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. 2009 Sep;77(9):3626-38.
doi: 10.1128/IAI.00219-09. Epub 2009 Jun 15.

Contribution of Autolysin and Sortase a During Enterococcus Faecalis DNA-dependent Biofilm Development

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Free PMC article

Contribution of Autolysin and Sortase a During Enterococcus Faecalis DNA-dependent Biofilm Development

Pascale S Guiton et al. Infect Immun. .
Free PMC article

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.

Figures

FIG. 1.
FIG. 1.
DNA is critical for OG1RF biofilms under static conditions. (A) Crystal violet staining of OG1RF biofilms grown statically in TSBG at 37°C in the presence or absence of 5 μg/ml DNase I and monitored for 72 h. The error bars indicate standard errors of the means for three to seven different experiments. *, P < 0.05, as determined by the Mann-Whitney test. (B) Seventy-two-hour static OG1RF biofilms without DNase I treatment (left panel) or treated with 5 μg/ml of DNase continuously (middle panel) or for 3 h postformation (right panel). Biofilms were then stained with SYTO9 (green) and PI (red) and visualized with CLSM. 3D reconstructions of z stacks were generated with the Volocity software. One unit on each side of each grid equals 14.3 μm. (C) Relative biomasses of 72-h OG1RF biofilms treated for 1 h, 2 h, 3 h, or 24 h with 5 μg/ml of DNase I or iDNase I at 37°C. The error bars indicate standard errors of the means for three different experiments. *, P < 0.05, as determined by the Mann-Whitney test; **, P < 0.01, as determined by the Mann-Whitney test; ***, P < 0.0005, as determined by the Mann-Whitney test.
FIG. 2.
FIG. 2.
Hydrodynamic conditions induce the production of a DNase I-sensitive extracellular fibrous network. The images are freeze-dry electron micrographs (magnification, ×20,000) (A and D) and CLSM images (B, C, and E) of 48-h biofilms of OG1RF stained with SYTO9 (green) and PI (red) that were not treated (A to C) or were treated for 1 h with 5 μg/ml of DNase I (D and E). The arrows indicate SYTO9-PI-stained fibers visible in untreated OG1RF biofilms but not in DNase I-treated biofilms by CLSM. (B and E) Scale bars = 20 μm. (C) Scale bar = 10 μm. (A and D) Scale bars = 500 nm.
FIG. 3.
FIG. 3.
Atn contributes to DNA release during biofilm formation. (A) Negative staining and immunoelectron microscopy images of 48-h OG1RF and OG1RFΔatn biofilms with or without 1 h of DNase I treatment and labeled with mouse anti-dsDNA monoclonal antibody and secondary immunogold. The images are representative images of 10 random fields. Scale bars = 200 nm. (B) Cell-associated or extracellular gold particles (indicated by arrowheads in panel A) were quantified for 10 independent fields of 48-h shaken biofilms at a magnification of ×30,000.
FIG. 4.
FIG. 4.
Atn is required for production of DNase-sensitive biofilms under hydrodynamic conditions. (A and B) Crystal violet staining of OG1RF (WT) and OG1RFΔatnatn) biofilms treated continuously for 24 h and 48 h (A) or for 1 h after 48 h of growth (B) with 5 μg/ml of DNase I under hydrodynamic conditions in TSBG. The error bars indicate standard errors of the means for three or four independent experiments. *, P < 0.05, as determined by the Mann-Whitney test; **, P < 0.005, as determined by the Mann-Whitney test; ***, P < 0.0005, as determined by the Mann-Whitney test. (C) CLSM-acquired z-stack images of 48-h biofilms of OG1RF and OG1RFΔatn that were not treated or were treated for 1 h with 5 μg/ml of DNase I and stained with SYTO9 and PI. Long chains of bacteria were evident in OG1RFΔatn mutant biofilms. DNase I treatment dramatically reduced the biomass of the OG1RF biofilm, but not the biomass of the OG1RFΔatn biofilm. Furthermore, few SYTO9-PI-positive bacterial cells (yellow) were visible in the OG1RFΔatn mutant biofilm compared to the wild-type biofilm. Scale bars = 20 μm.
FIG. 5.
FIG. 5.
Primary attachment requires srtA and atn, but not eDNA. (A) OG1RF (WT), OG1RFΔatnatn), OG1RFΔsrtAsrtA), and OG1RFΔsrtA srtAsrtA/srtA) were incubated statically on plastic coverslips for 4 to 6 h at 37°C in TSBG with or without 5 μg/ml of DNase I. Biofilms were stained with crystal violet and then solubilized. The degree of primary attachment was determined based on the OD595 of solubilized crystal violet. The error bars indicate the standard errors of the means for at least three independent experiments. *, P < 0.05, as determined by the Mann-Whitney test; **, P < 0.01, as determined by the Mann-Whitney test; ***, P < 0.0001, as determined by the Mann-Whitney test. (B) Representative CLSM images of 4-h and 6-h adherent cells in the presence or absence of DNase I following staining with SYTO9 and PI. The arrows indicate microcolonies in 6-h samples. One unit on each side of each grid equals 14.3 μm. (C) Quantification of the change in fluorescence of SYTO9 and PI relative to the total fluorescence from adhering cells from 4 h to 6 h postinoculation. The graph was generated from data obtained from 12 randomly chosen fields from two independent experiments using the Volocity software.
FIG. 6.
FIG. 6.
SrtA promotes high levels of DNA-dependent biofilm development. (A) Crystal violet-based quantification of OG1RF (WT) and OG1RFΔsrtAsrtA) biofilm development. (B) Crystal violet-based quantification of 72-h OG1RF, OG1RFΔsrtA, and OG1RFΔsrtA srtAsrtA/srtA) biofilms. The error bars indicate the standard errors of the means for three different experiments *, P < 0.05, as determined by the Mann-Whitney test; **, P < 0.01, as determined by the Mann-Whitney test. (C) 3D reconstruction of CLSM-acquired images of static 72-h-old biofilms of OG1RF, OG1RFΔsrtA, and OG1RFΔsrtA srtA grown statically and stained with SYTO9 (green) and PI (red). The CLSM images are representative images from the same experiment. One unit on each side of the grid equals 14.3 μm. (D) CLSM images of 48-h biofilms under hydrodynamic conditions for OG1RF, OG1RFΔsrtA, and OG1RFΔsrtA srtA stained with SYTO9 and PI. Scale bars = 10 μm.

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