Rickettsia cell envelope glycoconjugates are derived from the host cell amino sugar biosynthesis pathway

立克次体细胞包膜糖复合物源自宿主细胞氨基糖生物合成途径

• 批准号: 9804880
• 负责人: Joseph J Gillespie
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9804880
• 关键词: Abbreviations; Acetyltransferase; Amino Sugars; Anabolism; Arthropods; Bacteria; Biochemical; Bioinformatics; C-terminal; Carbohydrates; Cell Wall; Cells; Cellular Metabolic Process; Chemical Engineering; Chemistry; Chitin; Disaccharides; Endemic Flea-Borne Typhus; Enzymes; Epidemic; Evolution; Fever; Generations; Genes; Genetic; Genome; Glucosamine; Glycoconjugates; Glycolysis; Infection; Isotopes; Label; Life Style; Lipid A; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Metabolic; Metabolic Pathway; Metabolism; Molecular and Cellular Biology; Monitor; Mutagenesis; N-terminal; Parasites; Pathogenesis; Pathway interactions; Pentosephosphate Pathway; Peptide Nucleic Acids; Peptidoglycan; Proliferating; Records; Research; Rickettsia; Rickettsia Infections; Rickettsia typhi; Rocky Mountain Spotted Fever; Role; Sialic Acids; Small Interfering RNA; Supplementation; System; Techniques; Testing; Typhus; Vertebral column; Virulence; Work; cell envelope; combat; comparative; experimental study; extracellular; gene synthesis; genetic approach; genetic manipulation; human disease; innovation; knock-down; metabolomics; novel; parasitism; pathogen; protein expression; reconstruction; stem; sugar; targeted treatment; tool; trait; uptake

Project Summary The global impact of rickettsial diseases is highlighted by historical records, reemergence of fatal arthropod-borne spotted and typhus fever rickettsioses, and emergence of new pathogens. The intracellular lifestyle of Rickettsia spp. poses immense challenges to research; nevertheless, a transdisciplinary approach alleviates the near intractability of these bacteria to genetic manipulation. This application employs such an approach, targeting one of the most understudied aspects of rickettsial virulence, host-dependent metabolic parasitism. Our recent reconstruction of the Rickettsia metabolic and transport network identified 51 host-acquired metabolites needed to compensate for degraded biosynthesis pathways. Without glycolysis and the pentose phosphate pathway, peptidoglycan (PGN) and lipopolysaccharide (LPS) must be synthesized using host sugars. N-acetylglucosamine 1-P (NAG-1-P), a precursor of both PGN and lipid A disaccharide backbones (as well as other LPS sugars), is usually synthesized by bacteria using the bifunctional enzyme GlmU: the C-terminal acetyltransferase domain generates NAG-1-P from glucosamine-1-P (GlcN-1-P), while the N-terminal uridyltransferase domain converts NAG-1-P to UDP-NAG. Curiously, Rickettsia spp. contain a “halfling” GlmU enzyme with only the uridyltransferase domain, indicating rickettsiae do not synthesize GlcN-1-P. Given the eukaryotic pathway (synthesizing sialic acid or chitin) generates NAG-1-P from NAG-6-P (not GlcN-1-P), we speculate that rickettsiae import host NAG-1-P and convert it to UDP- NAG using a streamlined enzyme (GlmU-N) tailored to eukaryotic metabolism. Specifically, we hypothesize that rickettsiae uniquely utilize host-derived NAG-1-P for biosynthesis of cell envelope glycoconjugates, and that such metabolite thievery from the host amino sugar biosynthesis pathway is essential for rickettsial intracellular replication and survival. To test our hypothesis, we will characterize rickettsial uptake and metabolism of host NAG- 1-P using sophisticated biochemical techniques (AIM 1), and determine the essentiality of host NAG-1-P pilfering using genetic tools for silencing host and pathogen metabolic genes (AIM 2). Under AIM 1, we will augment host cell amino sugar metabolism (using chemical engineering and isotope supplementation) to monitor synthetic NAG-1-P incorporation into the rickettsial cell envelope. Specifically, we’ll measure azido sugars incorporated into PGN and LPS (via click chemistry and IFA), and 15N-labeled NAG-1-P-derived metabolites incorporated into PGN (via mass spectrometry). Under AIM 2, we will show that rickettsiae must acquire host NAG-1-P for synthesis of glycoconjugates (and thus replication) by using siRNAs to assess the impact of silencing host amino sugar synthesis genes on R. typhi infection, as well as peptide nucleic acid-mediated knockdown of protein expression to determine the functional significance of GlmU-N during early host infection. Collectively, our work will illuminate 1) a novel rickettsial trait (GlmU-N and a unique NAG-1-P transport system) that stands as a promising target for therapeutics aimed at combatting fatal rickettsioses, 2) the impact (pathogenesis) of NAG-1-P pilfering on host cell metabolism, and 3) the importance of NAG-1-P as a critical ingredient for developing a rickettsial axenic media.

