Identification of Metabolic Virulence Factors in the Epidemic Typhus Rickettsia

流行性斑疹伤寒立克次体代谢毒力因子的鉴定

• 批准号: 7305296
• 负责人: JONATHON PETER AUDIA
• 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: 2007
• 资助国家: 美国
• 起止时间: 2007-07-05 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7305296
• 关键词: Attenuated; Biochemical Pathway; Bioinformatics; Biological Assay; Bioterrorism; Carrier Proteins; Categories; Cells; Charge; Citric Acid Cycle; Code; Coenzyme A; Communities; Culture Media; Cytosol; DNA; DNA Library; Data; Development; Dihydroxyacetone; Dihydroxyacetone Phosphate; Enzymes; Epidemic; Equilibrium; Escherichia coli; Evolution; Expression Library; Family; Fatty Acids; Genetic; Genetic Complementation Test; Genome; Glycerol-3-Phosphate Dehydrogenase; Goals; Grant; Growth; Homologous Gene; Human; Hygiene; Incubated; Inorganic Phosphate Transporter; Knock-out; Lead; Lice; Longitudinal Studies; Membrane Proteins; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; Mutation; Open Reading Frames; Organism; Organophosphates; Orphan; Pathway interactions; Philosophy; Phosphatidic Acid; Phospholipids; Phosphotransferases; Physiology; Procedures; Proteins; Radiolabeled; Reaction; Research; Rewards; Rickettsia; Rickettsia prowazekii; Risk; Sanitation; Screening procedure; System; Therapeutic Agents; Trioses; Typhus; Vaccines; Virulence; Virulence Factors; alpha-glycerophosphoric acid; antimicrobial; auxotrophy; carrier mediated transport; dephosphocoenzyme A; design; enzyme substrate; genome sequencing; high throughput screening; human disease; inorganic phosphate; insight; interest; mitochondrial genome; mutant; novel; pantothenate; pathogen; protein expression; radiotracer; therapeutic target; uptake

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.

描述(由申请人提供)：这项R21提案的目标是启动一项研究，最终可能导致鉴定专性胞浆内病原体普氏立克次体中的代谢毒力因素，普氏立克次体是人类疾病流行性斑疹伤寒的虱媒病原体。专性的细胞内生长导致了普罗瓦泽克乳杆菌作为一种典型的代谢清除剂的进化；许多必需的代谢物是从宿主胞浆中运输出来的，而不是从头合成。虽然立克次体是典型的经历还原进化的生物体，但它们似乎在清除主要宿主细胞代谢物方面保持了代谢冗余的机制。对R.prowazekii基因组的注释揭示了“孤儿酶”的存在--生化途径的唯一残留物已经丢失，取而代之的是途径最终产物的转运体。此外，这些孤立的酶经常被预测催化酶途径的最后一步。我推测这些所谓的孤立酶是有功能的，立克次体已经为这些酶的底物进化出了新的运输系统，从而提供了从宿主获得必要代谢物的替代方法。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，因此对生长至关重要。本研究将集中于两个途径：1)立克次体通过转运辅酶A(CoA)和3‘脱磷辅酶A(DPC)获得辅酶A(CoA)；2)立克次体通过转运3-甘油-3-磷酸(G3P)和二羟丙酮磷酸(DHAP)获得sn-甘油-3-磷酸(G3P)。我推测，这些多余的途径可以代表治疗这种选择剂的新的免疫/治疗靶点，并被利用来生产普罗瓦泽克杆菌疫苗株。根据R21授予机制的理念，这一探索性项目将确定和表征参与CoA/DPC和G3P/DHAP摄取的立克次体运输系统，以确定适合进行长期研究的候选者，在该研究中，这些系统将被灭活并进行毒力分析。这些研究是制定应对生物恐怖主义威胁的对策的重要步骤。目的I：立克次体具有获得G3P的平行代谢途径。立克次体能够利用细菌GlpT转运蛋白的同源物运输G3P。然而，立克次体也能够在GPSA蛋白(一种G3P脱氢酶)的催化下将二羟丙酮磷酸(DHAP)转化为G3P。我将使用在大肠杆菌中表达的立克次体DNA文库的高通量筛选来鉴定和鉴定立克次体Dhap转运蛋白。目的II：立克次体获得CoA的途径是平行的。纯化的立克次体利用载体介导的转运系统运输CoA及其代谢前体3‘-脱磷辅酶A(DPC)。生物信息学已经确定立克次体具有一种注释的DPC激酶，可以将DPC转化为CoA。我将使用上述筛选方法来鉴定和表征这些转运蛋白。 普氏立克次体是人类疾病流行性斑疹伤寒的虱媒媒介因子，被归类为B类选择性因子，表明其作为生物恐怖主义因子具有潜在的颠覆性。这项研究的重点是确定和表征普罗瓦泽克杆菌特有的基本运输系统，从而成为开发治疗这种专性胞质内病原体的新型抗微生物药物的潜在靶点。