Metabolic determinants of Staphylococcus aureus skin colonization

项目总结 人的皮肤是人体最大的器官。这种身体屏障和皮肤的先天免疫力 这是抵御病原体的第一道防线。金黄色葡萄球菌是一种条件致病菌， 软组织感染的优势微生物。这些感染可迅速发展为全身性感染 感染和恶化的健康后果。越来越多的耐药金黄色葡萄球菌流行株(如： 耐甲氧西林金黄色葡萄球菌(耐甲氧西林金黄色葡萄球菌，MRSA)使这些感染的临床治疗复杂化。 尽管皮肤定植增加了感染MRSA的风险，但研究宿主病原体的这一方面 互动一直是具有挑战性的。目前的动物模型缺乏酸性、皮肤结构和分泌腺。 人类皮肤的分布，许多研究小组无法接触到。此外，在体外测试中使用 模仿人类皮肤表面的媒体尚未发表。为了解决这些差距，我开发了一个In 含有MRSA会在人体皮肤上遇到的代谢物、离子和pH的体外介质。我 对在这些条件下生长的MRSA进行了rna-seq，并将这些结果与以前的rna-seq进行了比较。 小鼠皮肤接种耐甲氧西林金黄色葡萄球菌的实验。在这两个数据集中，代谢尿素的基因来自 汗腺；以及从天然保湿因子中提取的尿毒酸显著上调。我 假设尿素和尿毒酸的代谢有助于金黄色葡萄球菌的pH动态平衡和 分别在皮肤上生长。这一假设将在以下目标中得到检验：目标1：确定 体外模型中尿素酶及其相关功能在耐甲氧西林金黄色葡萄球菌生长和pH稳态中的作用。 遗传和生化方法将用于体外培养和分化角质形成细胞模型中 研究尿素酶的作用，尿素和镍转运对尿素酶功能的贡献，以及治疗 抑制尿素酶活性的途径目的2：研究尿坎酸代谢在MRSA皮肤中的作用 殖民主义。将在前面提到的模型中使用遗传和生化方法来验证 预测该途径的编码功能，研究该途径的底物特异性，并更好地 了解监管控制。途径表达的流行病学将通过Western Blot分析进行评估 特应性皮炎患者和健康对照的金黄色葡萄球菌临床分离株。这些研究将 为今后皮肤微生物区系的研究奠定基础，扩大对耐甲氧西林金黄色葡萄球菌的认识 皮肤上的生理学，并确定未来治疗开发的潜在靶点。

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.