Chemical tools to understand and target Helicobacter pylori glycosylation

描述（申请人提供）：幽门螺杆菌（Hp）是世界范围内引起十二指肠溃疡和胃癌的主要原因。不幸的是，现有的抗生素不再能有效地根除Hp感染并治愈这些疾病。对Hp发病机制的深入了解将极大地帮助开发新的治疗方法。Hp的毒力似乎与病原体对糖基化蛋白的能力直接相关。尽管Hp的聚糖和糖基化机制与发病机制有关，但对Hp的糖基化机制仍知之甚少。该项目的长期目标是开发能够对细菌糖基化进行基础研究的化学工具，特别是与人类疾病有关的化学工具。本应用程序的目的是扩展最近引入的代谢寡糖工程（MOE）技术，以研究和干扰病原体Hp的糖蛋白。MOE是一种强大的化学方法，它将可检测的报告基因安装到聚糖中，最终允许它们的可视化、富集、识别和扰动。该申请的中心假设是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Hp聚糖的平台。提出这项研究的基本原理是双重的：一旦Hp的糖蛋白和糖基化机制被表征，新的治疗干预靶点将被揭示。此外，一旦根据其独特的聚糖靶向Hp，就可以进行临床前试验，以评估该策略作为治疗Hp感染的一种手段。因此，拟议的研究与NIH的使命有关，即发展有助于减轻人类疾病负担的基础知识。在强有力的初步数据的指导下，这一假设将通过追求两个具体目标来验证：1)表征Hp的糖蛋白和糖基化机制；2)基于其独特的聚糖化学靶向Hp。在第一个目标下，将利用已经证明的MOE方法来富集Hp的糖蛋白，然后通过质谱分析鉴定富集的蛋白质和修饰它们的聚糖。此外，将构建缺乏假定的糖基转移酶的Hp缺失突变体，并使用MOE评估其糖蛋白谱以确定负责糖蛋白合成的基因。在第二个目标下，将针对细胞表面糖伪氨基酸选择性灭活Hp，这是Hp发病所必需的，但在人类中却不存在。叠氮化物标记的假胺酸残基在Hp上将被靶向治疗性膦，然后评估对Hp的损害。这种方法是创新的，因为它利用了将成熟的技术应用于高度新颖的系统。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发展，以根除Hp感染。此外，该化学方法将广泛适用于其他细菌，并极大地促进了细菌糖蛋白的研究。

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.