GENETICS OF PSEUDOMONAS AERUGINOSA LIPOPOLYSACCHARIDES

铜绿假单胞菌的脂多糖（LPS）是一种 免疫显性抗原和这种机会的主要毒力因子 致病菌 我们正在研究LPS O的遗传基础 铜绿假单胞菌中的抗原产生。 显然， 了解这种病原体毒力的潜在机制， O抗原的产生和变异显然是这方面的最前沿 过程 虽然有20个公认的铜绿假单胞菌血清群， 其单糖组成和结构彼此不同 在O抗原重复单位中，这些血清群中有10个负责 对于绝大多数的感染。 制定有效 预防或治疗铜绿假单胞菌感染的疫苗接种策略 随着抗生素和目前治疗的极限达到， O-抗原特异性抗体对铜绿假单胞菌具有保护作用 然而，观察到的保护通常是血清群特异性的。 我们已经部分实现的长期目标之一是， 通过肠道微生物引起铜绿假单胞菌O抗原产生， 用作口服疫苗。 我们已经克隆了 铜绿假单胞菌菌株O抗原（rfb基因簇）的表达 PA103，血清群11菌株，因此，代表了大多数 在环境菌株中常见的血清群， 通常是导致感染的原因 我们已经表达了铜绿假单胞菌 血清群11 O抗原对大肠杆菌和沙门氏菌，以及， 将这些重组生物体口服给小鼠， 对随后用血清群11铜绿假单胞菌攻击的保护。 这些结果表明，这种方法的可行性，以建立一个 有效的疫苗，并代表的具体目标的基础上， 目前的建议：比较编码两种不同rf的克隆基因 铜绿假单胞菌的基因簇（来自血清组11和血清组5）， 遗传组织方面，O抗原所需的蛋白质数量 表达，以及共同和独特的基因的功能， 这些血清型 这一信息将促进我们的基本理解 铜绿假单胞菌LPS生产的生物学，并允许我们开发 实现我们长期目标所需的工具。 这些包括 确定铜绿假单胞菌的O抗原在 毒力，无论是在急性感染和慢性肺部感染， 囊性纤维化患者。 我们将能够创造出同基因的 仅在O抗原结构上不同的菌株，并检查这些菌株 在动物模型和体外，研究LPS与 宿主免疫系统效应物如补体和吞噬细胞。 我们 也希望能研制出抗铜绿假单胞菌的重组口服疫苗 基于我们克隆任何血清型的O抗原基因簇的能力， 这个有机体。 通过表达许多不同的铜绿假单胞菌血清型 在沙门氏菌菌株中，我们将制备重组口服"鸡尾酒" 对临床感染中重要的所有血清群具有保护作用。 与其他铜绿假单胞菌疫苗不同，这些重组口服疫苗将 具有遗传上确定、易于施用和 耐受性良好，并且应该能够在 各种粘膜部位。 这些研究还将为我们提供新的见解， 铜绿假单胞菌LPS O抗原的表达及其基因调控。

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa is an immunodominant antigen and a major virulence factor of this opportunisti pathogenic bacterium. We are investigating the genetic basis of LPS O antigen production in P. aeruginosa. There is an obvious interest in understanding the mechanisms underlying this pathogen's virulence, and O antigen production and variation are clearly at the forefront of this process. While there are twenty recognized serogroups of P. aeruginosa that differ from one another in monosaccharide composition and structure of the O antigen repeating units, ten of these serogroups are responsibl for the vast majority of infections. Development of effective vaccination strategies to prevent or treat P. aeruginosa infections are needed as the limits of antibiotic and current therapies are reached. O-antigen-specific antibodies are protective against P. aeruginosa infections, however the protection observed is often serogroup specific. One of our long-term objectives, already partially realized, will be to elicit P. aeruginosa O antigen production by enteric organisms that can be used as oral vaccines. We have cloned the genes required for expression of O antigen (the rfb gene cluster) from P. aeruginosa strain PA103, a serogroup 11 strain and, as such, representative of the most commonly found serogroup among environment strains, and one which is often responsible for infections. We have expressed the P. aeruginosa serogroup 11 O antigen on Escherichia coli and Salmonella, and, after oral delivery of these recombinant organisms to mice, have elicited protection against subsequent challenge with serogroup 11 P.aeruginosa. These results indicate the feasibility of such an approach to create an effective vaccine, and represent the basis for the specific aims of the current proposal: to compare the cloned genes encoding two different rf gene clusters of P. aeruginosa (from serogroup 11 and serogroup 5) in terms of genetic organization, number of proteins required for O antigen expression, and the functions of the genes that are common and unique to these serogroups. This information will advance our basic understanding of the biology of P. aeruginosa LPS production and allow us to develop the tools necessary to realize our long-term goals. These include determining the role that the O antigen of P. aeruginosa plays in virulence, both in acute infections and in chronic lung infections of patients with cystic fibrosis. We will be able to create isogenic strains differing only in O antigen structure, and examine these strains in animal models and in vitro, investigating the interaction of LPS with host immune system effectors such as complement and phagocyte cells. We also hope to develop a recombinant oral vaccine against P. aeruginosa based on our ability to clone the O antigen gene cluster of any serogrou of this organism. By expressing many different P. aeruginosa serogroups in Salmonella strains, we will prepare a recombinant oral 'cocktail' protective against all serogroups important in clinical infections. Unlike other P. aeruginosa vaccines, these recombinant oral vaccines wil have the advantage of being genetically defined, easily administered and well tolerated, and should be capable of eliciting immune responses at various mucosal sites. These studies will also provide new insights int the expression and genetic regulation of LPS O antigen by P. aeruginosa.