Chemical tools to understand and target Helicobacter pylori glycosylation

描述（由申请人提供）：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Hp感染和治愈这些疾病。对Hp发病机制的深入了解将大大有助于新治疗方法的开发。Hp的毒力似乎与病原体使蛋白质糖基化的能力直接相关。虽然Hp的聚糖和糖基化机制与发病机制有关，但对Hp的糖组仍知之甚少。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，特别是关于人类疾病。本申请的目的是扩展最近引入的代谢寡糖工程（莫伊）技术以研究和干扰病原体Hp的糖蛋白。莫伊是一种强大的化学方法，将可检测的报告分子安装到聚糖中，最终允许其可视化、富集、识别和扰动。该申请的中心假设是，莫伊将揭示新的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。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得到表征，治疗干预的新靶点将被揭示。此外，一旦Hp基于其独特的聚糖被靶向，将为临床前试验设置阶段，以评估该策略作为治疗Hp感染的手段。因此，拟议中的研究与NIH的使命的一部分有关，即开发基础知识，这将有助于减轻人类疾病的负担。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进行测试：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，将利用已经被证明的莫伊方法来富集Hp的糖蛋白，然后通过质谱分析来鉴定富集的蛋白质和修饰它们的聚糖。此外，将构建缺乏推定糖基转移酶的Hp缺失突变体，并使用莫伊评估其糖蛋白谱，以鉴定负责糖蛋白合成的基因。在第二个目标下，细胞表面糖假氨基酸（pseudaminic acid）将被靶向选择性地抑制Hp，假氨基酸是Hp发病机制所必需的，但在人类中却不存在。Hp上的叠氮化物标记的假氨基酸残基将用治疗性膦靶向，然后评估对Hp的损伤。这种方法是创新的，因为它将一种成熟的技术应用于一个高度新颖的系统。这项研究具有重要意义，因为它有望启动有效的基于糖基化的治疗策略的发展，以根除Hp感染。此外，本文所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。 公共卫生相关性：拟议的研究是细菌生物学的一个重要和未充分研究的领域，具有潜在的适用性，以了解幽门螺杆菌感染的发病机制，以及提供新的治疗干预措施，将帮助患有溃疡和胃癌的患者。拟议的研究与公共卫生有关，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消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.