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）：该R21提案的目的是启动一项研究，该研究最终可以导致强制性胞质内病原体Rickettsia prowazekii的代谢毒力因子，这是人类疾病流行病的诱人药物。强大的细胞内生长导致了R. prowazekii作为典型的代谢清除剂的演变。许多必需的代谢产物从宿主的胞质溶胶中运输而不是从头合成。尽管立克西亚（Rickettsiae）典型的生物正在经历还原性进化的生物，但它们似乎在清除基本宿主细胞代谢物方面保持了代谢冗余的机制。 R. prowazekii基因组的注释揭示了存在“孤儿酶”的存在 - 生化途径的唯一残留物，否则这些途径否则就丢失了途径的最终产物的转运蛋白。此外，通常预测这些孤立的酶会催化酶促途径的最后一步。我假设这些所谓的孤儿酶是功能性的，而人力素的人是为这些酶的底物进化了新型的传输系统，因此提供了从宿主那里获取必需代谢物的替代方法。这些平行的清除机制可能需要与宿主细胞酶进行关键代谢产物的宿主细胞竞争所必需的，因此对于生长至关重要。这项研究将重点介绍两种途径：1）Rickettsial通过运输COA和3'dephospocoA（DPC）和2）Rickettsial的Rickettsial获取（COA）通过G3P和Dihydroxyacetone Chophate（Dhydroxyacetone phosp）的运输（DHAP）（Dhydroxyacetone opt）（DHAP）。我认为这些冗余途径可以代表新型免疫/治疗靶标的目标，以治疗该精选剂并被利用以产生R. prowazekii疫苗菌株。遵循R21授予机制哲学，该探索性项目将识别并表征与COA/DPC和G3P/DHAP吸收相关的立克交通系统，以确定适合长期研究的合适候选者，在该研究中，这些系统将被灭活和分析用于毒力。这些研究代表了开发针对生物恐怖威胁的对策的重要步骤。目的I：立克西亚具有采购G3P的平行代谢途径。人力车能够使用细菌GLPT转运蛋白的同源物运输G3P。然而，立克人士还能够在由GPSA蛋白（G3P脱氢酶）催化的反应中转化二羟基丙酮（DHAP）为G3P。我将使用在大肠杆菌中表达的立克式DNA文库的高通量筛选来识别和表征立克的DHAP转运蛋白。 AIM II：Rickettsiae具有获得COA的平行途径。使用单独的载流子介导的运输系统，纯化的立克西亚转运COA及其代谢前体3'Dephophocoenzyme A（DPC）。生物信息学已经确定立克西亚具有带注释的DPC激酶将DPC转换为COA。我将使用上述筛选方法来识别和表征这些转运蛋白。 人力车prowazekii是人类疾病流行病的虱子载体药物，被归类为B类选择剂，表明其潜在的颠覆为生物恐怖主义。这项研究的重点是识别和表征prowazekii独有的基本运输系统，因此是开发新型抗微生物的潜在靶标，以治疗这种强制性的胞质内病原体。