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: 10113517
• 负责人: ALEXANDER R HORSWILL
• 金额: $ 19.38万
• 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 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10113517
• 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; opportunistic pathogen; 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条件致病菌，可引起广谱急性和慢性 感染.抗生素耐药性是一个日益严重的挑战，耐甲氧西林金黄色葡萄球菌（MRSA） 感染更难治疗，导致患者和医疗保健系统的负担增加。 社区耐甲氧西林沙门氏菌感染。金黄色葡萄球菌（CA-MRSA）的USA 300谱系原因 在美国尤其严重的疾病，影响其他健康个体并引起广泛坏死 在皮肤和肺中仍有大量的炎症。人类多形核白细胞（PMN）占主导地位的初始 先天性免疫细胞对入侵微生物如S.金黄色。最佳PMN杀菌剂 这种作用依赖于吞噬细胞NADPH氧化酶产生的氧化剂和一系列 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)在它们的颗粒中，因此具有氧化氯化物的独特能力，从而产生HOCl， 强效杀菌剂如何S。金黄色葡萄球菌感知并抵抗吞噬体的氧化微环境， 不清楚S.金黄色葡萄球菌主要使用双组分系统（TCS）感知细胞外信号，我们和 其他人已经表明agr群体感应TCS对S.金黄色葡萄球菌在PMN中的存活。我们的总目标 是研究S.金黄色葡萄球菌抗人中性粒细胞氧化杀伤作用及其机制 S的后果。金黄色葡萄球菌和中性粒细胞。为了解决这些问题，在具体目标1中，我们将研究 agr系统对SPIN调节和表达的贡献。SPIN是一种小的分泌蛋白， 最近显示直接结合到人类MPO和防止H2 O2进入活性位点，我们发现， 在初步研究中，SPIN受agr系统调节。我们假设agr依赖的 SPIN的表达对S.金黄色葡萄球菌在人PMN中的存活。我们将研究SPIN表达， WT和调节突变体，表征SPIN基因启动子，并确定SPIN对 agr依赖的S. PMN内的金黄色葡萄球菌。在具体目标2中，我们将确定S的新机制。金黄色 抗PMN氧化杀伤。在一个合理的方法中，我们灭活了USA 300中的YjiE（ORF 93）， 在E.大肠杆菌，并观察到增加敏感性的PMN特异性 氧化剂HOCl。此外，在初步筛选中，我们发现了USA 300突变株， 对HOCl具有抗性和敏感性。在这一目标中，我们将描述在USA 300的YjiE调节子。我们还将继续 调查HOCl抗性菌株以鉴定靶基因并完成新的转座子文库的筛选 HOCl响应目标。最后，我们将评估携带突变S的中性粒细胞的命运。金黄色葡萄球菌 对HOCl的不同反应。了解摄入S的机制。金黄色葡萄球菌阻碍PMN将 为创造新的干预措施以改善人类健康提供框架。