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: 7791569
• 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/7791569
• 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

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.

描述（由申请人提供）： 尽管有效的抗生素，但对金黄色葡萄球菌（SA）的严重感染仍然是一个重要的临床挑战。新的治疗进展正在等待分子碱基的持久性，慢性和转移扩散 - 即SA感染的标志。用强烈的菌株和增加的抗生素耐药性压倒性的感染是更好地了解宿主防御SA的强大激励措施。 多形核中性粒细胞（PMN）代表细胞介导的抗菌活性的基石，并在吞噬体中发挥所有抗菌作用，在吞噬体中，活性氧（ROS）和颗粒含量合作以杀死和降解微生物。重要的是，由PMN产生的过氧化氢（H2O2）被PMN颗粒蛋白骨髓过氧化物酶（MPO）扩增以产生HOCL（BLEACH）。除PMN外，特定的IIA磷脂酶A2（GPIIA-PLA2）存在于感染动物，眼泪和炎性液的血浆中，还具有杀死和降解SA的有效活性。 在VA值得支持的情况下，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-PLA2之间的协同作用，以杀死和降解SA，以及（B）MPO-H2O2-CL对SA对SA对Phagosomes中的MPO-H2O2-CL攻击的几种特征。此外，我们已经在吞噬作用后立即通过SA鉴定出转录和结构反应。我们怀疑这种变化有助于某些摄入的SA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一致。 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抵抗PMN-GPIIA-PLA2的作用以及HOCL和相关氧化剂的特异性毒性，并在PMN和永久性感染中生存并逃脱并逃脱。我们的具体目的是：1。定义PMN氧化酶衍生的氧化剂对人PMN和GPIIA-PLA2的协同作用的特定贡献，对SA蛋白质和磷脂的MPO介导的修饰发生了什么？ ROS 1 MPO在吞噬体中诱导的Sa磷脂和蛋白质的SA磷脂和蛋白质的修饰，其底物替代GPIIA-PLA2，其底物或两者兼而有之？细胞壁成分中的遗传突变体（包括D-丙二醇化或心脂蛋白合酶）是否可以更好地生存并逃离PMN吞噬体？ 2。为了确定MPO-H2O2-CL系统如何杀死大多数SA，相反，幸存的生物的子集如何适应吞噬体中克服MPO衍生的细胞毒素的反应。 SA中细胞质GFP的漂白是否可以准确评估PMN吞噬体中的HOCL活性？ SA中的蛋白质是MPO特异性修饰的靶标；哪个直接或间接地促进了SA对PMN的敏感性？被吞噬的SA修复了哪些目标？ SA是否在蛋氨酸亚氧化甲氧化物还原酶和对氧化还原敏感的伴侣HSP33中有突变，对HOCL诱导的氧化剂胁迫反应的系统，或多或少易受PMN吞噬体中细胞毒素的影响？对HOCL介导的损伤的抗性是否使SA持续或从PMN中逃脱？ 我们预计，我们的研究将提供重要而新颖的见解，以了解当摄入的SA符合PMN吞噬体的细胞毒性含量时发生的复杂生物学。此外，我们认为，由于我们提出的工作，可以确定治疗干预的新颖目标。 公共卫生相关性： SA感染通常发生在退伍军人中，包括门诊患者和住院患者。 VA医疗保健系统在2007年为500万人提供服务，其中有153个医疗中心的约580,000人出院，因此是美国最大的医疗保健系统之一。根据一项基于VA的研究，SA感染的患病率从2000年的每1,000次出院的26.93增加到2007年的30.59（14％）。因此，随之而来的发病率和死亡率上升，对我们的医疗保健系统构成了重大挑战。了解先天免疫因素（包括PMN和GPIIA-PLA2）的机制，可以调解SA的成功杀害和降解将提供基本的见解，这些见解可以转化为新型治疗目标的发展。我们认为，我们对允许SA在PMN中生存的反应以及在某些情况下逃脱的反应可能揭示了导致毒力但并不需要生存力本身的基因及其产物，从而避免了更多常规的测定以识别抗生素靶标。