The regulation of autolysis in Enterococcus faecalis

描述（申请人提供）：肠球菌是医院获得性感染的主要原因，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，导致显著的发病率和死亡率。此外，由于存在多重耐药性，这些感染对治疗构成重大挑战。本研究的目的是探讨粪肠球菌蛋白酶、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的形成。主要的自溶素AtlA是这两种蛋白酶的靶点，通过水解细胞壁导致细菌亚群死亡和随后释放细胞外DNA的能力，在生物膜的形成中起着关键作用。从裂解细胞中释放的DNA是肠球菌生物膜形成的重要生物膜基质。我们最近表明，这一过程是由一个自相残杀的机制控制的，其功能是由Fsr仲裁系统控制的。AtlA与这两种蛋白酶的相互作用，以及它与细胞壁的关联，将通过构建缺乏特异性LysM模块结构域的AtlA的等基因突变体，以及缺乏n端T/E富结构域的AtlA突变体来研究。这些突变体将评估其自溶能力和对生物膜形成的贡献。AtlA及其突变体与两种蛋白酶的相互作用也将在表面等离子体共振研究中使用纯化蛋白来评估，以确定结合亲和力，并且将通过免疫电子显微镜检查天然和突变形式的AtlA在细胞表面的定位。此外，通过生成和检测o -乙酰转移酶活性缺陷的细胞壁修饰，以及自溶和生物膜形成中壁酸的d -丙烯酰化缺陷，将探索调节AtlA自溶活性的细胞壁修饰。最后，为了确定dna依赖的生物膜过程在体内对疾病的贡献程度，将通过比较导管诱导的兔感染性心内膜炎模型中的突变体来检查粪肠球菌中参与自溶调节的蛋白质的相对贡献。

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.