Identification of Metabolic Virulence Factors in the Epidemic Typhus Rickettsia

描述（由申请人提供）：本R21提案的目标是启动一项研究，最终可能导致鉴定人类疾病流行性斑疹伤寒的虱媒病原体——专性胞浆内致病菌普拉兹立克次体的代谢毒力因子。专性的细胞内生长导致了proproazekii作为典型的代谢清道夫的进化；许多必需的代谢物由宿主细胞质转运而不是重新合成。虽然立克次体是典型的经历还原性进化的生物体，但它们似乎在清除必需的宿主细胞代谢物方面保持了代谢冗余机制。对R. prowazekii基因组的注释揭示了“孤儿酶”的存在，这是生化途径的唯一残余，否则它们就会丢失，从而有利于该途径最终产物的转运体。此外，这些孤立的酶通常被预测催化酶途径的最后一步。我假设这些所谓的孤儿酶是功能性的，立克次体已经进化出新的运输系统来运输这些酶的底物，从而提供了从宿主获取必需代谢物的替代途径。这些平行的清除机制可能是立克次体与宿主细胞酶竞争关键代谢物所必需的，因此是生长所必需的。本研究将重点研究两种途径：1)立克次体通过运输辅酶A和3'脱磷辅酶A （DPC）获得辅酶A (CoA); 2)立克次体通过运输G3P和磷酸二羟丙酮（DHAP）获得森-甘油-3-磷酸（G3P）。我认为这些冗余的途径可能代表新的免疫/治疗靶点的靶标，以治疗这种选择剂，并被利用来生产proproazekii疫苗株。根据R21授权机制，该探索性项目将确定和表征参与CoA/DPC和G3P/ dhp摄取的立克次体运输系统，以确定长期研究的合适候选系统，在该研究中，这些系统将被灭活并检测毒性。这些研究是制定应对生物恐怖主义威胁对策的重要步骤。目的一：立克次体具有平行的代谢途径来获取G3P。立克次体能够使用细菌GlpT转运体的同源物转运G3P。然而，立克次体也能够在GpsA蛋白（一种G3P脱氢酶）催化的反应中将二羟丙酮磷酸（DHAP）转化为G3P。我将使用高通量筛选在大肠杆菌中表达的立克次体DNA文库来鉴定和表征立克次体DHAP转运体。目的二：立克次体具有平行途径获得辅酶a。纯化立克次体运输辅酶A及其代谢前体，3'去磷辅酶A （DPC）使用单独的载体介导的运输系统。生物信息学已经确定立克次体具有一个带注释的DPC激酶来将DPC转化为辅酶a。我将使用上述筛选方法来识别和表征这些转运蛋白。

DESCRIPTION (provided by applicant): The goal of this R21 proposal is to initiate a study that could ultimately lead to the identification of metabolic virulence factors in the obligate intracytoplasmic pathogen Rickettsia prowazekii, the louse-vectored agent of the human disease epidemic typhus. Obligate intracellular growth has led to the evolution of R. prowazekii as a quintessential metabolic scavenger; many essential metabolites are transported from the host cytosol in lieu of de novo synthesis. Although rickettsiae typify organisms undergoing reductive evolution they appear to have maintained mechanisms of metabolic redundancy with respect to scavenging of essential host cell metabolites. Annotation of the R. prowazekii genome revealed the presence of 'orphaned enzymes' - the sole remnants of biochemical pathways that have been otherwise lost in favour of a transporter for the end product of the pathway. Further, these orphaned enzymes are often predicted to catalyze the last step in the enzymatic pathway. I hypothesize that these so-called orphaned enzymes are functional and that rickettsiae have evolved novel transport systems for the substrates of these enzymes, thus providing alternate means to acquire essential metabolites from the host. These parallel scavenging mechanisms could be required for rickettsial competition with host cell enzymes for critical metabolites and are, thus, essential for growth. This study will focus on two pathways: 1) rickettsial acquisition of Coenzyme A (CoA) via transport CoA and 3'dephosphoCoA (DPC) and 2) rickettsial acquisition of sn-glycerol-3-phosphate (G3P) via transport of G3P and dihydroxyacetone phosphate (DHAP). I posit that these redundant pathways could represent targets for novel immuno/therapeutic targets to treat this Select Agent and be exploited to produce a R. prowazekii vaccine strain. Following the R21 granting mechanism philosophy, this exploratory project will identify and characterize the rickettsial transport systems involved in CoA/DPC and G3P/DHAP uptake to identify suitable candidates for a long-term study in which these systems will be inactivated and assayed for virulence. These studies represent important steps in developing countermeasures against bioterrorism threats. Aim I: Rickettsiae possess parallel metabolic pathways to procure G3P. Rickettsiae are able to transport G3P using a homologue of the bacterial GlpT transporter. However, rickettsiae are also able to convert dihydroxyacetone phosphate (DHAP) to G3P in a reaction catalyzed by the GpsA protein (a G3P dehydrogenase). I will use high-throughput screening of a rickettsial DNA library expressed in Escherichia coli to identify and characterize the rickettsial DHAP transporter. Aim II: Rickettsiae possess parallel pathways to obtain CoA. Purified rickettsiae transport CoA and its metabolic precursor, 3'dephosphocoenzyme A (DPC) using separate carrier-mediated transport systems. Bioinformatics has determined that rickettsiae possess an annotated DPC kinase to convert DPC to CoA. I will use the above mentioned screening method to identify and characterize these transporters. Rickettsia prowazekii is the louse-vectored agent of the human disease epidemic typhus and is categorized as a Category B select agent indicative of its potential subversion as an agent of bioterrorism. The focus of this study is to identify and characterize essential transport systems that are unique to R. prowazekii and are, thus, potential targets for the development of novel antimicrobials to treat this obligate intracytoplasmic pathogen.