Regulation of Staphylococcus aureus colonization and disease

金黄色葡萄球菌定植和疾病的调节

基本信息

批准号：
10456281
负责人：
ALEXANDER R HORSWILL
金额：
$ 37.63万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456281
关键词：
Address Adherence Adult Antibiotic Resistance Bacterial Adhesins Binding Binding Sites Biochemical Biological Assay Cells Chemotaxis Cues DNA Binding Disease Environment Enzyme Kinetics Exhibits Family Goals Healthcare Systems Human Immune Evasion In Vitro Infection Infectious Skin Diseases Kinetics Laboratories Leucocidin Life Style Ligands Membrane Membrane Proteins Modeling Molecular Analysis Mus N-terminal Nasal cavity Output Patients Phenotype Phosphoric Monoester Hydrolases Phosphotransferases Play Population Property Quantitative Reverse Transcriptase PCR Regulation Regulon Reporter Risk Factors Role Signal Transduction Site Skin Skin colonization Staphylococcus aureus Staphylococcus aureus infection Surface System Testing Time Urease Virulence acute infection antibiotic resistant infections base chronic infection enzyme activity extracellular improved in vitro Model in vivo Model in vivo imaging inhibitor inorganic phosphate insight intravital imaging methicillin resistant Staphylococcus aureus mutant neutrophil opportunistic pathogen pH Homeostasis pathogen prevent promoter response sensor small molecule libraries trait transcriptome sequencing vaginal mucosa

项目摘要

Project Summary Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance levels are growing and methicillin-resistant S. aureus (MRSA) infections are more challenging to treat, resulting in increased burden on both patients and healthcare systems. Despite being such an effective pathogen, S. aureus can asymptomatically colonize approximately 20% of the healthy adult population, primarily in the nasal cavity and secondarily on the skin. There is a clear need to understand the transition from colonizer to invader, since the majority of S. aureus disease is the result of autoinfection from the colonized strain. Our recent findings indicate that the ArlRS TCS plays a critical role in the S. aureus transition to an invasive pathogen. The primary output of ArlRS is controlling the expression of MgrA, a cytoplasmic regulator that represses urease and large surface proteins and induces immune evasion factors. Our model is that S. aureus exhibits the traits of a commensal when MgrA levels are low, and exhibits the trait of an invasive pathogen when MgrA levels are high. To test this model, in Aim 1 we will determine the contribution of ArlRS to S. aureus adherence, colonization, and infection. Toward this end, we will compare WT, ∆arlRS, and ∆mgrA mutant strains using in vitro models of adherence and an in vivo model of skin colonization. Additionally, we will perform real-time in vivo imaging to track MgrA regulon expression, and carry out skin infection and immune evasion assessments of these strains. In Aim 2, we will perform a biochemical characterization of ArlS and identify of ArlR-dependent promoters. ArlR is a response regulator of the OmpR family, and the ArlR DNA- binding site and target promoters are unknown. The ArlS kinase has an N-terminal sensor domain with two membrane-spanning passes that flank a 14.1 kDa extracellular Cache domain. The goal of this aim is to determine the biochemical properties of ArlS and ArlR, which will provide critical insight into the mechanism of ArlS regulation and ArlR promoter targets. We will assess the kinetics of ArlS-ArlR phosphotransfer using mutants to determine the enzyme activity (kinase and phosphatase activity) that is important for regulating the ArlRS regulon. We will also use SELEX to identify the ArlR binding site and confirm target promoters with qRT- PCR and RNAseq. In Aim 3, we will perform molecular analysis of the ArlS sensing mechanism. We hypothesize that the ArlS extracellular Cache domain responds to an environmental cue that results in high urease and SasG expression to promote S. aureus colonization. To further investigate this mechanism, we will examine signal control over ArlS function using reporter strains, and we will make site-directed changes in ArlS Cache domain residues and assess function. We will also screen for additional ArlS Cache domain ligands from small-molecule libraries by thermal shift PCR assays and NMR. Discovering ligands that alter ArlRS function and prevent transition of S. aureus to an invasive pathogen could have potential in treating antibiotic-resistant infections.

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抗生素耐药性水平不断增加，耐甲氧西林金黄色葡萄球菌 (MRSA) 感染也在增加。治疗更具挑战性，导致患者和医疗系统的负担增加。金黄色葡萄球菌是一种如此有效的病原体，它可以无症状地定植于大约 20% 的健康成人中人群，主要是鼻腔，其次是皮肤，显然需要了解。从殖民者到入侵者的转变，因为大多数金黄色葡萄球菌疾病是自身感染的结果我们最近的研究结果表明，ArlRS TCS 在金黄色葡萄球菌转变中发挥着关键作用。 ArlRS 的主要输出是控制 MgrA（一种细胞质）的表达。抑制脲酶和大表面蛋白并诱导免疫逃避因子的调节器我们的模型是。当 MgrA 水平较低时，金黄色葡萄球菌表现出共生体的特征，并表现出侵入性的特征为了测试该模型，在目标 1 中，我们将确定 ArlRS 对 MgrA 水平的贡献。为此，我们将比较 WT、ΔarlRS 和 ΔmgrA。此外，我们将使用体外粘附模型和体内皮肤定植模型来研究突变菌株。进行实时体内成像追踪MgrA调节子表达，并进行皮肤感染和免疫在目标 2 中，我们将对 ArlS 和 ArlS 进行生化表征。 ArlR 依赖性启动子的鉴定 ArlR 是 OmpR 家族的反应调节因子，并且 ArlR DNA- 结合位点和靶启动子未知。ArlS 激酶的 N 端传感器结构域有两个。侧翼 14.1 kDa 细胞外缓存域的跨膜通道该目标的目的是确定 ArlS 和 ArlR 的生化特性，这将为深入了解 ArlS 和 ArlR 的机制提供重要见解 ArlS 调节和 ArlR 启动子靶点我们将使用 ArlS-ArlR 磷酸转移来评估动力学。突变体来确定酶活性（激酶和磷酸酶活性），这对于调节我们还将使用 SELEX 来识别 ArlR 结合位点并通过 qRT- 确认目标启动子。 PCR 和 RNAseq 在目标 3 中，我们将对 ArlS 传感机制进行分子分析。 ArlS 细胞外缓存域对导致高脲酶和 SasG 的环境线索做出反应为了进一步研究这一机制，我们将检查信号。使用报告菌株控制 ArlS 功能，我们将在 ArlS 缓存域中进行定向更改我们还将从小分子中筛选其他 ArlS Cache 结构域配体。通过热位移 PCR 测定和 NMR 发现改变 ArlRS 功能并防止其发生的配体。金黄色葡萄球菌向侵入性病原体的转变可能具有治疗抗生素耐药性感染的潜力。