Enterococcus faeciumBiofilm Formation: Identification of Major Autolysin AtlAEfm, Associated Acm Surface Localization, and AtlAEfm-Independent Extracellular DNA Release

ABSTRACTEnterococcus faeciumis an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faeciumbiofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faeciumE1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faeciumand renamed atlAEfm. In addition, AtlAEfmwas implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faeciumthat contributes to cell-cell interactions in the atlAEfmmutant and is important for cell separation. In conclusion, AtlAEfmis the major autolysin in E. faeciumand contributes to biofilm stability and Acm localization, making AtlAEfma promising target for treatment of E. faeciumbiofilm-mediated infections.IMPORTANCENosocomial infections caused by Enterococcus faeciumhave rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfmdisruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfmis associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfmmutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfmmutant. These findings indicate that AtlAEfmis important in biofilm and collagen binding in E. faecium, making AtlAEfma promising target for treatment of E. faeciuminfections.