Contribution of extracellular enzymes to Staphylococcus aureus biofilm development

项目摘要 金黄色葡萄球菌是我们医疗机构中最有问题的细菌病原体之一。S.金黄色 可以通过发展成被称为生物膜的封闭细胞群落而在宿主中存活和持续存在，并且 许多研究已经证明生物膜对宿主免疫防御和化疗具有抗性。 我们的中心PPG假设是S。金黄色葡萄球菌生物膜的发展创造了独特的代谢生态位， 免疫抑制环境在本提案（项目3）中，我们关注的是S。 金黄色葡萄球菌胞外酶对生物膜生长和成熟的影响，在宿主中的持久性， 传播到一个新的网站。在S.金黄色葡萄球菌分泌，我们将优先透明质酸酶（HysA） 和核酸酶（Nuc 1），因为它们在生物膜-宿主相互作用表型和调节 阴谋我们最近证实透明质酸在S.金黄色葡萄球菌生物膜感染， HysA可以将该宿主糖胺聚糖降解为二糖（HA-DS）。我们的初步研究表明， HA-DS可以通过未知的分解代谢途径作为碳源，这种二糖具有 额外的抗炎特性可能有助于S.金黄色葡萄球菌生物膜 感染.在具体目标1中，我们将确定透明质酸代谢在S.金黄色 生物膜成熟我们将使用遗传分析和标记研究来表征HA-DS分解代谢途径 与代谢组学核心合作。我们还将测试生物膜成熟中的分解代谢途径敲除 和异物感染，并研究CodY和CcpA对透明质酸调节的贡献 猫此外，S.金黄色葡萄球菌将在HA-DS上生长，并进行RNAseq以鉴定全球 转录组学变化。在具体目标2中，我们将研究S。金黄色葡萄球菌酶降解宿主 聚合物影响生物膜抗炎状态。与Tammy Kielian博士（PPG项目4）合作， 我们将评估HA-DS以及Nuc 1和HysA酶及其调节剂对免疫细胞的影响， 功能我们还将确定HA-DS和HysA抑制剂对生物膜感染的影响，并测试是否 HA-DS是S.金黄色葡萄球菌的人关节炎患者的关节液中的人工关节感染（PJI）。在 具体目标3，我们将检查S。金黄色葡萄球菌胞外酶在生物膜分散中的调节和功能。我们 假设CodY控制S.金黄色葡萄球菌生物膜中的酶和营养依赖 方式我们将研究Nuc 1和HysA沿着CodY和SaeRS调节剂对生物膜的贡献， 在体外和异物感染期间分散。我们还将研究营养状况对CodY的影响 与Ken Bayles博士（PPG项目1）和 生物成像核心。最后，我们将确定aureusimine分子在S。金黄色葡萄球菌生物膜形成。 总的来说，这些研究将确定S。金黄色葡萄球菌胞外酶对生物膜代谢的影响， 发展和持久性，可能导致生物膜感染的创新疗法。

PROJECT SUMMARY Staphylococcus aureus is one of the most problematic bacterial pathogens in our healthcare settings. S. aureus can survive and persist in the host by developing into an encased community of cells called a biofilm, and numerous studies have demonstrated that biofilms are resistant to host immune defenses and chemotherapies. Our central PPG hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In this proposal (Project 3), we are focusing on the contribution of S. aureus extracellular enzymes to biofilm growth and maturation, persistence in the host, and ultimately dissemination to a new site. Of the many enzymes that S. aureus secretes, we will prioritize hyaluronidase (HysA) and nuclease (Nuc1), as they have commonalities in biofilm-host interaction phenotypes and regulatory schemes. We recently demonstrated that hyaluronan accumulates in an S. aureus biofilm infection and that HysA can degrade this host glycosaminoglycan to disaccharides (HA-DS). Our preliminary studies indicate that HA-DS can serve as a carbon source through an unknown catabolic pathway, and this disaccharide has additional anti-inflammatory properties that could be contributing to the persistent nature of S. aureus biofilm infections. In Specific Aim 1, we will determine the role and regulation of hyaluronan metabolism in S. aureus biofilm maturation. We will characterize the HA-DS catabolic pathway using genetic analysis and labeling studies in collaboration with the Metabolomics Core. We will also test catabolic pathway knockouts in biofilm maturation and foreign-body infections, and investigate the contribution of CodY and CcpA to regulation of hyaluronan catabolism. Additionally, S. aureus will be grown on HA-DS and RNAseq performed to identify global transcriptomic changes. In Specific Aim 2, we will investigate how S. aureus enzymatic degradation of host polymers impacts the biofilm anti-inflammatory state. In collaboration with Dr. Tammy Kielian (PPG Project 4), we will assess the effect of HA-DS, as well as Nuc1 and HysA enzymes and their regulators, on immune cell function. We will also determine the impact of HA-DS and HysA inhibitors on biofilm infection, and test whether HA-DS is a biomarker for S. aureus in human synovial fluid from patients with prosthetic joint infection (PJI). In Specific Aim 3, we will examine S. aureus exo-enzyme regulation and function in biofilm dispersal. We hypothesize that CodY controls dissemination from S. aureus biofilms in an enzyme and nutrient dependent manner. We will investigate the contribution of Nuc1 and HysA, along with CodY and SaeRS regulators, to biofilm dispersal in vitro and during foreign body infection. We will also examine the impact of nutritional status on CodY activity during biofilm formation and dispersal in collaboration with Dr. Ken Bayles (PPG Project 1) and the Bioimaging Core. Finally, we will determine the role of aureusimine molecules in S. aureus biofilm development. Collectively these studies will define the contribution of S. aureus exo-enzymes to biofilm metabolism, development and persistence, potentially leading to innovative therapies for biofilm infections.