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)仍然是一个重要的临床挑战，尽管 抗生素。新的治疗进展有待阐明持久性、慢性性、 和转移性传播--即金黄色葡萄球菌感染的特征。以强毒力菌株压倒性感染 抗生素耐药性的增加是更好地了解宿主对金黄色葡萄球菌的防御的强大动力。 中性粒细胞(PMN)是细胞介导的抗菌活性的基石 并在吞噬体内发挥所有的抗微生物作用，其中活性氧物种(ROS)和 颗粒内容物协同杀灭和降解微生物。重要的是，过氧化氢(H2O2)产生 PMN是由PMN颗粒蛋白髓过氧化物酶(MPO)扩增生成HOCl(漂白剂)。在……里面 除PMN外，感染者血浆中还存在一种特异性的IIA磷脂酶A2(GpIIA-PLA2) 动物、泪水和炎性液体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，表明(A)PMN依赖的ROS之间的协同作用 和GpIIA-PLA2对SA的杀伤和降解，以及(B)MPO-H_2O_2-Cl攻击SA的几个特征 吞噬小体。此外，我们已经立即确定了SA的转录和结构反应 在吞噬之后。我们怀疑这样的变化有助于某些被摄取的SA 在PMN中存活并随后逃脱，这些现象我们已经检查过，并与 长期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和我们在 上一期退伍军人事务部资金用于扩展我们的新研究，并测试总体假设 中性粒细胞吞噬小体中SA对改变其细胞表面成分的反应(包括 D-丙氨酸和心磷脂)，并诱导细胞质抗氧化剂(例如蛋氨酸亚砜还原酶和 Hsp33)，使其具有抵抗PMN-GpIIA-PLA2作用的能力和HOCl2和HSP33的特异性毒性。 相关的氧化剂，并在中性粒细胞内存活和逃脱，并使感染永久化。我们的具体目标是： 1.明确PMN氧化酶衍生的氧化剂对人类协同作用的具体贡献 抗SA的PMN和GpIIA-PLA2 在吞噬过程中，会发生哪些MPO介导的SA蛋白和磷脂的修饰？ 吞噬小体中ROS？MPO诱导的SA磷脂和蛋白质的哪些修饰改变了GpIIA- PLA2，它的底物，还是两者都有？是细胞壁成分中的基因突变，包括D-丙氨酸化或 心磷脂合成酶，更好地在PMN吞噬小体中生存和逃脱？ 2.要确定MPO-H_2O_2-Cl系统如何杀死大多数SA，反之，如何 存活的生物体适应于克服吞噬小体中MPO衍生的细胞毒素。 唾液中胞质GFP的漂白是否能准确评估PMN中HOCl的活性 噬菌体？SA中的哪些蛋白质是MPO特异性修饰的目标；哪些蛋白质直接或 间接影响金黄色葡萄球菌对PMN的敏感性？吞噬的SA修复哪些靶点？SA与 蛋氨酸亚砜还原酶和氧化还原敏感的伴侣Hsp33系统的突变 对HOCl诱导的氧化应激有反应，对PMN吞噬小体中的细胞毒素有或多或少的敏感性？会吗？ 对HOCl2介导的损伤的抵抗使SA能够在PMN内存活或逃逸？ 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符合PMN吞噬小体的细胞毒含量时发生。此外，我们认为， 作为我们提议的工作的结果，治疗干预的新靶点可能被确定。