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.

项目概要 金黄色葡萄球菌是一种机会致病菌，可引起广谱的急性和慢性疾病 感染。抗生素耐药性是一个日益严峻的挑战，耐甲氧西林金黄色葡萄球菌 (MRSA) 感染更难以治疗，导致患者和医疗系统的负担增加。 USA300 谱系的社区相关耐甲氧西林金黄色葡萄球菌 (CA-MRSA) 感染 在美国尤其严重的疾病，影响健康个体并引起广泛坏死 尽管进行了抗生素治疗，但仍存在于皮肤和肺部。人类多形核白细胞（PMN）在最初的 对入侵微生物（如金黄色葡萄球菌）的先天免疫细胞反应。最佳 PMN 杀菌效果 作用依赖于吞噬细胞 NADPH 氧化酶产生的氧化剂和一系列氧化剂之间的协作 蛋白质储存在 PMN 颗粒中。在吞噬细胞中，PMN 是独特的，因为它们拥有髓过氧化物酶 (MPO) 在其颗粒中，因此具有氧化氯化物并从而生成 HOCl 的独特能力， 强效杀菌剂。金黄色葡萄球菌如何感知和抵抗吞噬体的氧化微环境 不清楚。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 对于金黄色葡萄球菌在 PMN 中的存活很重要。我们的总体目标 旨在研究金黄色葡萄球菌抵抗人类PMN氧化杀伤的机制及其作用 对金黄色葡萄球菌和 PMN 的影响。为了解决这些问题，在具体目标 1 中，我们将调查 agr系统对SPIN调节和表达的贡献。 SPIN 是一种小型分泌蛋白 最近被证明可以直接与人类 MPO 结合并阻止 H2O2 进入活性位点，我们发现 初步研究表明SPIN受agr系统调节。我们假设农业依赖性 SPIN 的表达对于金黄色葡萄球菌在人 PMN 中的存活至关重要。我们将研究 SPIN 表达 WT 和调控突变体，表征 SPIN 基因启动子，并确定 SPIN 对 PMN 内金黄色葡萄球菌的 agr 依赖性存活。在具体目标 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。通过理性的方法，我们灭活了 USA300 中的 YjiE (ORF 93)，这是一个保守的 在大肠杆菌中鉴定出 HOCl 响应转录因子，并观察到对 PMN 特异性的敏感性增加 氧化剂次氯酸。另外，在初步筛选中，我们还发现了USA300突变株，其特征是： 对次氯酸（HOCl）耐药且敏感。为此，我们将描述 USA300 中 YjiE 调节子的特征。我们也将继续 研究次氯酸抗性菌株以确定目标基因并完成转座子库的筛选以寻找新的 HOCl响应目标。最后，我们将评估携带突变金黄色葡萄球菌菌株的 PMN 的命运 对次氯酸的不同反应。了解摄入的金黄色葡萄球菌阻止 PMN 的机制 为创建新的干预措施以改善人类健康提供一个框架。