Mechanisms of Pseudomonas Resistance to Membrane Permeabilization by SP-A

描述(由申请人提供)：传统上，肺表面活性物质蛋白-A(SP-A)被认为能调理和促进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的伤害尚不清楚。特别是，我们发现野生型PA菌株PA01对SP-A的膜通透性具有抗性。我们的长期目标是了解SP-A的抗菌机制，并揭示细菌如何对SP-A产生耐药性/敏感性。有待检验的总体假设是，PA途径包括鞭毛、水杨酸盐和绿球蛋白，以及磷酸烯醇-丙酮酸磷酸转移酶，对于抵抗SP-A的膜通透性是重要的。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，缺乏鞭毛(FlgE)、缺乏水杨酸和绿球蛋白生物合成(PchA)、以及产生磷酸烯醇-丙酮酸磷酸转移酶(PtsP)的PA突变株，在SP-A+/+小鼠肺中优先被清除，但在SP-A-/-小鼠肺中存活。最引人注目的是，flgE、pchA和ptsP突变细菌对SP-A介导的膜通透性的敏感性显著增加，但没有调理作用。我们提出了三个目标，以研究鞭毛、PchA和PtsP途径维持内毒素和细胞膜完整性的机制，调节使SP-A失活的细菌过程，赋予对SP-A介导的膜通透性的杀伤抗性，以及对SP-A和其功能依赖或辅助的抗菌肽和蛋白(AMPP)的杀伤作用。目的1将确定PA的鞭毛、PchA和PtsP协调的“攻击性”策略，以提供对SP-A介导的膜通透性的抵抗。这些攻击性措施包括PA分泌降解SP-A的蛋白酶的能力，以及分泌水杨酸盐的能力，水杨酸盐剥离与SP-A相关的钙离子，这是SP-A活动所必需的。目的2将确定由PA的鞭毛、水杨酸盐/绿球蛋白和磷酸烯醇-丙酮酸磷酸转移酶协调的“防御”策略，以增强对SP-A介导的膜通透性的抗性。有待确定的防御措施包括增加内毒素的生物合成和修饰，以及趋化逃避。目标3将检验这一假设，即在最初的相互作用中，SP-A先发制人地“麻痹”PA，允许其他AMPP协同或相加地作用于杀死细菌。我们将使用“棋盘”分析来确定鞭毛、PchA和PtsP途径对单个AMPP、单独AMPP、AMMP不同组合以及AMMP与SP-A的协同和相加杀伤作用。完成拟议的目标将加强我们对SP-A的抗微生物机制的了解，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，也是免疫功能低下患者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拒绝肺移植、感染和死亡，这突显了迫切需要探索替代策略来管理铜绿假单胞菌感染。加强对表面活性蛋白A的抗菌机制的了解，以及铜绿假单胞菌对表面活性蛋白A的耐药性/敏感性的机制，可能会导致威胁生命的肺炎的新治疗策略。

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.