Dataset Information

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.

SUBMITTER: Guiton PS

PROVIDER: S-EPMC2738007 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development.

Guiton Pascale S PS Hung Chia S CS Kline Kimberly A KA Roth Robyn R Kau Andrew L AL Hayes Ericka E Heuser John J Dodson Karen W KW Caparon Michael G MG Hultgren Scott J SJ

Infection and immunity 20090615 9

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 forma ...[more]

PMID: 19528211

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Project description:UnlabelledEnterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that--in early E. faecalis biofilms--eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix.ImportanceThis paper reports the production of extracellular DNA during early biofilm formation in Enterococcus faecalis. The work is significant because the mechanism of eDNA (extracellular DNA) production is independent of cell lysis and the DNA is confined to well-defined structures, suggesting a novel form of DNA secretion by viable cells. Previous models of biofilm formation in enterococci and related species propose cell lysis as the mechanism of DNA release.

Project description:Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with (75)Se is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production.

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Project description:Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

Dataset Information

Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development.

Publications

Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development.

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