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，这些感染构成了巨大的治疗挑战。本研究的目的是研究协同调节粪肠球菌蛋白水解酶、明胶酶(GLE)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。主要的自溶酶ATLA是这两种酶的靶标，它在生物膜的形成中发挥着关键作用，因为它能够降解细胞壁，导致细菌亚群死亡，随后释放胞外DNA。从裂解细胞释放的DNA是肠球菌生物膜形成的重要生物膜基质。我们最近已经证明，这一过程是由兄弟会机制控制的，其功能由FSR Quorum系统控制。ATLA与这两种蛋白酶的相互作用以及它与细胞壁的联系将通过构建缺乏特定LysM模块结构域的ATLA的等基因突变体以及缺乏N-末端T/E富含结构域的突变体来检测。将评估这些突变体的自溶能力和对生物膜形成的贡献。ATLA及其突变体与这两种酶的相互作用也将使用表面等离子共振研究中的纯化蛋白来评估，以确定结合亲和力，并将通过免疫电子显微镜检查ATLA的天然和突变形式在细胞表面的定位。此外，将通过产生和检查O-乙酰转移酶活性缺陷的突变体以及磷壁酸在自溶和生物膜形成中的D-丙氨化来探索对细胞壁的修饰以调节ATLA的自溶活性。最后，为了确定依赖DNA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来检查参与粪肠球菌自溶调节的蛋白质的相对贡献。 与公共卫生相关：抗生素耐药性在美国和世界各地是一个日益严重的问题，特别是在医院遇到的感染中。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。人们越来越需要开发新的战略和治疗方法来治疗这些类型的感染。

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.