Mechanisms of Pseudomonas Resistance to Membrane Permeabilization by SP-A

假单胞菌对 SP-A 膜透化的抗性机制

• 批准号: 8049617
• 负责人: Gee W Lau
• 金额: $ 39.01万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049617
• 关键词: A Mouse; Anabolism; Antibiotic Resistance; Bacteria; Binding; Biological Assay; Calcium; Carbohydrates; Cause of Death; Cell membrane; Cessation of life; Chelating Agents; Chemotaxis; Collectins; Data; Employee Strikes; Flagella; Goals; Health; Human; Individual; Infection; Lead; Libraries; Life; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Lung; Measures; Mediating; Membrane; Microbe; Modification; Mutagenesis; Nosocomial Infections; Paralysed; Pathway interactions; Peptide Hydrolases; Permeability; Phosphotransferases; Pneumonia; Predisposition; Process; Proteins; Pseudomonas; Pseudomonas aeruginosa; Publishing; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Pyruvate; Relative (related person); Reporting; Resistance; Role; Sepsis; Testing; Transplantation; antimicrobial; antimicrobial peptide; comparative; cystic fibrosis patients; killings; macrophage; microbial; mutant; pathogen; pyochelin; salicylate; treatment strategy

DESCRIPTION (provided by applicant): Traditionally, the pulmonary surfactant protein-A (SP-A) is thought to opsonize and enhance the clearance of microbial pathogens. Recently, we have reported that SP-A also directly kills Pseudomonas aeruginosa (PA) in a macrophage-independent manner, by increasing the permeability of bacterial membranes. However, the mechanism by which SP-A disrupts PA cell membranes and its relative importance in lung defense are poorly defined. In addition, how microbes protect themselves against SP-A is unknown. Especially, we have shown that wild-type PA strain PA01 is resistant to membrane permeabilization by SP-A. Our long-term goal is to understand the antimicrobial mechanisms of SP-A, and to reveal how bacteria confer resistance/susceptibility to SP-A. The overall hypothesis to be tested is that PA pathways including flagellum, salicylate and pyochelin, and phosphoenol-pyruvate phosphotransferase, are important to resist membrane permeabilization by SP-A. Our hypothesis is supported by our published and preliminary data which show that PA mutant strains deficient in flagellum (flgE), deficient in salicylate and pyochelin biosynthesis (pchA), and defective in producing phosphoenol-pyruvate phosphotransferase (ptsP), are preferentially cleared in the SP-A+/+ mouse lungs, but survived in the SP-A-/- mouse lungs. Most strikingly, the flgE, pchA and ptsP mutant bacteria show significant increase in susceptibility to SP-A mediated membrane permeabilization, but not opsonization. We propose three aims to examine the mechanisms by which flagellum, PchA and PtsP pathways maintain LPS and cell membrane integrity, and regulate the bacterial processes that inactivate SP-A, to confer resistance to killing by SP-A-mediated membrane permeabilization and to killing by SP-A and antimicrobial peptides and proteins (AMPPs) whose functions that are either dependent or aided by SP-A. Aim 1 will determine the "offensive" strategies orchestrated by PA's flagellum, PchA and PtsP to confer resistance to SP-A-mediated membrane permeabilization. These offensive measures to be tested include the ability of PA to secrete proteases that degrade SP-A, and to secrete salicylate that strips Ca2+ associated with SP-A, which is required for the activity of SP-A. Aim 2 will determine the "defensive" strategies orchestrated by PA's flagellum, salicylate/pyochelin and phosphoenol-pyruvate phosphotransferase to confer resistance to SP-A-mediated membrane permeabilization. The defensive measures to be determined include increased LPS biosynthesis and modification, and chemotaxis evasion. Aim 3 will examine the hypothesis that during the initial interactions, SP-A preemptively "paralyzes" PA, allowing other AMPPs to act synergistically or additively to kill the bacteria. We will use "checker board" assays to determine the roles of flagellum, PchA and PtsP pathways against individual, synergistic and additive killing by individual AMPP alone, different combinations of AMMPs, and AMMPs with SP-A. Completion of the proposed aims will enhance our understanding of the antimicrobial mechanisms of the SP-A and lead to new treatment strategies for pneumonias. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections in humans, lung infections in cystic fibrosis patients, and a primary cause of death and sepsis in immuno-compromised individuals. The continuous emergence of antibiotic resistant P. aeruginosa, which can lead to denial for lung transplant, infection and death, emphasize the urgent need to explore alternative strategies to manage P. aeruginosa infections. Enhance understanding of the antimicrobial mechanisms of the Surfactant Protein A, and the mechanisms by which P. aeruginosa confers resistance/susceptibility to Surfactant Protein A may lead to new treatment strategies for life-threatening pneumonias.

