The regulation of autolysis in Enterococcus faecalis

粪肠球菌自溶的调控

• 批准号: 8458148
• 负责人: Lynn E Hancock
• 金额: $ 12.29万
• 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 2013-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458148
• 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.

描述（由申请人提供）：作为医院获得感染的主要原因，肠球菌占所有医院感染的近10％。此外，肠球菌是社区中亚急性心内膜炎的主要原因，导致发病率明显和死亡率。此外，由于存在多药耐药性，这些感染引起了重大的治疗挑战。这项研究的目的是调查核心肠球菌蛋白酶，明胶酶（GELE）和丝氨酸蛋白酶（SPRE）如何通过调节自动释放过程的生物膜发展的潜在机制。主要的自脂素ATLA是蛋白酶的靶标，并且通过水解细胞壁导致细胞壁死亡以及随后释放细胞外DNA而在生物膜发展中起关键作用。从裂解细胞释放的DNA是肠球菌生物膜发展的重要生物膜基质。我们最近表明，此过程受术机制的控制，其功能由FSR Quorum系统控制。 ATLA与两个蛋白酶的相互作用及其与细胞壁的关联将通过构造缺乏特定Lysm模块化结构域的ATLA的同源突变体以及缺乏N端T/E丰富结构域的突变体。这些突变体将根据其自溶能力和对生物膜形成的贡献进行评估。 ATLA及其突变体与两种蛋白酶的相互作用也将使用表面等离子体共振研究中的纯化蛋白质进行评估，以确定结合亲和力，并且将ATLA的天然和突变体形式与细胞表面进行了定位。此外，将通过对O-乙酰转移酶活性有缺陷的突变体以及在自溶和生物膜形成中的T-D-丙二醇化来探索对ATLA自溶活性的修改。最后，为了确定DNA依赖性生物膜过程在多大程度上导致体内疾病，将通过比较导管诱导的感染性心内膜炎的兔模型中的突变体来检查参与粪肠球菌调节的蛋白质的相对贡献。