The Fsr quorum-sensing system of Enterococcus faecalis modulates surface display of the collagen-binding MSCRAMM Ace through regulation of gelE - PubMed (original) (raw)
The Fsr quorum-sensing system of Enterococcus faecalis modulates surface display of the collagen-binding MSCRAMM Ace through regulation of gelE
Kenneth L Pinkston et al. J Bacteriol. 2011 Sep.
Abstract
Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalisis associated with host cell adherence and endocarditis. The Fsr quorum-sensing system of E. faecalis, a two-component signal transduction system, has also been repeatedly linked to virulence in E. faecalis, due in part to the transcriptional induction of an extracellular metalloprotease, gelatinase (GelE). In this study, we discovered that disruption of the Fsr pathway significantly increased the levels of Ace on the cell surface in the latter phases of growth. Furthermore, we observed that, in addition to fsrB mutants, other strains identified as deficient in GelE activity also demonstrated a similar phenotype. Additional experiments demonstrated the GelE-dependent cleavage of Ace from the surface of E. faecalis, confirming that GelE specifically reduces Ace cell surface display. In addition, disruption of the Fsr system or GelE expression significantly improved the ability of E. faecalis to adhere to collagen, which is consistent with higher levels of Ace on the E. faecalis surface. These results demonstrate that the display of Ace is mediated by quorum sensing through the action of GelE, providing insight into the complicated world of Gram-positive pathogen adhesion and colonization.
Figures
Fig. 1.
Screening of a Tn insertion library identified an fsrB_mutant (▾_fsrB) that had significant levels of surface-expressed Ace in the stationary phase. Each member of the Tn library is represented by gene identification (ID) number (ef number) on the _x_axis. Relative whole-cell ELISA signals are represented on the _y_axis. Each mutant was labeled with anti-Ace MAb, followed by a goat anti-mouse IgG-HRP conjugate and developed with TMB substrate. Absorbance values were determined by using a Thermo Multiskan EX plate reader.
Fig. 2.
The surface expression of Ace is affected by _fsrB_and EDTA. (A) Ace expression on the cell surface of OG1RF (with or without EDTA) and _ΔfsrB_cells at various growth phases were determined by flow cytometry using anti-Ace MAb as the primary antibody. Mean fluorescence intensity (MFI) was determined by flow cytometry. (B) The growth of E. faecalisOG1RF in BHI alone or BHI + 0.1 mM EDTA and of _ΔfsrB_cells was determined by measuring the OD600at the indicated time points. The data are presented as the averages of three independent experiments.
Fig. 3.
Gelatinase activity correlates with maintenance of Ace surface expression. The GelE activity (A) and Ace surface expression (B) in selected E. faecalisstrains (spots 1 to 7) and mutants (spots 8 to 10) of E. faecalisOG1RF were determined. In panel A, gelatinase activity was detected by inoculating overnight-cultured E. faecalisstrains on a TH agar plate containing 3% gelatin, followed by growth overnight at 37°C. The gelatinase activities of different strains were determined by assessing the “halo” formation. In panel B, selected strains were cultured overnight, diluted to an OD600of 0.05, and cultured for 4 h. Surface Ace expression was confirmed via anti-Ace MAb labeling. Histograms representing strains that demonstrated gelatinase activity are represented in white, while those without gelatinase activity are shaded.
Fig. 4.
(A) Cleavage of Ace from surface of cells with CM containing GelE. OG1RF Δ_gelE_Δ_sprE_cells were collected and centrifuged. The pellets were resuspended into selected CM from the indicated strains (OG1RF wild type or E. faecalis ΔgelE, Δ_sprE_, or Δ_gelE_Δ_sprE_strains). These CM-treated cells were centrifuged, and the resulting supernatants were used in Western blots for the detection of Ace or GelE. (B) Flow cytometry analysis of Δ_gelE_Δ_sprE_cells treated with CM from the strains indicated on the histogram demonstrated that the loss of surface Ace expression correlates with the presence of GelE in CM.
Fig. 5.
GelE cleavage of rAce analyzed by SDS-PAGE. Sample lanes from left to right represent reaction mixtures containing 30 μg of rAce/ml with various amounts of purified GelE (0.015, 0.045, 0.15, 0.45. 1.5, 4.5, and 15 μg/ml), rAce (30 μg/ml) alone, or GelE (15 μg/ml) alone. Molecular mass standards are indicated to the right in kilodaltons.
Fig. 6.
Mutants that retain Ace expression in the stationary phase maintain the ability to adhere to collagen. OG1RF and selected mutants were cultured overnight in BHI. The overnight cultures were subsequently diluted to an OD600of 0.05 and incubated for an additional 1 h (early phase) or 4 h (late phase). Adhesion and standard deviation calculations were averaged from four wells per experiment, and the data are representative of three independent experiments.
References
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