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

描述（由申请人提供）： 金黄色葡萄球菌（Staphylococcus aureus，SA）的严重感染仍然是一个重要的临床挑战，尽管使用了有效的抗生素。新的治疗进展等待阐明的分子基础的持久性，慢性和转移性扩散-即SA感染的标志。强毒株的压倒性感染和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。多形核中性粒细胞（PMN）代表细胞介导的抗微生物活性的基石，并且在吞噬体内发挥其全部抗微生物作用，其中活性氧（ROS）和颗粒内容物协作杀死和降解微生物。重要的是，由PMN产生的过氧化氢（H2 O2）被PMN颗粒蛋白髓过氧化物酶（MPO）放大以产生HOCl（漂白剂）。除了PMN之外，存在于感染动物的血浆、泪液和炎性流体中的特异性IIA族磷脂酶A2（GpIIA-PLA 2）表现出杀死和降解SA的有效活性。在VA Merit的支持下，我们在阐明摄入的SA和PMN之间相互作用的两个互补方面的特征方面取得了进展，证明了（a）PMN依赖性ROS和GpIIA-PLA 2之间的协同作用，以杀死和降解SA，以及（B）MPO-H2 O2-Cl攻击吞噬体中SA的几个特征。此外，我们已经确定了转录和结构的反应，SA后立即吞噬。我们怀疑这种变化有助于一些摄入的SA在PMN中存活并随后逃逸的能力，我们已经研究了这种现象，并且与长期的临床观察和实验数据一致。我们现在建议使用我们在VA资助的前一时期开发的工具和分析方法来扩展我们的新研究，并测试PMN吞噬体中SA对改变其细胞表面组成的反应的总体假设。（包括D-丙氨酸和心磷脂的含量）和诱导细胞质抗氧化剂（例如，甲硫氨酸亚砜还原酶和hsp 33）导致它们抵抗PMN-GpIIA-PLA 2的作用和HOCl和相关氧化剂的特异性毒性的能力，以及在PMN中存活和逃离PMN并使感染永久化的能力。我们的具体目标是：1。为了确定PMN氧化酶衍生的氧化剂对人PMN和GpIIA-PLA 2对抗SA的协同作用的具体贡献，在吞噬过程中发生了什么MPO介导的SA蛋白和磷脂修饰？吞噬体中ROS 1 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？细胞壁成分的遗传突变体，包括D-丙氨酰化或心磷脂合酶，是否更适合在PMN吞噬体中生存和逃逸？2.确定MPO-H2 O2-Cl系统如何杀死大多数SA，以及相反，存活生物体的子集如何适应以克服吞噬体中MPO衍生的细胞毒素。SA中胞质GFP的漂白是否提供了PMN吞噬体中HOCl活性的准确评估？SA中的哪些蛋白质是MPO特异性修饰的靶点，直接或间接地导致SA对PMN的易感性？被吞噬的SA修复哪些靶点？SA与突变的蛋氨酸亚砜还原酶和氧化还原敏感的伴侣hsp 33，系统响应HOCl诱导的氧化应激，或多或少容易受到细胞毒素在PMN吞噬体？对HOCl介导的损伤的抵抗是否允许SA持续存在于PMN中或从PMN中逃逸？我们预计，我们的研究将提供重要的和新的见解时发生的复杂的生物学摄入SA满足PMN吞噬体的细胞毒性内容。此外，我们认为，新的治疗干预的目标可能会被确定为我们提出的工作的结果。

DESCRIPTION (provided by applicant): 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 1 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.