Human neutrophils, phospholipase A2 and S.aureus: microbial targets and responses

金黄色葡萄球菌（SA）的严重感染仍然是一个重要的临床挑战， 抗生素新的治疗进展等待阐明的分子基础的持久性，慢性， 和转移扩散-即SA感染的标志。强毒株的压倒性感染， 抗生素耐药性的增加是更好地了解宿主对SA防御的有力诱因。 多形性中性粒细胞（PMN）代表细胞介导的抗菌活性的基石 并在吞噬体内发挥其所有的抗菌作用，其中活性氧（ROS）和 颗粒内容物协同杀死和降解微生物。重要的是，产生的过氧化氢（H2 O2） 通过PMN颗粒蛋白髓过氧化物酶（MPO）扩增以产生HOCl（漂白剂）。在 除了PMN外，还存在于感染者血浆中的特异性IIA族磷脂酶A2（GpIIA-PLA 2）。 动物、泪液和炎性液体，表现出杀死和降解SA的有效活性。 在VA Merit的支持下，我们在阐明以下两个互补方面的特征方面取得了进展： 摄入的SA和PMN之间的相互作用，证明（a）PMN依赖性ROS之间的协同作用 （B）MPO-H_2 O_2-Cl对SA的几种攻击特性 吞噬体此外，我们已经确定了转录和结构反应的SA立即 吞噬作用后。我们怀疑，这种变化有助于一些摄入的SA的能力， 在中性粒细胞中存活，随后逃逸，我们已经研究过的现象， 长期的临床观察和实验数据。 我们现在建议使用我们在此期间创建的工具和开发的分析方法 前一时期的VA资金，以扩大我们的新的研究和测试的整体假设， 中性粒细胞吞噬体中SA改变其细胞表面组成的反应（包括 D-丙氨酸和心磷脂）和诱导细胞质抗氧化剂（例如甲硫氨酸亚砜还原酶和 hsp 33）导致其抵抗PMN-GpIIA-PLA 2的作用和HOCl的特异性毒性的能力， 相关的氧化剂，并在PMN中生存和逃脱，并使感染持续下去。我们的具体目标是： 1.目的：明确PMN氧化酶衍生的氧化剂对人体免疫系统协同作用的具体贡献。 PMN和GpIIA-PLA 2抗SA 在吞噬过程中，MPO介导的SA蛋白和磷脂的修饰发生了什么？ 吞噬体中ROS和MPO诱导的SA磷脂和蛋白质的哪些修饰改变了GpIIA- PLA 2，其底物，或两者？是细胞壁成分的遗传突变体，包括D-丙氨酰化或 心磷脂合酶，更好地装备生存和逃离PMN吞噬体？ 2.为了确定MPO-H2 O2-Cl系统如何杀死大多数SA，以及相反， 存活的生物体适应于应答以克服吞噬体中MPO衍生的细胞毒素。 SA中胞浆GFP的漂白是否提供了PMN中HOCl活性的准确评估 吞噬体？SA中的哪些蛋白质是MPO特异性修饰的靶点;哪些蛋白质直接或 间接影响SA对PMN的敏感性。被吞噬的SA修复哪些靶点？SA与 甲硫氨酸亚砜还原酶和氧化还原敏感性分子伴侣HSP 33中的突变， 对HOCl诱导的氧化应激有反应，或多或少容易受到中性粒细胞吞噬体中的细胞毒素的影响？并 抗HOCl介导的损害允许SA坚持或逃离PMN？ 我们预计，我们的研究将为复杂的生物学提供重要而新颖的见解， 当摄入的SA满足PMN吞噬体的细胞毒性内容物时发生。此外，我们认为， 作为我们提出的工作的结果，可以鉴定用于治疗干预的新靶点。

Serious infection with Staphylococcus aureus (SA) remains an important clinical challenge despite potent antibiotics. Novel therapeutic advances await elucidation of the molecular bases for persistence, chronicity, and metastatic spread - i.e. the hallmarks of SA infection. Overwhelming infection with virulent strains and increasing antibiotic resistance are powerful incentives to understand better the host defense against SA. Polymorphonuclear neutrophils (PMN) represent the cornerstone of cell-mediated antimicrobial activity and exert ~ all of their antimicrobial effort within phagosomes, where reactive oxygen species (ROS) and granule contents collaborate to kill and degrade microbes. Importantly, hydrogen peroxide (H2O2) produced by PMN is amplified by the PMN granule protein myeloperoxidase (MPO) to generate HOCl (bleach). In addition to PMN, a specific Group IIA phospholipase A2 (GpIIA-PLA2), which is present in plasma of infected animals, tears, and inflammatory fluid, exhibits potent activity to kill and degrade SA. With VA Merit support, we have made progress in elucidating features of two complementary aspects of interactions between ingested SA and PMN, demonstrating (a) a synergy between PMN-dependent ROS and GpIIA-PLA2 to kill and degrade SA, and (b) several characteristics of MPO-H2O2-Cl attack on SA in phagosomes. Furthermore, we have identified transcriptional and structural responses by SA immediately following phagocytosis. We suspect that such changes contribute to the capacity of some ingested SA to survive in PMN and subsequently escape, phenomena we have examined and are consistent with longstanding clinical observations and experimental data . We propose now to use tools that we have created and analytical methods we have developed during the previous period of VA funding to extend our novel studies and test the overall hypothesis that the responses of SA in the PMN phagosome to modify the composition of their cell surface (including content of D-alanine and cardiolipin) and to induce cytoplasmic anti-oxidants (e.g. methionine sulfoxide reductase and hsp33) result in their capacity to resist actions of PMN- GpIIA-PLA2 and the specific toxicity of HOCl and related oxidants, and to survive in, and escape from, PMN and perpetuate infection. Our Specific Aims are: 1. To define the specific contributions of the PMN oxidase-derived oxidants to the synergy of human PMN and GpIIA-PLA2 against SA What MPO-mediated modifications of SA proteins and phospholipids occur during phagocytosis? What modifications in SA phospholipids and proteins induced by ROS ¿ MPO in the phagosome alter GpIIA- PLA2, its substrates, or both? Are genetic mutants in cell wall constituents, including D-alanylation or cardiolipin synthase, better equipped to survive in and escape from the PMN phagosome? 2. To determine how the MPO-H2O2-Cl system kills most SA and, conversely, how the subset of surviving organisms adapt to respond to overcome MPO-derived cytotoxins in the phagosome. Does bleaching of cytoplasmic GFP in SA provide accurate assessment of HOCl activity in PMN phagosome? What proteins in SA are targets for MPO-specific modifications; which contribute, directly or indirectly, to susceptibility of SA to PMN? What targets are repaired by phagocytosed SA? Are SA with mutations in methionine sulfoxide reductases and the redox-sensitive chaperone hsp33, systems that respond to HOCl-induced oxidant stress, more or less vulnerable to cytotoxins in PMN phagosomes? Does resistance to HOCl-mediated damage allow SA to persist in or escape from PMN? We anticipate that our studies will provide important and novel insights into the complex biology that occurs when ingested SA meet the cytotoxic contents of the PMN phagosome. In addition, we believe that novel targets for therapeutic intervention may be identified as a result of our proposed work.