Pathogenic Mechanism for Lung Infection in Mucoid Pseudomonas

 描述（由申请人提供）： 许多VA患者易感染并死于机会性病原体铜绿假单胞菌的感染。这可能是肺气肿、慢性支气管炎、癌症和免疫抑制药物治疗的并发症。暴露于这种无处不在的细菌可导致医院感染，这是常见的通过呼吸道呼吸机，导管，腰椎穿刺和普通外科手术。铜绿假单胞菌对大多数抗生素具有高度耐受性或耐药性，使得难以控制此类感染，从而导致高死亡率。本研究的目的是提高我们对一种称为藻酸盐的保护性囊状多糖的生物合成的理解，藻酸盐是铜绿假单胞菌产生的毒力因子。在慢性呼吸道感染期间（例如，COPD），适应性突变在体内发生，导致这种胞外多糖的过度产生，其赋予对吞噬细胞杀伤的抗性。此类临床分离株表现出粘液样菌落形态。提高我们对铜绿假单胞菌致病机制的认识将加强对该细菌引起的肺部疾病的管理。大多数用于产生藻酸盐的酶聚集在12个基因的操纵子中。在这项研究中，我们将：（1）确定第二信使环二GMP在藻酸盐聚合中的作用，（2）表征Alg 44中的膜融合蛋白（MFP）结构域的作用，和（3）表征藻酸盐分泌蛋白之间的蛋白质-蛋白质相互作用。此外，作为药物发现计划的一部分，我们将（4）筛选那些拮抗藻酸盐乙酰化的小化合物库，这是抵抗吞噬作用所必需的。与结构生物学家合作，我们将确定所有藻酸盐生物合成蛋白的结构，这将导致对定点突变体的分析，以更好地了解聚合-分泌复合物。这项研究的长期目标是了解海藻酸盐生物合成所需的所有组分的功能，海藻酸盐现在被认为是肺部感染期间的关键毒力因子。所获得的信息对于开发治疗铜绿假单胞菌感染的新治疗方法至关重要。这些研究的结果也将有助于我们对细菌荚膜生物合成的全面了解，这是细菌毒力避免宿主免疫反应的常见机制。

 DESCRIPTION (provided by applicant): Many VA patients are susceptible and succumb to infections with the opportunistic pathogen, Pseudomonas aeruginosa. This can occur as a complication of emphysema, chronic bronchitis, cancer and immunosuppressive drug therapy. Exposure to this ubiquitous bacterium can result in nosocomial infections, which are common via respiratory ventilators, catheters, lumbar punctures and general surgery. P. aeruginosa is highly tolerant or resistant to most antibiotics, making it difficult to control such infections, which leads to a high mortality rate. The goal of tis research is to improve our understanding of the biosynthesis of a protective capsule-like polysaccharide called alginate, which is produced as a virulence factor by P. aeruginosa. During chronic respiratory infections (e.g., COPD), adaptive mutations occur in vivo that lead to the over production of this exopolysaccharide, which confers resistance to phagocytic killing. Such clinical isolates demonstrate mucoid colony morphology. Improving our understanding of this pathogenic mechanism in P. aeruginosa will enhance the management of pulmonary disease caused by this bacterium. Most of the enzymes for the production of alginate are clustered in an operon of 12 genes. In this study, we will: (1) Determine the role of the second messenger cyclic di-GMP in the polymerization of alginate, (2) Characterize the role of the membrane fusion protein (MFP) domain in Alg44, and (3) Characterize the protein-protein interactions among the alginate secretion proteins. In addition, as part of a drug discovery plan, we will (4) screen smal compound libraries for those that antagonize the acetylation of alginate, which is required for resistance to phagocytosis. In collaboration with a structural biologist, we will determine the structures of all the alginate biosynthetic proteins, which will lead to the analysis of site-direced mutants to better understand the polymerization-secretion complex. The long-term goal of this research is to understand the functions of all of the components required for the biosynthesis of alginate, which is now recognized as a critical virulence factor during pulmonary infection. The information gained could be vital for the development of new therapeutic approaches in the treatment of P. aeruginosa infections. The results of these studies will also contribute to our overall understanding of bacterial capsule biosynthesis, which is a common mechanism of bacterial virulence for avoiding the host immune response.