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

人中性粒细胞、磷脂酶 A2 和金黄色葡萄球菌：微生物靶标和反应

• 批准号: 8195608
• 负责人: William M. Nauseef
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8195608
• 关键词: Abbreviations; Alanine; Animals; Antibiotic Resistance; Antibiotics; Antioxidants; Biological Assay; Biology; Cardiolipins; Caring; Cell Wall; Cell surface; Cells; Characteristics; Clinical; Complex; Cytoplasmic Granules; Cytotoxin; Data; Development; Elements; Epidemic; Event; Exhibits; Funding; Generations; Genes; Genetic; Green Fluorescent Proteins; Healthcare Systems; Host Defense; Human; Hydrogen Peroxide; Immune; Immune system; Incentives; Infection; Inflammatory; Liquid substance; Mediating; Medical; Medical center; Methionine; Microbe; Modification; Molecular; Molecular Chaperones; Morbidity - disease rate; Mutation; Organism; Outpatients; Oxidants; Oxidases; Oxidation-Reduction; Pathogenesis; Patients; Peroxidases; Phagocytosis; Phagosomes; Phosphatidyl glycerol; Phospholipase A2; Phospholipids; Plasma; Predisposition; Prevalence Study; Proteins; Reactive Oxygen Species; Resistance; Sodium Azide; Staphylococcus aureus; System; Testing; Therapeutic Intervention; Toxic effect; Translating; Veterans; Virulence; Virulent; Work; analytical method; antimicrobial; base; cardiolipin synthase; cytotoxic; diphenyleneiodonium; group IIA phospholipase A2; human PLA2G2A protein; insight; killings; meetings; methicillin resistant Staphylococcus aureus; methionine sulfoxide; methionine sulfoxide reductase; microbial; mortality; mutant; neutrophil; novel; novel therapeutics; oxidant stress; public health relevance; repaired; response; tool

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.

严重感染金黄色葡萄球菌（SA）仍然是重要的临床挑战目的地 抗生素。新型的热进步等待分子碱基的持久性，慢性， 和转移扩散 - 即SA感染的标志。用强烈的菌株和 抗生素耐药性的增加是强大的激励措施，以更好地了解宿主对SA的防御。 多形核中性粒细胞（PMN）代表细胞介导的抗菌活性的基石 并在吞噬体中施加所有抗菌作用，其中活性氧（ROS）和 颗粒内容协作以杀死和降解微生物。重要的是，产生过氧化氢（H2O2） PMN通过PMN颗粒蛋白髓过氧化物酶（MPO）扩增以产生HOCL（漂白剂）。在 除了特定的IIA磷脂酶A2（GPIIA-PLA2）的PMN，该基因在感染的血浆中呈现 动物，眼泪和炎性液表现出潜在的活动，以杀死和降解SA。 在VA优点支持下，我们在阐明两个完整方面的特征方面取得了进步 摄入的SA和PMN之间的相互作用，证明了（a）依赖PMN的ROS之间的协同作用 和gpiia-pla2杀死和降解sa，以及（b）MPO-H2O2-CL对SA攻击SA中的几个特征 吞噬体。此外，我们立即通过SA确定了转录和结构响应 吞噬作用。我们怀疑这种变化有助于某些摄入的SA的能力 在PMN中生存并随后逃脱，我们已经检查了现象，并且与 长期存在的临床观察和实验数据。 我们现在建议使用我们创建的工具以及在此期间开发的分析方法 上一个VA资金时期，以扩展我们的新研究并检验总体假设 SA在PMN吞噬体中的反应以修饰其细胞表面的组成（包括 D-丙氨酸和心磷脂）并诱导细胞质抗氧化剂（例如，甲硫氨酸硫酸还原酶和 Hsp33）导致它们能够抵抗PMN-GPIIA-PLA2的作用以及HOCL和HOCL和 相关的氧化物，以生存并逃离PMN和永久感染。我们的具体目的是： 1。定义PMN氧化物衍生的氧化物对人协同作用的特定贡献 PMN和GPIIA-PLA2对SA 在吞噬作用期间，发生了哪些MPO介导的SA蛋白和磷脂的修饰？ 吞噬体中ROS诱导的SA磷脂和蛋白质的修饰改变了GPIIA- pla2，其底物，还是两者？细胞壁中的遗传突变体构成，包括丙苯或 Cardiolipin合酶，可以更好地生存并逃离PMN吞噬体？ 2。确定MPO-H2O2-CL系统如何杀死大多数SA，相反 幸存的生物适应吞噬体中克服MPO衍生的细胞毒素的反应。 SA中细胞质GFP的漂白是否提供了PMN中HOCL活性的准确评估 吞噬体？ SA中的蛋白质是MPO特异性修饰的靶标；直接贡献的 间接地，SA对PMN的敏感性？被吞噬的SA修复了哪些目标？与SA在一起 甲基二亚氧化物降低的突变和对氧化还原敏感的伴侣HSP33的突变，系统的系统 应对HOCL诱导的氧化应激，或多或少易受PMN吞噬体中细胞毒素的影响？做 对HOCL介导的损害的抗性使SA可以持续或从PMN中逃脱？ 我们预计我们的研究将为复杂的生物学提供重要和新颖的见解 当摄入的SA符合PMN吞噬体的细胞毒性含量时，就会发生。此外，我们相信 由于我们提出的工作，可以确定热干预的新目标。