Metabolic determinants of Staphylococcus aureus skin colonization

项目摘要 皮肤是人体最大的器官。这种物理屏障和皮肤的先天免疫力 这是对抗病原体的重要第一防御。金黄色葡萄球菌是一种条件致病菌， 软皮肤和组织感染的优势微生物。这些感染可以迅速发展成全身性的 感染和恶化的健康结果。越来越多的耐药流行性S。金黄色葡萄球菌菌株（例如， 耐甲氧西林表皮葡萄金黄色葡萄球菌，MRSA）使这些感染在临床环境中的治疗复杂化。 虽然皮肤定植增加了MRSA感染的风险，但研究宿主-病原体的这一方面， 互动是一个挑战。目前的动物模型缺乏酸性、皮肤结构和外分泌腺 人类皮肤的分布，许多研究小组无法获得。此外，体外试验， 模仿人类皮肤表面的介质尚未公开。为了解决这些差距，我开发了一个 包含MRSA在人体皮肤上遇到的代谢物、离子和pH值的体外培养基。我 对在这些条件下生长的MRSA进行RNA-seq，并将这些结果与先前的RNA-seq进行比较。 小鼠皮肤接种MRSA实验。在这两个数据集中，代谢尿素的基因，来自 汗腺;和尿刊酸，来源于天然保湿因子，显著上调。我 假设尿素和尿刊酸的代谢有助于S.金黄色葡萄球菌pH稳态和 分别在皮肤上生长。将在以下目标中检验这一假设：目标1：确定 尿素酶和相关功能在人皮肤样体外模型中MRSA生长和pH稳态中的作用。 遗传和生物化学方法将用于体外培养基和分化的角质形成细胞模型， 研究尿素酶的作用，尿素和镍转运对尿素酶功能的贡献， 目的2：研究尿刊酸在MRSA皮肤中代谢的作用 殖民化遗传学和生物化学方法将用于前面提到的模型，以验证 该途径的预测编码功能，研究该途径的底物特异性， 了解监管控制。将通过蛋白质印迹分析评估途径表达的流行病学 临床S.从特应性皮炎患者和健康对照的金黄色葡萄球菌皮肤分离物。这些研究将 为未来的皮肤微生物群研究提供基础，扩大我们对MRSA的了解 皮肤上的生理学，并确定未来治疗发展的潜在目标。

PROJECT SUMMARY The human skin is the largest organ of the body. This physical barrier and the skin's innate immunity are an essential first defense against pathogens. Staphylococcus aureus is an opportunistic pathogen and the dominant microorganism of soft skin and tissue infections. These infections can quickly develop into systemic infections and worsen health outcomes. Increasingly, antibiotic-resistant epidemic S. aureus strains (e.g. methicillin-resistant S. aureus, MRSA) complicate treatment of these infections in the clinical setting. Although skin colonization increases risk of a MRSA infection, studying this aspect of host-pathogen interactions has been challenging. Current animal models lack the acidity, skin structure, and eccrine gland distribution of human skin and are not accessible to many research groups. Additionally, in vitro testing with media mimicking the human skin surface have not been published. To address these gaps, I developed an in vitro media that incorporates the metabolites, ions, and pH that MRSA would encounter on the human skin. I performed RNA-seq of MRSA grown in these conditions and compared these results to a previous RNA-seq experiment of MRSA inoculated on mouse skin. In both datasets, genes for metabolizing urea, derived from sweat glands; and urocanic acid, derived from the natural moisturizing factor, were significantly upregulated. I hypothesize that the metabolism of urea and urocanic acid contributes to S. aureus pH homeostasis and growth, respectively, on the skin. This hypothesis will be tested in the following aims: Aim 1: Ascertain the role of urease and related functions in MRSA growth and pH homeostasis in human skin-like in vitro models. Genetic and biochemical approaches will be used in an in vitro media and a differentiated keratinocyte model to investigate the role of urease, contribution of urea and nickel transport to urease function, and therapeutic approaches to inhibit urease activity Aim 2: Investigate the role of urocanic acid metabolism in MRSA skin colonization. Genetic and biochemical approaches will be used in the previously mentioned models to validate the predicted encoded functions of this pathway, investigate substrate specificity of the pathway, and better understand regulatory control. Epidemiology of pathway expression will be assessed by Western Blot analysis of clinical S. aureus skin isolates from atopic dermatitis patients and healthy controls. These studies will provide a foundation for future research of the skin microbiota, expand our understanding of MRSA physiology on the skin, and identify potential targets for future therapeutic development.