Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

Abstract

Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.Biofilms are bacterial communities encased within an extracellular matrix composed of carbohydrates, proteins, and nucleic acids (10). They are an ideal environment for exchange of genetic materials, such as genes encoding virulence factors and antibiotic resistance determinants, among bacteria within a community (61, 76), while allowing the flow of water and nutrients, as well as ions and various small molecules, to bacteria within the community (8) and providing a protective shield against antibiotics, antimicrobial substances, and phagocytosis (33, 80). Development of a biofilm is a complex multistage process. It is initiated by primary adhesion of the bacteria to a substratum, which is followed by the formation of microcolonies and production of an exopolysaccharide matrix, and it finally culminates with the formation of a three-dimensional (3D) multicellular mature structure (48). Bacterial biofilms are important medically because they have been associated with the pathogenesis of chronic and device-related persistent infections, such as cystic fibrosis, urinary tract infections, and endocarditis (11).Enterococcus faecalis is a gram-positive bacterium that is a commensal of the gastrointestinal tract of healthy individuals. However, it is also an important opportunistic pathogen that is responsible for a wide variety of nosocomial infections, including endocarditis, urinary tract infections, and bacteremia (14,17,23,44). E. faecalis accounts for approximately 65 to 80% of all enterococcal nosocomial infections (16). The ability of E. faecalis to adhere to and develop biofilms on medical devices, such as intravascular and urinary catheters (25,26,36,37,72), is thought to contribute to its pathogenes...

Enterococcus faecalis Overcomes Foreign Body-Mediated Inflammation To Establish Urinary Tract Infections

“…In a murine model of cystitis, E. faecalis induces minimal inflammation and is rapidly eliminated from the bladder (35). However, with the introduction of silicone tubing implants into the bladder, E. faecalis becomes adept at causing CAUTI despite the edema and the robust inflammatory response induced by the catheter (36). Thus, we investigated whether E. faecalis virulence was altered in animals preimplanted 24 h before infection to determine the effect of a preexisting catheter-induced inflammatory response on the outcome of infection.…”

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

“…The 196-nucleotide intragenic region between EF1117 and EF1116 contains a stem-loop transcriptional terminator (32), which has been duplicated in the cloning site of pCJK141 so that sequences integrated into the chromosome are flanked by terminators to prevent transcriptional read-through in either direction. To generate the knock-in p 23 construct, the ahrC gene-including its native ribosome binding site (RBS)-was PCR amplified from OG1RF genomic DNA using primer pair 0983 RBS BamHI F/0983 st cod NcoI R, and ligated into pCJK141 at the BamHI and NcoI restriction sites in the multiple cloning site of the vector. A PCR product containing the p 23 promoter, originally from Lactococcus sp.…”

“…To generate the knock-in p 23 construct, the ahrC gene-including its native ribosome binding site (RBS)-was PCR amplified from OG1RF genomic DNA using primer pair 0983 RBS BamHI F/0983 st cod NcoI R, and ligated into pCJK141 at the BamHI and NcoI restriction sites in the multiple cloning site of the vector. A PCR product containing the p 23 promoter, originally from Lactococcus sp. (33), was generated with primer pair NotI P23/BamHI P23 and was introduced upstream of ahrC in pCJK141 at the NotI and BamHI restriction sites.…”

“…Of these, srtA, coding for the housekeeping sortase, and the ebp pilus genes and their associated sortase gene, srtC, have also been implicated in enterococcal virulence (12,16,21,22). In contrast, disruption of the major autolysin gene (atlA), an important mediator of DNA release during biofilm formation in vitro (23), did not result in reduced virulence in either peritonitis (24) or catheter-associated urinary tract infection (12). To the best of our knowledge, the other seven loci have not been studied directly in virulence models.…”

Innovative Solutions to Sticky Situations: Antiadhesive Strategies for Treating Bacterial Infections

“…Indeed, virulence of Grampositive pathogens, including E. faecalis and Staphylococcus aureus, is severely attenuated in animal models when the class A sortase is deleted (164,165). These findings have resulted in the pursuit of many strategies to discover inhibitors of sortase A function, including screening of natural products, high-throughput screening of chemical libraries, and structure-based in silico screening of compounds (Table 2).…”