How Staphylococcus aureus resists killing by human neutrophlls

金黄色葡萄球菌如何抵抗人类中性粒细胞的杀伤

• 批准号: 9976306
• 负责人: ALEXANDER R HORSWILL
• 金额: $ 24.64万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-24 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976306
• 关键词: Active Sites; Acute; Address; Affect; Antibiotic Resistance; Antibiotic Therapy; Applications Grants; Bacterial Infections; Bacterial Proteins; Binding; Cessation of life; Chlorides; Chronic; Collaborations; Communities; Community Hospitals; Cytoplasmic Granules; Development; Disease; Environment; Escherichia coli; Exhibits; Extensive Necrosis; Genes; Genetic; Genus staphylococcus; Goals; Health; Healthcare Systems; Human; Hydrogen Peroxide; Immune; Individual; Infection; Ingestion; Intervention; Invaded; Knowledge; Letters; Libraries; Link; Lung; Mediating; Membrane; Microbe; NADPH Oxidase; Open Reading Frames; Organism; Oxidants; Oxides; Pathogenesis; Patients; Peroxidases; Phagocytes; Phagocytosis; Phagosomes; Process; Protein Array; Proteins; Recovery; Regulation; Regulon; Research; Resistance; Signal Transduction; Skin; Staphylococcus aureus; Staphylococcus aureus infection; System; Time; Transcript; acute infection; chlorination; chronic infection; extracellular; improved; inhibitor/antagonist; methicillin resistant Staphylococcus aureus; microbicide; microorganism; mutant; neutrophil; novel; pathogen; prevent; promoter; quorum sensing; recruit; resistant strain; response; screening; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance is a growing challenge and methicillin-resistant Staphylococcus aureus (MRSA) infections are more difficult to treat, resulting in increased burden for both patients and healthcare systems. Infections with community-associated methicillin-resistant S. aureus (CA-MRSA) of the USA300 lineage cause especially severe disease in the USA, affecting otherwise healthy individuals and provoking extensive necrosis in skin and lung despite antibiotic treatment. Human polymorphonuclear leukocytes (PMN) dominate the initial innate immune cellular response to invading microorganisms such as S. aureus. Optimal PMN microbicidal action relies on collaboration between oxidants generated by the phagocyte NADPH oxidase and an array of proteins stored in PMN granules. Among phagocytes, PMN are unique, as they possess myeloperoxidase (MPO) in their granules and thus have the singular capacity to oxidize chloride and thereby generate HOCl, a potent microbicide. How S. aureus senses and resists the oxidative microenvironment of the phagosome is unclear. S. aureus primarily perceives extracellular signals using two-component systems (TCS), and we and others have shown the agr quorum-sensing TCS is important for S. aureus survival in PMNs. Our overall goal is to investigate the mechanisms through which S. aureus resists human PMN oxidative killing and their consequences for S. aureus and for PMN. To address these questions, in Specific Aim 1 we will investigate the contribution of the agr system to SPIN regulation and expression. SPIN is a small secreted protein that was recently shown to directly bind to human MPO and prevent H2O2 from entering the active site, and we discovered in preliminary studies that SPIN is regulated by the agr system. We hypothesize that the agr-dependent expression of SPIN is critical for S. aureus survival within human PMN. We will investigate SPIN expression in WT and regulatory mutants, characterize the SPIN gene promoter, and determine the contribution of SPIN to agr-dependent survival of S. aureus within PMN. In Specific Aim 2, we will identify new mechanisms of S. aureus resistance to PMN-oxidative killing. In a rational approach, we inactivated YjiE (ORF 93) in USA300, a conserved HOCl-responsive transcription factor identified in E. coli, and observed increased sensitivity to the PMN-specific oxidant HOCl. Additionally, in preliminary screening, we have discovered USA300 mutant strains that are more resistant and sensitive to HOCl. In this aim we will characterize the YjiE regulon in USA300. We will also continue investigating HOCl resistant strains to identify target genes and finish screening the transposon library for new HOCl-responsive targets. Finally, we will assess the fate of PMN harboring mutant S. aureus strains with differential response to HOCl. Understanding of mechanisms by which ingested S. aureus thwart PMN will provide a framework for creating novel interventions to improve human health.

项目摘要 金黄色葡萄球菌是一种机会性病原体，可引起急性和慢性频谱 感染。抗生素耐药性是日益增长的挑战，耐甲氧西林金黄色葡萄球菌（MRSA） 感染更难治疗，从而增加患者和医疗保健系统的负担。 美国300谱系的耐甲氧西林的金黄色葡萄球菌（CA-MRSA）的感染原因 在美国特别是严重的疾病，影响其他健康的个体并引起广泛的坏死 尽管治疗抗生素，但在皮肤和肺中。人类多形核白细胞（PMN）主导着初始 先天免疫细胞对入侵微生物（例如金黄色葡萄球菌）的反应。最佳PMN微生物 作用依赖于吞噬细胞NADPH氧化酶产生的氧化剂与一系列氧化剂之间的合作 储存在PMN颗粒中的蛋白质。在吞噬细胞中，PMN是独特的，因为它们具有髓过氧化物酶 （MPO）在其颗粒中，因此具有氧化氯化物的奇异能力，从而产生HOCL，A 有效的菌心。金黄色葡萄球菌如何感觉到吞噬体的氧化微环境是 不清楚。金黄色葡萄球菌主要使用两个组件系统（TCS）感知细胞外信号，我们和我们和 其他人则表明，Agr Quorum敏感性TC对于PMN中的金黄色葡萄球菌存活很重要。我们的总体目标 是为了研究金黄色葡萄球菌抵抗人PMN氧化杀死及其的机制 金黄色葡萄球菌和PMN的后果。为了解决这些问题，在特定的目标1中，我们将调查 AGR系统对自旋调节和表达的贡献。旋转是一种小的分泌蛋白 最近显示的可以直接与人类MPO结合并防止H2O2进入活动位点，我们发现 在初步研究中，自旋受AGR系统调节。我们假设AGR依赖性 自旋的表达对于人类PMN中的金黄色葡萄球菌存活至关重要。我们将研究中的自旋表达 WT和调节突变体，表征自旋基因启动子，并确定自旋对 Agr依赖性金黄色葡萄球菌在PMN中的生存。在特定目标2中，我们将确定金黄色葡萄球菌的新机制 对PMN氧化杀伤的抵抗力。以合理的方法，我们在USA300中灭活了Yjie（ORF 93）， HOCL反应性转录因子在大肠杆菌中鉴定出来，并观察到对PMN特异性的敏感性提高 氧化剂HOCL。此外，在初步筛选中，我们发现了更多的USA300突变菌株 对HOCL的抗性和敏感。在此目标中，我们将描述USA300的Yjie Regulon。我们还将继续 研究HOCL抗性菌株以识别靶基因并完成筛选新的新的 HOCL响应目标。最后，我们将评估带有突变体S.金黄色葡萄球菌菌株的PMN的命运 对HOCL的差异反应。了解摄入的金黄色葡萄球菌阻碍PMN的机制 提供一个框架来创建新的干预措施以改善人类健康。