Role of the enterococcal site 2 protease in biofilm formation, adaptation, and host-pathogen interactions

项目摘要/摘要 粪肠球菌是造成医疗费用上升的主要原因 与医疗保健相关的感染。粪肠球菌的耐药性及其形成能力 生物被膜需要对感染采取长期和复杂的治疗策略。因此，有一个关键的 需要确定治疗和预防肠球菌疾病的新方法。我们之前已经证明了 保守的膜内金属蛋白水解酶EEP是S2P家族的一部分，是一种重要的致病因子。 粪肠球菌在体外生物膜形成和体内生物膜相关感染。这在很大程度上没有特征 EEP在粪肠球菌生物膜形成中的作用除了其已知的细胞-细胞信号转导功能外，还包括 对先天免疫系统效应分子溶菌酶攻击的适应，以及 携带抗生素耐药性的质粒。然而，尽管它在这些细胞过程中很重要，但还有 关于EEP的生化活动以及这种活动如何影响的许多基本问题尚未解答 粪肠球菌病原菌与宿主的相互作用。更广泛地说，我们对EEP和EEP的机制和生物学理解 其他革兰氏阳性病原菌中的同源S2P也是有限的。该项目将调查以下内容 问题：(1)EEP的底物和产物是什么？(2)EEP的效应器如何影响生物膜的形成和 适应细胞表面压力？反过来，这些依赖EEP的过程如何影响寄主病原体 相互作用？；以及(3)EEP的结构如何影响其识别和切割底物的能力？我们的 实验设计将检验EEP的蛋白分解活性导致在 在致病环境中影响粪肠球菌与哺乳动物宿主相互作用的细胞表面。我们将配对 蛋白质组、分子遗传学和微生物学方法与体内生物膜形成的两种动物模型 确定生物膜中候选的EEP底物和下游效应因子(目标1)，表征遗传和 使粪肠球菌细胞对溶菌酶产生抗药性的细胞表面改变的生化基础(目标2)，以及 确定EEP中与其功能有关的关键结构区和氨基酸(目标3)。完成 将提供关于一种新的功能和机制的基本新知识。 保守的酶，可翻译给与抗生素相关的其他病原菌 耐药性和生物被膜感染，如耐甲氧西林金黄色葡萄球菌和艰难梭菌。

PROJECT SUMMARY/ABSTRACT Enterococcus faecalis significantly contributes to the burden of escalating healthcare costs as a leading cause of healthcare-associated infections. The antimicrobial-recalcitrant nature of E. faecalis and its ability to form biofilms necessitates prolonged and complex treatment strategies for infections. Therefore, there is a critical need to identify new approaches for treating and preventing enterococcal disease. We previously showed that the conserved intramembrane metalloprotease Eep, part of the site 2 protease (S2P) family, is a critical factor in E. faecalis for both in vitro biofilm formation and in vivo biofilm-associated infection. This largely uncharacterized role of Eep in E. faecalis biofilm formation is in addition to its documented functions in cell-cell signaling, cellular adaptation in response to attack by the innate immune system effector molecule lysozyme, and the spread of antibiotic resistance-carrying plasmids. However, despite its importance in these cellular processes, there are numerous unanswered fundamental questions about Eep’s biochemical activity and how that activity influences E. faecalis pathogen-host interactions. More broadly, our mechanistic and biological understanding of Eep and orthologous S2Ps in other Gram-positive pathogens is also limited. This project will investigate the following questions: (1) What are Eep’s substrates and products?; (2) How do Eep’s effectors affect biofilm formation and adaptation to cell surface stress? In turn, how do these Eep-dependent processes affect host-pathogen interactions?; and (3) How does Eep’s structure influence its ability to recognize and cleave substrates? Our experimental design will test the hypothesis that the proteolytic activity of Eep leads to coordinated changes at the cell surface that influence E. faecalis interactions with mammalian hosts in pathogenic settings. We will pair proteomic, molecular genetic, and microbiological approaches with two animal models of in vivo biofilm formation to identify candidate Eep substrates and downstream effectors in biofilms (Aim 1), characterize the genetic and biochemical basis of cell surface alterations that render E. faecalis cells resistant to lysozyme (Aim 2), and determine the key structural regions and amino acids in Eep that contribute to its function (Aim 3). Completion of the proposed experiments will provide fundamental new knowledge about the functions and mechanism of a conserved enzyme that will be translatable to other pathogenic bacteria that are associated with antibiotic resistance and biofilm infections, such as methicillin-resistant Staphylococcus aureus and Clostridium difficile.