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

描述(由申请人提供)： 严重感染金黄色葡萄球菌(SA)仍然是一个重要的临床挑战，尽管有强大的抗生素。新的治疗进展等待阐明持久性、慢性化和转移性传播的分子基础--即SA感染的特征。强毒株的压倒性感染和不断增加的抗生素耐药性是更好地了解宿主对SA的防御的强大动力。中性粒细胞(PMN)是细胞介导的抗菌活性的基石，在吞噬体内发挥其所有的抗菌作用，在吞噬体内，活性氧(ROS)和颗粒内容物协同作用杀死和降解微生物。重要的是，PMN产生的过氧化氢(H_2O_2)被PMN颗粒蛋白髓过氧化物酶(MPO)放大产生HOCl(漂白剂)。除了PMN外，感染动物的血浆、泪液和炎性液体中都存在一种特异性的IIA磷脂酶A2(GpIIA-PLA2)，它具有很强的杀灭和降解SA的活性。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依赖的ROS和GpIIA-PLA2之间的协同作用可以杀死和降解SA，以及(B)MPO-H_2O_2-Cl攻击吞噬体内的SA的几个特征。此外，我们已经确定了SA在吞噬后立即产生的转录和结构反应。我们怀疑这样的变化有助于一些摄入的SA在PMN中存活并随后逃脱，这一现象我们已经检查过，并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创建的工具和我们在VA资助的前一段时间开发的分析方法来扩展我们的新研究，并测试总体假设，即PMN吞噬小体中SA的反应改变其细胞表面的组成(包括D-丙氨酸和心磷脂的含量)并诱导细胞质抗氧化剂(例如蛋氨酸亚砜还原酶和Hsp33)导致其抵抗PMN-GpIIA-PLA2的作用和HOCl及相关氧化剂的特定毒性，以及在PMN中生存和逃脱PMN并持续感染。我们的具体目标是：1.为了明确PMN氧化酶衍生的氧化剂在人PMN和GpIIA-PLA2协同抗SA中的具体作用，在吞噬过程中发生了什么MPO介导的SA蛋白和磷脂的修饰？吞噬体内ROS 1MPO诱导的SA磷脂和蛋白质的哪些修饰改变了GpIIA-PLA2及其底物，或者两者都改变了？细胞壁成分中的基因突变，包括D-丙氨酰化或心磷脂合成酶，是否更适合在PMN吞噬小体中生存和逃脱？2.确定MPO-H_2O_2-Cl系统如何杀死大多数SA，相反，存活的生物亚群如何适应对克服吞噬小体中MPO衍生的细胞毒素的反应。唾液中胞浆GFP漂白是否能准确评估PMN吞噬小体中的HOCl活性？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地导致SA对PMN的易感性？吞噬的SA修复哪些靶点？甲硫氨酸亚砜还原酶和氧化还原敏感的伴侣Hsp33突变的SA是对HOCl2诱导的氧化应激做出反应的系统，还是更容易受到PMN吞噬小体中的细胞毒素的影响？对HOCl介导的损伤的抵抗是否允许SA在PMN内存活或逃逸？我们预计，我们的研究将为摄取SA遇到PMN吞噬小体的细胞毒性内容时发生的复杂生物学提供重要和新颖的见解。此外，我们认为，作为我们拟议的工作的结果，我们可能会确定治疗干预的新靶点。 公共卫生相关性： SA感染通常发生在退伍军人中，无论是门诊患者还是住院患者。2007年，退伍军人管理局医疗保健系统从153个医疗中心为500万人提供服务，出院人数约为58万人，因此是美国最大的医疗保健系统之一。根据一项以退伍军人事务部为基础的研究，金黄色葡萄球菌感染率从2000年的每千人26.93人增加到2007年的30.59人(14%)。因此，随之而来的发病率和死亡率的上升对我们的医疗保健系统构成了重大挑战。了解包括PMN和GpIIA-PLA2在内的先天免疫元件介导SA成功杀伤和降解的机制将提供基本的见解，可转化为开发新的治疗靶点。我们认为，我们对允许金黄色葡萄球菌在PMN中存活并在某些情况下逃脱的反应的逃避，可能揭示了有助于毒力但本身并不是生存所必需的基因及其产物，从而避开了识别抗生素靶标的更传统的检测方法。

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. PUBLIC HEALTH RELEVANCE: SA infections occur commonly in Veterans, both in outpatients as well as hospitalized patients. The VA healthcare system served > 5 million people in 2007 with ~580,000 discharges from 153 medical centers and is thus one of the largest healthcare systems in the US. According to a VA-based study, the prevalence of SA infections increased from 26.93 per 1,000 discharges in 2000 to 30.59 in 2007 ( 14%). Consequently, the attendant rises in morbidity and mortality present a significant challenge to our health care system. Understanding the mechanisms by which innate immune elements, including PMN and GpIIA-PLA2, mediate successful killing and degradation of SA will provide fundamental insights that can be translated into the development of novel targets for therapy. We believe that our eludication of responses that allow SA to survive in PMN, and in some cases escape, may reveal genes and their products that contribute to virulence but are not required for viability per se, thus eluding more conventional assays to identify antibiotic targets.