Mechanisms of Pseudomonas Resistance to Membrane Permeabilization by SP-A

描述（由申请人提供）：传统上认为肺表面活性蛋白-A（SP-A）可调理和增强微生物病原体的清除。最近，我们报道了SP-A也直接杀死铜绿假单胞菌（PA）的巨噬细胞非依赖性的方式，通过增加细菌膜的渗透性。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。特别地，我们已经表明野生型PA菌株PA 01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对SP-A的抗性/敏感性。待测试的总体假设是PA途径（包括鞭毛、水杨酸盐和绿脓菌螯铁蛋白以及磷酸烯醇-丙酮酸磷酸转移酶）对于抵抗SP-A的膜透化作用是重要的。我们的假设得到了我们发表的和初步的数据的支持，这些数据表明，鞭毛缺陷（flgE）、水杨酸盐和绿脓菌螯铁蛋白生物合成缺陷（pchA）以及产生磷酸烯醇丙酮酸磷酸转移酶缺陷（ptsP）的PA突变株在SP-A+/+小鼠肺中优先清除，但在SP-A-/-小鼠肺中存活。最引人注目的是，flgE、pchA和ptsP突变体细菌显示对SP-A介导的膜透化的敏感性显著增加，但不显示调理作用。我们提出了三个目标，以检查鞭毛，PchA和PtsP途径维持LPS和细胞膜完整性的机制，并调节细菌的过程，使SP-A，赋予抗性杀死SP-A介导的膜透化和杀死SP-A和抗菌肽和蛋白质（AMPPs），其功能是依赖或辅助SP-A。目的1将确定由PA的鞭毛，PchA和PtsP编排的“进攻”策略，以赋予对SP-A介导的膜透化的抗性。待测试的这些攻击性措施包括PA分泌降解SP-A的蛋白酶的能力，以及分泌剥离SP-A活性所需的与SP-A相关的Ca 2+的水杨酸盐的能力。目的2将确定由PA的鞭毛、水杨酸盐/绿脓菌螯铁蛋白和磷酸烯醇丙酮酸磷酸转移酶协调的“防御”策略，以赋予对SP-A介导的膜透化的抗性。待确定的防御措施包括增加LPS的生物合成和修饰，以及趋化性逃避。目的3将检验在最初的相互作用期间，SP-A抢先“麻痹”PA，允许其他AMPs协同或相加地作用以杀死细菌的假设。我们将使用“棋盘”测定法来确定鞭毛、PchA和PtsP通路对单独AMPP、AMMP的不同组合以及AMMP与SP-A的单独、协同和相加杀伤的作用。完成拟议的目标将提高我们对SP-A抗菌机制的理解，并导致新的肺炎治疗策略。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的最常见原因之一，也是免疫功能低下个体死亡和败血症的主要原因。抗生素耐药铜绿假单胞菌的不断出现，可能导致肺移植、感染和死亡的拒绝，强调迫切需要探索管理铜绿假单胞菌感染的替代策略。加强对表面活性剂蛋白A的抗菌机制以及铜绿假单胞菌对表面活性剂蛋白A产生耐药性/敏感性的机制的理解，可能会为危及生命的肺炎带来新的治疗策略。

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.