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

项目摘要 立克次体病的全球影响突出表现在历史记录中， 斑点热和斑疹伤寒立克次体病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;然而，跨学科的方法使研究变得更加困难。 这些细菌的基因操作。该应用程序采用了这样的方法，针对的是最受欢迎的应用程序之一 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确定了51种宿主获得的代谢物， 降解的生物合成途径。没有糖酵解和戊糖磷酸途径，肽聚糖（PGN） 脂多糖（LPS）必须使用宿主糖合成。N-乙酰葡糖胺1-P（NAG-1-P），a PGN和脂质A二糖主链（以及其他LPS糖）的前体通常通过以下方法合成： 使用双功能酶GlmU的细菌：C-末端乙酰转移酶结构域产生NAG-1-P 葡糖胺-1-P（GlcN-1-P），而N-末端尿苷酰转移酶结构域将NAG-1-P转化为UDP-NAG。 奇怪的是，立克次体属（Rickettsia spp.）含有仅具有尿苷酰转移酶结构域的“半身”GlmU酶，表明 立克次氏体不合成GlcN-1-P。 NAG-1-P从NAG-6-P（而不是GlcN-1-P），我们推测立克次体输入宿主NAG-1-P并将其转化为UDP-1。 NAG使用针对真核生物代谢的流线型酶（GlmU-N）。具体来说，我们假设， 立克次氏体独特地利用宿主来源的NAG-1-P用于细胞包膜糖缀合物的生物合成， 宿主氨基糖生物合成途径的代谢物窃取是立克次体细胞内 复制和生存。为了验证我们的假设，我们将描述宿主NAG的立克次体摄取和代谢， 利用先进的生物化学技术（AIM 1）对NAG-1-P进行了初步的研究，并确定了宿主NAG-1-P偷窃的必要性 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），以监测合成NAG-1-P 整合到立克次体细胞包膜中。具体来说，我们将测量PGN中的叠氮糖， LPS（通过点击化学和IFA）和掺入PGN中的15 N标记的NAG-1-P衍生的代谢物（通过质谱） 光谱法）。在AIM 2中，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评估沉默宿主氨基糖的影响， R.伤寒感染，以及肽核酸介导的蛋白质表达敲低， 确定GlmU-N在宿主感染早期的功能意义。总的来说，我们的工作将阐明1）a 一种新的立克次体性状（GlmU-N和独特的NAG-1-P转运系统），是一种有前途的靶点， 2）NAG-1-P偷窃对宿主细胞的影响（发病机理） 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。

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.