Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction - PubMed (original) (raw)

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction

Sreedhar R Nallapareddy et al. Infect Immun. 2006 Sep.

Abstract

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

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Figures

FIG. 1.

RT-PCR analysis of ace transcripts in E. faecalis OG1RF. (A) Growth patterns of OG1RF at 37 or 46°C. Time points at which total RNA was isolated are marked with gray (37°C) and black (46°C) arrowheads. (B and C) RT-PCR-amplified bands for ace or control gene gdh. The total amounts of RNA per reaction used were 5 ng for each of the samples in panel B and 250 ng for each of the samples in panel C. A 100-bp DNA ladder (Invitrogen) was used as the size marker (lanes 1 and 14). For the samples in lanes 10 to 13, as well as lanes 19 and 20, the reverse transcriptase was omitted (negative control for RT). Growth temperatures, as well as different growth phases from which RNA was extracted, are given above each lane of panel B, and the genes analyzed are shown below panel C. ML, mid-log phase; LL, late-log phase, SE, stationary-phase entry; and S, stationary phase.

FIG. 2.

E. faecalis OG1RF ace mRNA expression after growth in the presence of ECM proteins. The results are presented as amplified products electrophoresed on ethidium bromide-stained agarose gels. RT-PCR amplification of the gdh (housekeeping gene) was performed to ensure that similar amounts of input RNA and similar RT efficiencies were being compared. (A) Effect of different ECM proteins and serum on ace transcription. Mid-exponential-growth-phase cultures in BHI medium were split into aliquots and incubated with 0.3 mg of CI, CIV, fibrinogen (FG), or BSA/ml. Samples were removed after 1 h, and total RNA was prepared as described in Materials and Methods. For cultures grown in 40% horse serum in BHI, total RNA was extracted from the late exponential growth phase. (B) Dose-dependent induction of ace expression by OG1RF. OG1RF cells were grown in the presence of 0.05 mg/ml to 0.3 mg of CIV/ml for 1 h. The samples in lanes 3 and 10 of panel B are RT-PCR amplicons of RNA isolated from cultures grown in BHI buffered with CH3COOH and HCl, respectively. RT-PCRs performed with two independent sample preparations showed similar results.

FIG. 3.

Quantitative ace mRNA levels in E. faecalis OG1RF exposed to different milieu. Relative levels of ace transcripts from OG1RF grown in the presence or absence of CIV or FG or grown at 46°C were quantified with reference to transcripts of OG1RF grown in BHI at 37°C. The 23S rRNA transcript was used as an endogenous control. Primer pairs were checked for primer-dimer formation by dissociation curve and also by using the primers without the addition of RNA template. The results are presented as means ± the standard deviations. In the insert, the real-time amplification plots of RNA samples obtained from cultures grown in the presence or absence of CIV were shown.

FIG. 4.

Immunofluorescence images of surface-exposed Ace on E. faecalis strains. Panels A, C, E, G, I, and K (phase contrast) and B, D, F, H, J, and L (fluorescent visualization) are E. faecalis OG1RF cells stained with anti-Ace rabbit sera. Panels M and O (phase contrast) and N and P (fluorescent visualization) are E. faecalis strain MC02152 cells stained with anti-Ace rabbit sera. Rhodamine red-labeled goat anti-rabbit antibodies were used as the secondary antibody. Culture conditions are marked on the side of the respective panel pair. Preimmune rabbit sera with OG1RF and anti-Ace sera with ace mutant (TX5256) served as controls (data not shown).

FIG. 5.

Adherence assay with wild-type E. faecalis strain OG1RF and its isogenic ace disruption mutant (TX5256). The data shown in panels A and B represent the adherence of OG1RF cells to CIV and LN, respectively. Median and interquartile range values are shown. Adherent bacterial cells quantified by using 60 fields of phase-contrast microscope represent four independent experiments. Sample medians generated from the adherent bacteria quantifications were compared by using the Mann-Whitney test. Tests were performed by using GraphPad Prism v4 for Windows.

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Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction - PubMed (original) (raw)