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

肠球菌位点 2 蛋白酶在生物膜形成、适应和宿主-病原体相互作用中的作用

• 批准号: 10318166
• 负责人: Kristi L Frank
• 金额: $ 37万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-13 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318166
• 关键词: Affect; Aging; Amino Acids; Animal Model; Antibiotic Resistance; Attenuated; Biochemical; Biological; Biological Assay; Biological Process; Catalytic Domain; Cell physiology; Cell surface; Cells; Cellular Structures; Characteristics; Clostridium difficile; Code; Complex; Data; Defect; Development; Disease; Drug resistance; Enterococcus; Enterococcus faecalis; Enzymes; Etiology; Experimental Designs; Exposure to; Family; Felis catus; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Transcription; Goals; Health Care Costs; Healthcare; In Vitro; Infection; Infective endocarditis; Innate Immune Response; Innate Immune System; Knowledge; Lead; Membrane; Metalloproteases; Microbial Biofilms; Microbiology; Modeling; Modification; Molecular Genetics; Muramidase; Mutation; Nature; Open Reading Frames; Oryctolagus cuniculus; Oxidation-Reduction; Pathogenicity; Peptide Hydrolases; Peptide Signal Sequences; Phenotype; Plasmids; Population; Predisposition; Process; Proteolysis; Proteomics; Resistance; Resistance to infection; Role; Sigma Factor; Signal Pathway; Signal Transduction; Site; Source; Stress; Structure; Surface; Surface Properties; System; Testing; Time; Transmembrane Domain; United States; Virulence; Work; antimicrobial; experimental study; extracellular; healthcare-associated infections; in vivo; in vivo Model; infection rate; insight; intercellular communication; methicillin resistant Staphylococcus aureus; mutant; neutrophil; new therapeutic target; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; prevent; response; treatment strategy

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.

项目总结/摘要 粪肠球菌作为一种主要原因，对医疗保健费用不断上升的负担起着重要作用 与医疗相关的感染。E.粪便及其形成能力 生物膜需要延长和复杂的感染治疗策略。因此，存在一个关键的 需要确定治疗和预防肠球菌病的新方法。我们之前已经证明， 保守的膜内金属蛋白酶Eep是位点2蛋白酶（S2 P）家族的一部分，是 E. faecalis用于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E.粪便生物膜的形成除了其在细胞-细胞信号传导、细胞免疫应答、细胞免疫应答中的功能外， 适应性反应攻击的先天免疫系统效应分子溶菌酶，和传播 携带抗生素抗性的质粒。然而，尽管它在这些细胞过程中的重要性， 关于Eep的生化活动以及这种活动如何影响 E.粪便病原体-宿主相互作用更广泛地说，我们对Eep的机械和生物学理解， 在其它革兰氏阳性病原体中的邻位S2 Ps也有限。本项目将调查以下内容 问题：（1）EEP的基材和产品是什么？(2)Eep效应子如何影响生物膜形成， 适应细胞表面的压力反过来，这些依赖Eep的过程如何影响宿主-病原体 相互作用？（3）Eep的结构如何影响其识别和切割底物的能力？我们 实验设计将检验Eep的蛋白水解活性导致协同变化的假设， 影响E.在致病环境中粪便与哺乳动物宿主的相互作用。我们会配对 蛋白质组学、分子遗传学和微生物学方法，采用两种体内生物膜形成的动物模型 为了鉴定生物膜中的候选Eep底物和下游效应物（目的1），表征生物膜的遗传和 细胞表面改变的生化基础，使E。抗溶菌酶的粪菌细胞（Aim 2），以及 确定Eep中有助于其功能的关键结构区域和氨基酸（目标3）。完成 的建议实验将提供新的基本知识的功能和机制， 保守的酶，将被翻译到与抗生素相关的其他病原菌 耐药性和生物膜感染，如耐甲氧西林金黄色葡萄球菌和艰难梭菌。