How Staphylococcus aureus resists killing by human neutrophlls

项目总结 金黄色葡萄球菌是一种条件致病菌，可引起多种急性和慢性疾病。 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) 感染更难治疗，导致患者和医疗系统的负担增加。 USA300谱系社区相关耐甲氧西林金黄色葡萄球菌(CA-MRSA)感染 在美国特别严重的疾病，影响其他健康的人，并引起广泛的坏死 在皮肤和肺部，尽管接受了抗生素治疗。人多形核白细胞(PMN)在初始阶段占优势 对金黄色葡萄球菌等入侵微生物的先天免疫细胞反应。PMN的最佳杀菌效果 作用依赖于吞噬细胞NADPH氧化酶产生的氧化剂与一系列 储存在PMN颗粒中的蛋白质。在吞噬细胞中，PMN是独一无二的，因为它们具有髓过氧化物酶。 (MPO)在其颗粒中，因此具有奇特的氧化氯的能力，从而生成HOCl，a 强效杀微生物剂。金黄色葡萄球菌如何感知和抵抗吞噬小体的氧化微环境 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，而我们和 其他研究表明，agr群体感应TCS对于金黄色葡萄球菌在PMN中的存活是重要的。我们的总目标是 目的是研究金黄色葡萄球菌抵抗人中性粒细胞氧化杀伤的机制及其机制 对金黄色葡萄球菌和PMN的后果。为了解决这些问题，在具体目标1中，我们将调查 Agr系统对自旋调控和表达的贡献。Spin是一种小的分泌蛋白质， 最近发现可以直接与人MPO结合并阻止过氧化氢进入活性部位，我们发现 在初步研究中，自旋受到AGR系统的调节。我们假设依赖农业的 Spin的表达对金黄色葡萄球菌在人PMN内的存活至关重要。我们将研究自旋表达在 WT和调节突变体，表征Spin基因启动子，并确定Spin对 依赖AGR的金黄色葡萄球菌在PMN内的存活。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的抵抗力--氧化杀死。在一种理性的方法中，我们灭活了USA300中的YjiE(ORF 93)，一个保守的 在大肠杆菌中发现了HOCl反应转录因子，并观察到对PMN特异性的敏感性增加 氧化剂HOCl。此外，在初步筛选中，我们发现了USA300突变株 对高氯酸盐具有抗性和敏感性。在这个目标中，我们将描述USA300中的YjiE规则。我们也会继续 调查抗氯菌以确定目的基因并完成新基因转座子文库的筛选 HOCl2反应靶标。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。 对高氯酸盐的不同反应。对摄取金黄色葡萄球菌抑制中性粒细胞的机制的理解 为创造新的干预措施以改善人类健康提供框架。

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.