Inhibition of prokaryote-specific saccharide biosynthesis in microbial pathogens

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的新方法。就在过去几年中，细菌细胞表面糖缀合物，包括许多医学上相关的革兰氏阴性细菌病原体的外细胞壁脂多糖成分和细胞表面N-和o -连接糖蛋白，已经在分子细节上进行了表征，并发现对毒力和致病性至关重要。该提案旨在进一步定义和利用产生这些糖缀合物中发现的不寻常的微生物特异性碳水化合物构建块的途径，从而提供一种对抗细菌病原体的新方法。本研究的重点是在空肠梭菌和淋病奈索菌的N-和o -连接蛋白糖基化途径中，对参与将UDP- glcnac转化为UDP-二-N-乙酰基-杆菌胺（UDP- diNAcBac）的酶进行抑制剂的开发和体外和体内验证。由于UDP-diNAcBac是导致细菌糖缀合物生物合成途径的关键中间体，因此这些抑制剂可以作为选择性化学工具来阐明高度修饰的糖类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。结构导向片段筛选（FBS）策略在开发强效UDP-diNAcBac生物合成抑制剂中的应用2. 优化抑制剂在空肠梭菌和淋病奈瑟菌体内糖蛋白生物合成、细胞毒性及抑制糖蛋白生物合成对原生生物运动、粘附和侵袭的影响的评价3. 秀丽隐杆线虫模型对空肠隐杆线虫和淋病奈瑟菌感染性和毒力的影响。如果成功，该动物模型系统将对评估简单宿主的抑制活性有价值；4. 空肠梭菌UDP-diNAcBac生物合成抑制剂在鸡传染性模型中的作用评估（与阿尔伯塔大学的Szymanski合作）。这项研究解决了一个中心假设，即致病菌中的生物合成途径导致高度修饰的糖构建块，如二n -乙酰-杆菌胺，是可以在对抗传染病的战斗中利用的“阿喀琉斯之踵”。我们在这些研究中发展的一般原则将适用于其他将原核特异性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，这项研究将在面对不断升级的抗生素耐药性的全球对抗传染病危机中确定新的酶靶点和策略。

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.