Chemical tools to understand and target Helicobacter pylori glycosylation

描述(申请人提供)：幽门螺杆菌(Hp)是全球十二指肠溃疡和胃癌的主要原因。不幸的是，现有的抗生素不再有效地根除幽门螺杆菌感染和治愈这些疾病。对幽门螺杆菌发病机制的深入了解将极大地帮助开发新的治疗方法。幽门螺杆菌的毒力似乎与病原体糖基化蛋白质的能力直接相关。虽然幽门螺杆菌的多糖和糖基化机制与发病机制有关，但幽门螺杆菌的糖蛋白仍然知之甚少。该项目的长期目标是开发化学工具，使细菌糖基化的基础研究成为可能，特别是在人类疾病方面。这一应用的目的是扩展新近引进的代谢寡糖工程(MOE)技术，以研究和干扰病原菌Hp的糖蛋白。MOE是一种强大的化学方法，它将可检测到的记者安装到多糖中，最终允许他们可视化、浓缩、鉴定和干扰。该应用的中心假设是，MOE将揭示新的HP糖蛋白，促进糖基化机制的鉴定，并作为靶向独特的HP多糖的平台。这项拟议研究的理由有两个：一旦惠普的糖蛋白和糖基化机制得到表征，治疗干预的新靶点将被揭示。此外，一旦惠普根据其独特的多糖成为靶点，将为临床前试验奠定基础，以评估这一策略作为治疗幽门螺杆菌感染的手段。因此，拟议的研究与NIH使命中与发展基础知识相关的部分相关，这可能有助于减轻人类疾病的负担。在强大的初步数据的指导下，这一假说将通过追求两个具体目标来检验：1)表征HP的糖蛋白和糖基化机制；2)根据HP独特的多糖进行化学靶向。在第一个目标下，将利用已经证明的MOE方法来浓缩HP的糖蛋白，然后通过质谱分析鉴定浓缩的蛋白质和修饰它们的多糖。此外，还将构建缺少假定糖基转移酶的HP缺失突变体，并将使用MOE对其糖蛋白谱进行评估，以确定与糖蛋白合成有关的基因。在第二个目标下，细胞表面糖伪氨基酸将被选择性地灭活幽门螺杆菌，它在幽门螺杆菌的发病机制中是必不可少的，但在人类中还没有。幽门螺杆菌上的叠氮标记的伪氨基酸残基将被治疗性的磷化氢靶向，然后将评估对幽门螺杆菌的损害。这种方法是创新的，因为它利用了一种成熟的技术来应用于一个非常新颖的系统。这项拟议的研究意义重大，因为它有望启动基于糖基化的有效治疗策略的开发，以根除幽门螺杆菌感染。此外，这里开发的化学方法将广泛适用于其他细菌，并极大地促进细菌糖蛋白的研究。 与公共卫生相关：拟议的研究涉及一个重要且未被充分研究的细菌生物学领域，该领域可能适用于了解幽门螺杆菌感染的发病机制，并为帮助溃疡和胃癌患者的治疗干预提供新的靶点。这项拟议的研究与公共健康相关，因为要研究的分子与发病机制有关。因此，这些发现最终有望适用于根除细菌感染和改善人类健康。

DESCRIPTION (provided by applicant): Helicobacter pylori (Hp) is the leading cause of duodenal ulcers and gastric cancer worldwide. Unfortunately, existing antibiotics no longer effectively eradicate Hp infection and cure these ailments. The development of new treatments will be greatly aided by insights into the pathogenesis of Hp. Virulence of Hp appears to be directly linked to the pathogen's ability to glycosylate proteins. Although Hp's glycans and glycosylation machinery are linked to pathogenesis, Hp's glycome remains poorly understood. The long-term goal of this project is to develop chemical tools that enable fundamental studies of bacterial glycosylation, particularly with respect to human disease. The objective of this application is to expand the recently introduced technique of metabolic oligosaccharide engineering (MOE) to study and perturb glycoproteins of the pathogen Hp. MOE is a powerful chemical method that installs detectable reporters into glycans, ultimately permitting their visualization, enrichment, identification, and perturbation. The central hypothesis of the application is that MOE will reveal new Hp glycoproteins, facilitate the identification of glycosylation machinery, and serve as a platform for targeting unique Hp glycans. The rationale for the proposed research is twofold: once Hp's glycoproteins and glycosylation machinery have been characterized, new targets of therapeutic intervention will be revealed. Further, once Hp is targeted based on its unique glycans, the stage will be set for pre-clinical trials to evaluate this strategy as a means to treat Hp infection. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Characterize Hp's glycoproteins and glycosylation machinery; and 2) Chemically target Hp based on its unique glycans. Under the first aim, an already proven MOE approach will be utilized to enrich Hp's glycoproteins, and then the enriched proteins and the glycans that modify them will be identified by mass spectrometry analysis. Further, Hp deletion mutants lacking putative glycosyltransferases will be constructed, and their glycoprotein profile will be assessed using MOE to identify the genes responsible for glycoprotein synthesis. Under the second aim, the cell surface sugar pseudaminic acid, which is essential for Hp's pathogenesis yet absent from humans, will be targeted to selectively inactivate Hp. Azide-labeled pseudaminic acid residues on Hp will be targeted with therapeutic phosphines, and then damage to Hp will be assessed. This approach is innovative, because it capitalizes on applying a well-established technique to a highly novel system. The proposed research is significant, because it is expected to initiate the development of effective glycosylation-based therapeutic strategies to eradicate Hp infections. Moreover, the chemical approach developed here will be broadly applicable in other bacteria and greatly facilitate the study of bacterial glycoproteins. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of bacterial biology that has potential applicability to understanding the pathogenesis of Helicobacter pylori infection, as well as providing new targets for therapeutic interventions that will aid patients suffering from ulcers and gastric cancer. The proposed research has relevance to public health, because the molecules to be investigated are tied to pathogenesis. Thus, the findings are ultimately expected to be applicable to eradicating bacterial infection and improving human health.