title: Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA Release.
abstract: Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm 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. faecium E1162 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. faecium and renamed atlAEfm. In addition, AtlAEfm was 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. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have 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. atlAEfm disruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfm is associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfm mutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfm mutant. These findings indicate that AtlAEfm is important in biofilm and collagen binding in E. faecium, making AtlAEfm a promising target for treatment of E. faecium infections.
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Fernanda L
authors.initials: FL
authors.lastname: Paganelli
authors.forename: Rob J L
authors.initials: RJ
authors.lastname: Willems
authors.forename: Pamela
authors.initials: P
authors.lastname: Jansen
authors.forename: Antoni
authors.initials: A
authors.lastname: Hendrickx
authors.forename: Xinglin
authors.initials: X
authors.lastname: Zhang
authors.forename: Marc J M
authors.initials: MJ
authors.lastname: Bonten
authors.forename: Helen L
authors.initials: HL
authors.lastname: Leavis
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis*; D001422:Bacterial Adhesion; D018441:Biofilms / Q000254:growth & development*; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D019295:Computational Biology; D004269:DNA, Bacterial / Q000378:metabolism*; D016984:Enterococcus faecium / Q000737:chemistry* / Q000502:physiology*; D055786:Gene Knockout Techniques; D006801:Humans; D016254:Mutagenesis, Insertional; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000032:analysis
chemical_list: C473793:Acm protein, Enterococcus faecium; D018829:Adhesins, Bacterial; D004269:DNA, Bacterial; D037521:Virulence Factors; D003094:Collagen; D009238:N-Acetylmuramoyl-L-alanine Amidase