供洞察力。

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.