Mechanisms of Pseudomonas Resistance to Membrane Permeabilization by SP-A

描述（由申请人提供）：传统上，肺表面活性剂蛋白- a （SP-A）被认为是调理和增强微生物病原体的清除。最近，我们报道SP-A还通过增加细菌膜的通透性，以一种不依赖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的膜渗透具有抗性。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/易感性。需要验证的总体假设是，PA途径包括鞭毛、水杨酸和绿嘌呤，以及磷酸烯醇-丙酮酸磷酸转移酶，对抵抗SP-A的膜透性是重要的。我们的假设得到了我们已发表和初步数据的支持，这些数据表明，PA突变株鞭毛（flgE）缺陷、水杨酸和绿嘌呤生物合成（pchA）缺陷以及产生磷酸烯醇-丙酮酸磷酸转移酶（ptsP）缺陷，在SP-A+/+小鼠肺中被优先清除，但在SP-A-/-小鼠肺中存活。最引人注目的是，flgE、pchA和pgp突变菌对SP-A介导的膜渗透的敏感性显著增加，但对opsonization的敏感性没有增加。我们提出了三个目标来研究鞭毛、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，赋予SP-A介导的膜渗透杀伤和SP-A和抗菌肽和蛋白（AMPPs）的杀伤的抗性，其功能依赖或辅助SP-A。目的1将确定由PA的鞭毛、PchA和psp精心策划的“进攻”策略，以赋予对sp - a介导的膜渗透的抗性。要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，以及分泌去除SP-A相关的Ca2+的水杨酸盐的能力，这是SP-A活性所必需的。目的2将确定由PA的鞭毛、水杨酸/吡啶酸和磷酸烯醇-丙酮酸磷酸转移酶精心策划的“防御”策略，以赋予对sp - a介导的膜渗透的抗性。需要确定的防御措施包括增加LPS的生物合成和修饰，以及趋化性规避。目的3将检验在最初的相互作用中，SP-A先发制人地“麻痹”PA的假设，允许其他AMPPs协同或添加作用以杀死细菌。我们将使用“检查板”分析来确定鞭毛、PchA和PtsP途径对单个、协同和单独AMPP、不同amps组合以及与SP-A一起使用的amps的作用。完成所提出的目标将增强我们对SP-A的抗菌机制的理解，并导致肺炎的新治疗策略。公共卫生相关性：铜绿假单胞菌是人类院内感染、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。耐药铜绿假单胞菌的不断出现，可导致肺移植拒绝，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。进一步了解表面活性剂蛋白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/易感性的机制，可能会为危及生命的肺炎提供新的治疗策略。

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.