描述（由申请人提供）：传统上，肺表面活性蛋白-A（SP-A）被认为具有调理作用并增强微生物病原体的清除。最近，我们报道了 SP-A 还通过增加细菌膜的通透性，以不依赖于巨噬细胞的方式直接杀死铜绿假单胞菌 (PA)。然而，SP-A 破坏 PA 细胞膜的机制及其在肺防御中的相对重要性尚不清楚。此外，微生物如何保护自己免受 SP-A 的侵害尚不清楚。特别是，我们已经证明野生型 PA 菌株 PA01 对 SP-A 的膜透化具有抗性。我们的长期目标是了解 SP-A 的抗菌机制，并揭示细菌如何赋予 SP-A 耐药性/敏感性。待测试的总体假设是，PA 途径（包括鞭毛、水杨酸和绿脓杆菌素）以及磷酸烯醇丙酮酸磷酸转移酶对于抵抗 SP-A 的膜透化作用非常重要。我们的假设得到了我们已发表的初步数据的支持，这些数据表明，鞭毛缺陷（flgE）、水杨酸和绿脓素生物合成（pchA）缺陷以及磷酸烯醇丙酮酸磷酸转移酶（ptsP）产生缺陷的PA突变株优先在SP-A+/+小鼠肺中被清除，但在SP-A+/+小鼠肺中存活。 SP-A-/- 小鼠肺。最引人注目的是，flgE、pchA 和 ptsP 突变细菌对 SP-A 介导的膜透化作用的敏感性显着增加，但调理作用却没有显着增加。我们提出了三个目标，即检查鞭毛、PchA 和 PtsP 途径维持 LPS 和细胞膜完整性的机制，并调节使 SP-A 失活的细菌过程，以赋予对 SP-A 介导的膜透化作用的杀伤作用以及对 SP-A 和抗菌肽和蛋白质 (AMPP) 的杀伤作用的抵抗力，这些作用依赖于或辅助于 SP-A 和抗菌肽和蛋白质 (AMPP)。 SP-A。目标 1 将确定 PA 鞭毛、PchA 和 PtsP 精心策划的“进攻”策略，以赋予对 SP-A 介导的膜透化的抵抗力。这些待测试的进攻性措施包括PA分泌降解SP-A的蛋白酶的能力，以及分泌剥离与SP-A相关的Ca2+的水杨酸的能力，这是SP-A活性所必需的。目标 2 将确定 PA 鞭毛、水杨酸/绿脓素和磷酸烯醇丙酮酸磷酸转移酶精心策划的“防御”策略，以赋予对 SP-A 介导的膜透化的抵抗力。待确定的防御措施包括增加LPS生物合成和修饰以及趋化性逃避。目标 3 将检验以下假设：在初始相互作用期间，SP-A 先发制人地“瘫痪”PA，从而允许其他 AMPP 协同或相加地发挥作用来杀死细菌。我们将使用“棋盘”测定法来确定鞭毛、PchA 和 PtsP 途径对单独 AMPP、AMMP 的不同组合以及 AMMP 与 SP-A 的个体、协同和加性杀伤的作用。完成所提出的目标将增强我们对 SP-A 抗菌机制的理解，并带来新的肺炎治疗策略。公共卫生相关性：铜绿假单胞菌是人类医院感染、囊性纤维化患者肺部感染以及免疫功能低下个体死亡和败血症的主要原因之一。抗生素耐药性铜绿假单胞菌的不断出现，可能导致肺移植失败、感染和死亡，强调迫切需要探索管理铜绿假单胞菌感染的替代策略。增强对表面活性蛋白 A 抗菌机制以及铜绿假单胞菌对表面活性蛋白 A 产生耐药性/敏感性的机制的了解，可能会为危及生命的肺炎带来新的治疗策略。