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.

项目概要 历史记录强调了立克次体疾病的全球影响，致命的节肢动物传播的致命立克次体疾病的重新出现 斑点病和斑疹伤寒立克次体，以及新病原体的出现。立克次体的细胞内生活方式。 对研究提出了巨大的挑战；尽管如此，跨学科的方法缓解了近乎棘手的问题 对这些细菌进行基因操纵。该应用程序采用了这样的方法，针对最常见的之一 对立克次体毒力、宿主依赖性代谢寄生等方面的研究不足。我们最近重建的 立克次体代谢和运输网络鉴定出 51 种宿主获得性代谢物，以补偿 生物合成途径退化。没有糖酵解和磷酸戊糖途径，肽聚糖 (PGN) 脂多糖（LPS）必须使用宿主糖合成。 N-乙酰氨基葡萄糖 1-P (NAG-1-P)， PGN 和脂质 A 二糖主链（以及其他 LPS 糖）的前体通常由以下合成： 使用双功能酶 GlmU 的细菌：C 端乙酰转移酶结构域从 葡萄糖胺-1-P (GlcN-1-P)，而 N 末端尿苷基转移酶结构域将 NAG-1-P 转化为 UDP-NAG。 奇怪的是，立克次体属。含有仅具有尿苷基转移酶结构域的“半身人”GlmU 酶，表明 立克次体不合成 GlcN-1-P。鉴于真核途径（合成唾液酸或几丁质）产生 NAG-1-P来自NAG-6-P（不是GlcN-1-P），我们推测立克次体导入宿主NAG-1-P并将其转换为UDP- NAG 使用专为真核代谢定制的精简酶 (GlmU-N)。具体来说，我们假设 立克次氏体独特地利用宿主衍生的 NAG-1-P 来生物合成细胞包膜糖复合物，并且这样的 宿主氨基糖生物合成途径中的代谢物盗窃对于立克次体细胞内传播至关重要 复制和生存。为了检验我们的假设，我们将表征立克次体对宿主 NAG 的摄取和代谢 使用复杂的生化技术 (AIM 1) 检测 1-P，并确定宿主 NAG-1-P 盗窃的必要性 使用遗传工具沉默宿主和病原体代谢基因（AIM 2）。在 AIM 1 下，我们将增强宿主细胞 氨基糖代谢（使用化学工程和同位素补充）监测合成 NAG-1-P 掺入立克次体细胞包膜中。具体来说，我们将测量 PGN 中的叠氮糖并 LPS（通过点击化学和 IFA）和 15N 标记的 NAG-1-P 衍生代谢物并入 PGN（通过质量 光谱测定）。在 AIM 2 下，我们将证明立克次体必须获得宿主 NAG-1-P 才能合成 通过使用 siRNA 来评估沉默宿主氨基糖的影响，从而进行糖缀合物（以及复制） 伤寒杆菌感染的合成基因，以及肽核酸介导的蛋白质表达敲低 确定 GlmU-N 在早期宿主感染期间的功能意义。总的来说，我们的工作将阐明 1) a 立克次体的新特征（GlmU-N 和独特的 NAG-1-P 转运系统）是一个有希望的目标 旨在对抗致命立克次体病的疗法，2) NAG-1-P 盗窃对宿主细胞的影响（发病机制） 代谢，3) NAG-1-P 作为开发立克次体无菌培养基的关键成分的重要性。