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，抗生素耐药性是与微生物病原体作斗争的关键问题。鲜为人知的是抗菌治疗中新方法的前进道路。仅在过去的几年中，细菌细胞表面糖偶联物，包括外部细胞壁和细胞表面N和O链状糖蛋白的脂多糖成分的许多医学相关的革兰氏阴性细菌病原体，已经以分子细节表征，并且是对毒素和毒素的必不可少的表征。该提案旨在进一步定义和利用产生在这些糖缀合物中发现的异常微生物特异性碳水化合物的构建块的途径，从而为对抗细菌病原体提供了一种新颖的方法。这项研究专门针对抑制剂的开发，体外和体外验证，以在N-和O-O-C. jejuni和N.N.N.N.N.N.N.N.N.GONORHO的N-和O-Oinked蛋白蛋白糖基途径中转化为UDP-GLCNAC转化为UDP-DI-N-乙酰基 - 巴基洛胺（UDP-Dinacbac）。由于UDP-DinacBAC是导致细菌糖缀合物生物合成的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，以阐明高度修饰的糖水在微生物病原体中的基本作用。提出的研究的实验方法涉及：1。在结构引导的基于片段的筛选（FBS）策略中的应用来开发有效的UDP-Dinacbac生物合成抑制剂； 2。评估优化抑制剂的评估，即在探针糖蛋白生物合成，细胞毒性以及抑制糖蛋白生物合成对运动生物体运动的运动性，Jejuni和N. Gonorrhoeae的运动生物体的运动，粘附和侵袭的影响； 3。建立了秀丽隐杆线虫模型，用于Jejuni和N. Gonorrhoeae的感染力和毒力。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。评估Jejuni C. udp-dinacbac生物合成抑制剂在雏鸡感染模型中与艾伯塔大学的Szymanski合作的评估。这项研究探讨了以下中心假设：致病细菌中的生物合成途径导致高度改良的糖构建块，例如Di-n-乙酰基 - 巴基洛胺，代表了一种“ acchilles'heel”，可以在反感染疾病的战斗中被利用。我们在这些研究中开发的一般原则将适用于实施原核生物特异性N和O-连接糖蛋白作为毒力因子的其他微生物病原体。如果成功，这项研究将确定面对抗生素耐药性的全球危机中的新酶目标和策略。公共卫生相关性：通常意味着人类已经使用了半个世纪来击败细菌敌人，因为普通微生物病原体的抗生素耐药性对公共卫生构成了日益增长的威胁。最近的研究阐明了细胞表面糖蛋白的产生途径，这些途径在致命的革兰氏阴性细菌中起着关键作用，从而使其访问并攻击人类细胞。我们的建议将支持通过阻止糖缀合物组件中关键构件的产生来利用这些基本毒力相关途径的工作，从而开发了一套全新的抗菌治疗靶标。