Regulation of Staphylococcus aureus colonization and disease

金黄色葡萄球菌定植和疾病的调节

• 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
• 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

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。抗生素抗性水平正在增长，耐甲氧西林的金黄色葡萄球菌（MRSA）感染是 治疗的更多挑战，导致患者和医疗保健系统的燃烧增加。尽管是 这种有效的病原体，金黄色葡萄球菌可以不对称地定居约20％的健康成人 人口主要在鼻腔腔，在皮肤上次要。显然需要了解 从殖民者到侵略者的过渡，因为大多数金黄色葡萄球菌疾病是自身感染的结果 定植菌株。我们最近的发现表明，ARLRS TCS在金黄色葡萄球菌过渡中起关键作用 进入侵入性病原体。 ARLR的主要输出是控制MGRA的表达，一种细胞质 反映脲酶和大表面蛋白并诱导免疫进化因子的调节剂。我们的模型是 金黄色葡萄球菌在MGRA水平较低时表现出共生的特征，并表现出侵入性的特征 MGRA水平高的病原体。为了测试该模型，在AIM 1中，我们将确定ARLR对 S.金黄色葡萄球菌依从性，定殖和感染。为此，我们将比较wt，∆arlrs和∆mgra 使用粘附的体外模型和皮肤定植的体内模型。此外，我们会的 在体内成像中实时进行实时跟踪MGRA正则表达式，并进行皮肤感染和免疫 这些菌株的逃避评估。在AIM 2中，我们将对ARL进行生化特征 识别依赖ARLR的启动子。 ARLR是OMPR家族的响应调节剂，ARLR DNA- 结合位点和目标启动子尚不清楚。 ARLS激酶具有一个N末端传感器结构域，有两个 跨膜跨度为14.1 kDa细胞外缓存域。这个目标的目的是 确定ARLS和ARLR的生化特性，这将提供对机制的重要见解 ARLS调节和ARLR启动子靶标。我们将使用使用ARLS-ARLR磷酸转移的动力学 突变体确定酶活性（激酶和磷酸酶活性），这对于调节 Arlrs Regulon。我们还将使用SELEX识别ARLR结合位点，并用QRT-确认目标启动子 PCR和RNASEQ。在AIM 3中，我们将对ARLS感应机制进行分子分析。我们假设 ARLS细胞外缓存域对导致高尿素和SASG的环境提示做出反应 表达促进金黄色葡萄球菌定植。为了进一步研究这种机制，我们将检查信号 使用记者菌株控制ARLS函数，我们将在ARLS缓存域进行站点定向的更改 残留和评估功能。我们还将从小分子中筛选其他ARLS缓存域配体 通过热移PCR分析和NMR进行的库。发现改变arlrs功能并防止的配体 金黄色葡萄球菌向侵入性致病机构的过渡可能具有治疗抗生素耐药性感染的潜力。