Contribution of extracellular enzymes to Staphylococcus aureus biofilm development

项目总结 金黄色葡萄球菌是我们医疗保健环境中最有问题的细菌病原体之一。金黄色葡萄球菌 可以通过发育成一个被包裹的称为生物膜的细胞群落而在宿主体内生存和持续存在，以及 大量研究表明，生物膜对宿主的免疫防御和化疗具有抵抗力。 我们的中心PPG假设是金黄色葡萄球菌生物膜的发育创造了独特的代谢生态位，促进了 免疫抑制的环境。在本提案(项目3)中，我们重点关注S。 金黄色葡萄球菌胞外酶对生物膜的生长和成熟、在宿主中的持久性，并最终 传播到一个新的站点。在金黄色葡萄球菌分泌的众多酶中，我们将优先考虑透明质酸酶(HySA)。 和核酸酶(Nuc1)，因为它们在生物膜-宿主相互作用表型和调控方面具有共性 阴谋。我们最近证明了透明质酸在金黄色葡萄球菌生物被膜感染中积累，并且 HySA可将宿主糖胺聚糖降解为二糖(HA-DS)。我们的初步研究表明 HA-DS可以通过一种未知的分解代谢途径作为碳源，这种二糖具有 其他抗炎特性可能有助于金黄色葡萄球菌生物被膜的持久性 感染。在特定的目标1中，我们将确定透明质酸代谢在金黄色葡萄球菌中的作用和调节。 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 与新陈代谢组学核心合作。我们还将测试生物膜成熟过程中分解代谢途径的敲除。 和异物感染，并研究Cody和CCPA在调节透明质酸中的作用 分解代谢。此外，金黄色葡萄球菌将在HA-DS和RNAseq上培养，以鉴定全球 转录改变。在特定目标2中，我们将研究金黄色葡萄球菌如何对宿主进行酶降解 聚合物影响生物膜的抗炎状态。与Tammy Kielian博士(PPG项目4)合作， 我们将评估HA-DS以及Nuc1和HySA酶及其调节因子对免疫细胞的影响 功能。我们还将确定HA-DS和HySA抑制剂对生物被膜感染的影响，并测试 HA-DS是人工关节感染(PJI)患者关节液中金黄色葡萄球菌的生物标志物。在……里面 具体目标3，我们将研究金黄色葡萄球菌胞外酶在生物膜扩散中的调节和作用。我们 假设Cody控制金黄色葡萄球菌生物膜的扩散依赖于酶和营养 举止。我们将调查Nuc1和HySA以及Cody和SaeRS调节剂对生物膜的贡献 在体外和异物感染期间扩散。我们还将研究营养状况对Cody的影响 生物膜形成和扩散过程中的活动，与Ken Bayles博士(PPG项目1)和 生物成像核心。最后，我们将确定金红素分子在金黄色葡萄球菌生物膜发育中的作用。 总的来说，这些研究将确定金黄色葡萄球菌外酶对生物膜代谢的贡献， 发展和持久性，有可能导致生物被膜感染的创新疗法。

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.