Inhibition of prokaryote-specific saccharide biosynthesis in microbial pathogens

微生物病原体中原核生物特异性糖生物合成的抑制

基本信息

批准号：
8235459
负责人：
Barbara Imperiali
金额：
$ 23万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8235459
关键词：
Address Adherence Alberta province Anabolism Animal Model Anti-Bacterial Agents Antibiotic Resistance Bacterial Model Biochemical Biological Assay Biological Models Caenorhabditis elegans Campylobacter jejuni Carbohydrates Cell Adhesion Cell Wall Cell surface Cells Chemicals Collaborations Communicable Diseases Complement Development Disease Elements Enzymes Epithelial Cells Evaluation Foundations Genetic Glycoconjugates Glycoproteins Goals Gram-Negative Bacteria Human In Vitro Indium Infection Inhibitory Concentration 50 Lead Ligands Link Lipopolysaccharides Mammalian Cell Membrane Glycoproteins Methods Microbe Modeling Modification Molecular Neisseria gonorrhoeae Organism Pathogenesis Pathogenicity Pathway interactions Play Polysaccharides Production Prokaryotic Cells Property Protein Glycosylation Pseudomonas aeruginosa Public Health Recombinants Research Role Screening procedure Series Severities Solubility Structure Study models Surface Time Toxic effect Transaminases Transferase Universities Validation Virulence Virulence Factors Work X-Ray Crystallography antimicrobial assay development bacillosamine base cell motility combat enzyme activity glycoprotein biosynthesis glycosylation in vivo inhibitor/antagonist innovation link protein microbial microbial host novel strategies pathogen pathogenic bacteria scale up small molecule sugar tool

项目摘要

DESCRIPTION (provided by applicant): It is well known that antibiotic resistance is a critical issue in the battle against microbial pathogens. Less well known is the way forward to new approaches in antibacterial therapy. Just in the last few years bacterial cell surface glycoconjugates, including the lipopolysaccharide component of the outer cell wall and cell surface N- and O-linked glycoproteins of numerous medically relevant Gram-negative bacterial pathogens, have been characterized in molecular detail and found to be essential for virulence and pathogenicity. This proposal aims to further define and exploit the pathways that produce the unusual microbe-specific carbohydrate building blocks that are found in these glycoconjugates, thus providing a novel approach to combat bacterial pathogens. This research focuses specifically on the development and in vitro and in vivo validation of inhibitors to enzymes involved in the conversion of UDP-GlcNAc into UDP-di-N-acetyl-bacillosamine (UDP- diNAcBac) in the N- and O-linked protein glycosylation pathways of C. jejuni and N. gonorrhoeae. Since UDP-diNAcBac is a critical intermediate in the pathways that result in the biosynthesis of the bacterial glycoconjugates, these inhibitors could be employed as selective chemical tools to elucidate the fundamental roles of highly modified saccharides in microbial pathogenesis. The experimental approach of the proposed research involves: 1. Application of a structure-guided fragment-based screening (FBS) strategy for the development of potent UDP-diNAcBac biosynthesis inhibitors; 2. Evaluation of optimized inhibitors in assays that probe glycoprotein biosynthesis, cell toxicity and the effects of inhibiting glycoprotein biosynthesis on motility, adherence and invasion in the native organism in vivo in C. jejuni and N. gonorrhoeae; 3. Establishment of a C. elegans model for C. jejuni and N. gonorrhoeae infectivity and virulence. If successful, this animal model system will be valuable for to assessing inhibitory activity in a simple host; 4. Assessment of the effect of C. jejuni UDP-diNAcBac biosynthesis inhibitors in the chick infectivity model in collaboration with Szymanski at the University of Alberta. This research addresses the central hypothesis that the biosynthetic pathways in pathogenic bacteria that lead to highly modified sugar building blocks, such as di-N-acetyl-bacillosamine, represent an "Achilles' heel" that can be exploited in the battle against infectious diseases. The general principles that we develop in these studies will be applicable to other microbial pathogens that implement prokaryote-specific N- and O-linked glycoproteins as virulence factors. If successful, the research will identify new enzyme targets and strategies in the global crisis of combating infectious diseases in the face of escalating antibiotic resistance. PUBLIC HEALTH RELEVANCE: The usual means that humans have used for half a century to defeat their bacterial foes have faltered, as antibiotic resistance of common microbial pathogens presents a growing threat to public health. Recent research has clarified pathways in the production of cell surface glycoproteins that play key roles for deadly Gram-negative bacteria, enabling their access and attack on human cells. Our proposal will support work to exploit these essential virulence-associated pathways by blocking production of critical building blocks in the glycoconjugate assemblies, thus developing an entirely new set of targets for antimicrobial therapy.

描述（由申请人提供）：众所周知，抗生素耐药性是对抗微生物病原体的关键问题。不太为人所知的是抗菌治疗新方法的发展方向。就在最近几年，细菌细胞表面糖复合物，包括外细胞壁的脂多糖成分以及许多医学相关革兰氏阴性细菌病原体的细胞表面 N- 和 O- 连接糖蛋白，已在分子细节上进行了表征，并发现对于毒力和致病性至关重要。该提案旨在进一步定义和开发在这些糖复合物中发现的不寻常的微生物特异性碳水化合物构建模块的产生途径，从而提供一种对抗细菌病原体的新方法。这项研究特别侧重于 N- 和 O- 连接蛋白中参与 UDP-GlcNAc 转化为 UDP-二-N-乙酰基-杆菌胺 (UDP-diNAcBac) 的酶抑制剂的开发和体外和体内验证空肠弯曲菌和淋病奈瑟菌的糖基化途径。由于 UDP-diNAcBac 是细菌糖复合物生物合成途径中的关键中间体，因此这些抑制剂可以用作选择性化学工具来阐明高度修饰的糖在微生物发病机制中的基本作用。本研究的实验方法包括： 1. 应用结构引导片段筛选（FBS）策略来开发有效的 UDP-diNAcBac 生物合成抑制剂； 2. 在空肠弯曲菌和淋病奈瑟菌体内探索糖蛋白生物合成、细胞毒性以及抑制糖蛋白生物合成对体内天然生物体运动、粘附和侵袭的影响的测定中评估优化抑制剂； 3. 建立空肠弯曲菌和淋病奈瑟菌感染性和毒力的线虫模型。如果成功，该动物模型系统对于评估简单宿主的抑制活性将很有价值。 4. 与阿尔伯塔大学的 Szymanski 合作，评估空肠弯曲菌 UDP-diNAcBac 生物合成抑制剂在雏鸡感染模型中的效果。这项研究提出了一个核心假设，即致病菌中产生高度修饰的糖结构单元（例如二-N-乙酰基-芽孢杆菌胺）的生物合成途径是可用于对抗传染病的“阿喀琉斯之踵”。我们在这些研究中开发的一般原则将适用于其他以原核生物特异性 N 和 O 连接糖蛋白作为毒力因子的微生物病原体。如果成功，该研究将在面临抗生素耐药性不断升级的全球抗击传染病危机中确定新的酶靶点和策略。公共健康相关性：半个世纪以来，人类用来击败细菌敌人的常用手段已经动摇，因为常见微生物病原体的抗生素耐药性对公共健康构成了越来越大的威胁。最近的研究阐明了细胞表面糖蛋白的产生途径，这些糖蛋白对致命的革兰氏阴性细菌发挥着关键作用，使其能够进入并攻击人类细胞。我们的提案将支持通过阻断糖复合物组件中关键构件的产生来利用这些重要的毒力相关途径的工作，从而开发一套全新的抗菌治疗靶点。