Mechanisms investigations of O antigen synthesis in gram negative bacteria

机会细菌铜绿假单胞菌(PA)可导致肺部、肾脏或伤口感染死亡，并出现在许多医院获得性感染中。这些细菌通过一层碳水化合物来保护自己，碳水化合物在细菌与宿主环境之间的相互作用中发挥着重要作用。PA合成两种外部抗原性碳水化合物(O抗原)，即共同的A带和血清型特异的B带。负责组装这些碳水化合物的基因是已知的，但它们的确切功能尚未确定，主要是因为缺乏适当的方法。我们最近在NSERC的支持下开发了这些方法，并建立了一些肠道细菌、志贺氏菌和沙门氏菌基因产物的酶功能。在一个相关的项目中，我们已经开发出减少PA与肺细胞黏附的方法，这将有助于减少感染。我们这项提议的目标是了解PA中组装外层碳水化合物(O抗原)的酶的生化机制和功能。我们将使用负责O抗原合成的基因来产生酶，这些酶将被提纯并进行详细的研究。私家侦探是这一领域的专家。我们将与在PA中克隆这些基因的微生物学家合作，与几位化学家合作，帮助为这些研究合成新的碳水化合物，并与帮助分析酶的蛋白质结构专家合作。因此，我们将开发新的方法，以了解病原菌的功能有多重要。我们的长期目标是开发无毒的抑制剂来阻断特定病原体的O抗原合成，从而降低它们的毒力。四名研究生和几名本科生将接受微生物学、酶学、碳水化合物化学和蛋白质生物化学领域的培训。这些受训人员将拥有独特的专业知识，可应用于学术领域、生物技术、医学工程和药物开发。这些专业知识将对加拿大的大学、研究实验室和工业有价值。

The opportunistic bacteria Pseudomonas aeruginosa (PA), can cause death due to lung, kidney or wound infections and occur in many hospital-acquired infections. These bacteria protect themselves with an outer coat of carbohydrates that play a major role in the interactions between bacteria and their host environment. PA synthesize two types of outer antigenic carbohydrates (O antigens), the common A band and the serotype-specific B band. The genes responsible for the assembly of these carbohydrates are known but their exact functions have not been established, mainly due to lack of appropriate methods. We have recently developed these methods supported by NSERC, and have established the enzymatic functions of a number of gene products from intestinal bacteria E. coli, Shigella and Salmonella. In a related project, we have developed methods to reduce the adhesion of PA to lung cells, which will help to reduce infections. Our goal for this proposal is to understand the biochemical mechanisms and the functions of the enzymes that assemble the outer carbohydrates (O antigens) in PA. We will use the genes responsible for O antigen synthesis to produce the enzymes which will be purified and studied in detail. The P.I. is an expert in this field. We will collaborate with the microbiologist that has cloned these genes in PA, with several chemists that will help to synthesize new carbohydrates for these studies, and with a protein structure expert that will help to analyze the enzymes. We will therefore develop new methods in order to understand how important pathogenic bacteria function. Our long term goal is to develop non-toxic inhibitors to block O antigen synthesis in specific pathogens, thus reducing their virulence. Four graduate students and several undergraduate students will be trained in the fields of microbiology, enzymology, carbohydrate chemistry and protein biochemistry. These trainees will have a unique expertise that can be applied in the academic field, in biotechnology, medical engineering and drug development. This expertise and knowledge will be valuable for Universities, research laboratories and industry of Canada.