项目摘要 人力车疾病的全球影响由历史记录，致命性节肢动物传播的重新出现，强调 被发现和斑疹伤寒立克sios，以及新病原体的出现。立克属的细胞内生活方式。 对研究构成巨大挑战；然而，跨学科的方法减轻了 这些细菌进行遗传操作。该申请员工这样一种方法，针对最大的方法之一 人力体病毒的研究方面是宿主依赖的代谢寄生虫。我们最近重建 立克代谢和运输网络确定了51种宿主获得的代谢物，需要补偿 降解的生物合成途径。没有糖酵解和戊糖磷酸途径，辣椒粉（PGN） 必须使用宿主糖合成脂多糖（LPS）（LPS）。 N-乙酰葡萄糖1-P（NAG-1-P），A PGN和脂质的前体A二糖骨架（以及其他LPS糖）通常由 使用双功能酶GLMU的细菌：C末端乙酰转移酶结构域可从中产生NAG-1-P 葡萄糖胺-1-P（GLCN-1-P），而N末端尿苷转移酶结构域将NAG-1-P转换为UDP-NAG。 奇怪的是，立克属。仅包含仅带有尿苷转移酶结构域的“半身” GLMU酶 人力体不合成Glcn-1-P。鉴于真核途径（合成唾液酸或几丁质）会产生 NAG-1-P来自NAG-6-P（不是GLCN-1-P），我们推测Rickettsiae Import Host nag-1-p并将其转换为UDP- 使用基于真核代谢的简化酶（GLMU-N）NAG。具体来说，我们假设 立克的唯一利用宿主衍生的NAG-1-P用于细胞包膜的生物合成，并且这样 来自宿主氨基糖生物合成途径的代谢产物对立克细胞内至关重要 复制和生存。为了检验我们的假设，我们将表征宿主na的人力体的摄取和代谢 1-P使用复杂的生化技术（AIM 1），并确定宿主NAG-1-P窃取的重要性 使用遗传工具沉降宿主和病原体代谢基因（AIM 2）。在AIM 1下，我们将增强宿主单元 氨基糖代谢（使用化学工程和同位素补充）监测合成NAG-1-P 掺入立克细胞包膜中。具体而言，我们将测量掺入PGN和 LPS（通过单击化学和IFA）和15N标记的NAG-1-P衍生的代谢物掺入PGN（通过质量） 光谱法）。在AIM 2下，我们将证明Rickettsiae必须获取宿主NAG-1-P的合成 通过使用siRNA评估沉默宿主氨基糖的影响，糖缀合物（因此复制） 鼠伤寒红菌感染的合成基因，以及肽核酸介导的蛋白表达敲低 确定早期宿主感染期间GLMU-N的功能意义。总的来说，我们的工作将阐明1） 新颖的人力素性特质（GLMU-N和独特的NAG-1-P传输系统）是有望的目标 旨在打击致命的立克西奥的治疗，2）NAG-1-P对宿主细胞的影响（发病机理） 代谢和3）NAG-1-P是开发立克式轴突介质的关键要素的重要性。