The regulation of autolysis in Enterococcus faecalis

粪肠球菌自溶的调控

• 批准号: 8259836
• 负责人: Lynn E Hancock
• 金额: $ 29.3万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259836
• 关键词: Accounting; Acetylation; Acute Endocarditis; Address; Affinity; Amino Acid Sequence; Antibiotic Resistance; Antibiotic Therapy; Antibodies; Antimicrobial Resistance; Autolysin; Autolysis; Bacteria; Bacterial Infections; Binding; Biological Assay; C-terminal; Catalytic Domain; Catheters; Cell Wall; Cell surface; Cells; Cessation of life; Communities; Complement; Country; Cytolysis; DNA; Data; Development; Disease; Disease model; Endocarditis; Endophthalmitis; Enterococcus; Enterococcus faecalis; Flow Cytometry; Gelatinases; Genes; Goals; Homologous Gene; Hospitals; Immunoelectron Microscopy; In Vitro; Infection; Infective endocarditis; Link; Lupinus; Maps; Mass Spectrum Analysis; Microbial Biofilms; Modality; Modeling; Modification; Morbidity - disease rate; Multi-Drug Resistance; Muramic Acid; Mus; N-terminal; Nosocomial Infections; Operative Surgical Procedures; Oryctolagus cuniculus; Peptide Hydrolases; Peptide Sequence Determination; Peptidoglycan; Peritonitis; Play; Population; Predisposition; Process; Proteins; Proteolytic Processing; Regulation; Relative (related person); Role; Sepsis; Serine Protease; Severity of illness; Signal Transduction; Site; Staphylococcus aureus; Surface Plasmon Resonance; System; Teichoic Acids; Testing; Therapeutic; Transferase; Urinary tract; base; extracellular; improved; in vivo; infectious disease model; interest; killings; lipoteichoic acid; mortality; mutant; public health relevance

DESCRIPTION (provided by applicant): As a leading cause of hospital acquired infection, enterococci account for nearly 10 % of all nosocomial infections. In addition, enterococci are a leading cause of sub-acute endocarditis in the community resulting in significant morbidity and mortality. Furthermore these infections pose a significant treatment challenge due to the presence of multi-drug resistance. The goal of this study is to investigate the underlying mechanisms of how the coregulated Enterococcus faecalis proteases, gelatinase (GelE) and the serine protease (SprE) contribute to the development of biofilms through the regulation of autolytic processes. The major autolysin, AtlA, is a target of both proteases and plays a key role in the development of biofilms by its ability to hydrolyze the cell wall leading to the death of sub-populations of bacteria and subsequent release of extracellular DNA. The DNA released from lysed cells serves as an important biofilm matrix upon which enterococcal biofilms develop. We have recently shown that this process is governed by a fratricidal mechanism, whose function is controlled by the Fsr quorum system. The interaction of AtlA with both proteases, as well as its association with the cell wall will be examined by constructing isogenic mutants of AtlA that lack specific LysM modular domains, as well as mutants that lack the N-terminal T/E rich domain. These mutants will be assessed for their autolytic capacity and contribution to biofilm formation. The interaction of AtlA and its mutants with both proteases will also be assessed using purified proteins in surface plasmon resonance studies to determine binding affinities, and the localization of native and mutant forms of AtlA to the cell surface will be examined by immunoelectron microscopy. In addition, modifications to the cell wall thought to modulate the autolytic activity of AtlA will be explored by generating and examining mutants defective for O-acetyl transferase activity, as well as D-alanylation of teichoic acids in autolysis and biofilm formation. Lastly, to determine the extent to which DNA-dependent biofilm processes contribute to disease in vivo, the relative contribution of the proteins involved in the regulation of autolysis in E. faecalis will be examined by comparing mutants in the catheter- induced rabbit model of infectious endocarditis. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is a growing problem in this country and around the world, particularly among infections encountererd in hospitals. For some bacterial infections, including a number caused by enterococci, there are no antibiotic treatment options available. There is an increasing need to develop new strategies and therapeutics to treat these types of infections.

描述（由申请人提供）：作为医院获得性感染的主要原因，肠球菌占所有医院感染的近10%。此外，肠球菌是社区亚急性心内膜炎的主要原因，导致显著的发病率和死亡率。此外，由于存在多药耐药性，这些感染构成了重大的治疗挑战。本研究的目的是探讨共同调节的粪肠球菌蛋白酶明胶酶（GelE）和丝氨酸蛋白酶（SprE）如何通过调节自溶过程促进生物膜形成的潜在机制。主要的自溶素，CITRA，是两种蛋白酶的靶标，并且通过其水解细胞壁的能力在生物膜的形成中起关键作用，导致细菌亚群的死亡和随后的细胞外DNA的释放。从裂解的细胞释放的DNA充当肠球菌生物膜在其上形成的重要生物膜基质。我们最近表明，这个过程是由一个自相残杀的机制，其功能是由FSR法定人数系统控制。将通过构建缺乏特异性LysM模块结构域的CRAMA的同基因突变体以及缺乏N-末端富含T/E结构域的突变体来检查CRAMA与两种蛋白酶的相互作用以及其与细胞壁的关联。将评估这些突变体的自溶能力和对生物膜形成的贡献。还将在表面等离子体共振研究中使用纯化的蛋白质来评估BRA及其突变体与两种蛋白酶的相互作用，以确定结合亲和力，并将通过免疫电子显微镜检查BRA的天然和突变形式在细胞表面的定位。此外，修改细胞壁被认为是调节自溶活性的BTAA将探索通过产生和检查突变体缺陷的O-乙酰转移酶活性，以及D-丙氨酰化的磷壁酸在自溶和生物膜形成。最后，为了确定DNA依赖性生物膜过程在体内对疾病的贡献程度，研究了参与调节大肠杆菌自溶的蛋白质的相对贡献。将通过比较导管诱导的感染性心内膜炎兔模型中的突变体来检查粪便。 公共卫生相关性：抗生素耐药性在美国和世界各地都是一个日益严重的问题，特别是在医院里发现的感染病例中。对于一些细菌感染，包括一些由肠球菌引起的感染，没有可用的抗生素治疗选择。越来越需要开发新的策略和疗法来治疗这些类型的感染。