EbpA vaccine antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-associated bladder infection in mice

Abstract

Enterococci bacteria are a frequent cause of catheter-associated urinary tract infections, the most common type of hospital-acquired infection. Treatment has become increasingly challenging because of the emergence of multiantibiotic-resistant enterococcal strains and their ability to form biofilms on catheters. We identified and targeted a critical step in biofilm formation and developed a vaccine that prevents catheter-associated urinary tract infections in mice. In the murine model, formation of catheter-associated biofilms by Enterococcus faecalis depends on EbpA, which is the minor subunit at the tip of a heteropolymeric surface fiber known as the endocarditis- and biofilm-associated pilus (Ebp). We show that EbpA is an adhesin that mediates bacterial attachment to host fibrinogen, which is released and deposited on catheters after introduction of the catheter into the mouse bladder. Fibrinogen-binding activity resides in the amino-terminal domain of EbpA (EbpA(NTD)), and vaccination with EbpA and EbpA(NTD), but not its carboxyl-terminal domain or other Ebp subunits, inhibited biofilm formation in vivo and protected against catheter-associated urinary tract infection. Analyses in vitro demonstrated that protection was associated with a serum antibody response that blocked EbpA binding to fibrinogen and the formation of a fibrinogen-dependent biofilm on catheters. This approach may provide a new strategy for the prevention of catheter-associated urinary tract infections.

Fig. 1. EbpA^NTD is critical for biofilm formation and fibrinogen recognition

(A) E. faecalis strains with deletions (Δ) of Ebp pilus subunits and Gram-positive bacterial assembly proteins were evaluated for biofilm formation in a standard in vitro polyvinyl chloride coverslip assay after 48 hours by staining with crystal violet. The EbpA MIDAS motif mutant was designated as EbpA^AWAGA. (B and C) Adherence of the indicated whole bacterial strains to (B) collagen I (Col I)–coated or (C) fibrinogen (Fg)–coated surfaces was assessed by ELISA using a rabbit anti–group D streptococcal antibody. (D) EbpA domain structure, as predicted by PHYRE2 software (47). S, signal sequence; CWSS, C-terminal cell wall sorting signal; vWA, von Willebrand factor A domain containing a MIDAS motif. Shown below the figure are the regions included in the indicated EbpA subdomain proteins. (E) ELISA assay to quantitate binding of the indicated purified proteins to immobilized fibrinogen using a mouse anti-EbpA^Full antisera. All assays used human collagen I and fibrinogen. Data represent means ± SEM derived from at least three independent experiments with differences between mean values evaluated for significance using a paired t test: *P < 0.05; **P < 0.005; ***P < 0.0005; ns (differences not significant), P > 0.05.

Fig. 2. Time course of fibrinogen release and deposition on catheters implanted in mouse bladders

Mice were implanted with catheters and challenged by 1 × 10⁷ CFU E. faecalis OG1RF. (A and B) At the indicated time points after infection, bladder tissue (A) and catheters (B) were recovered and subjected to analysis by immunofluorescence using antibody staining to detect fibrinogen (anti-Fg; green). Samples were costained with antibody to detect uroplakin III (anti-UpIII; red) or with DAPI (blue) to delineate the urothelium and cell nuclei, respectively (representative images). The white broken line separates the bladder lumen (L) from the urothelium surface (U).

Fig. 3. Colocalization of E. faecalis EpbA with fibrinogen in catheter-implanted mouse bladders

Catheter-implanted mice were challenged with the indicated E. faecalis OG1RF (infected) or were unchallenged (uninfected). (A and B) Bladder tissue (A) and catheters (B) were analyzed after 24 hours of implantation by immunofluorescence using antibody staining to detect fibrinogen (anti-Fg; green) and E. faecalis (anti–group D; pink). Antibody staining for uroplakin III (anti-UpIII; red) or with DAPI (blue) delineated the urothelium and cell nuclei, respectively. (A) Mice were infected with E. faecalis OG1RF (wild type) (representative images). (B) Mice were infected with E. faecalis OG1RF (wild type) or by mutants that did not express pili (ΔEbpABCΔSrtC) or that expressed EbpA with a defective MIDAS motif (EbpA^AWAGA). Bacterial EbpA colocalization with fibrinogen was compared to that for mock-infected mice (PBS control) (representative images). The white broken line separates the bladder lumen (L) from the urothelium surface (U).

Fig. 4. E. faecalis OG1RF biofilm formation in human urine requires fibrinogen

Bacterial growth was determined by the number of CFU after 24 hours (or otherwise indicated) of culture in human urine alone or in urine supplemented with the indicated concentrations of fibrinogen (Fg), bovine serum albumin (BSA), or casamino acids (CA). (A) Cultures were inoculated to an initial density of ~5 × 10⁵ CFU/ml. (B to D) Growth curves in urine over a range of concentrations of fibrinogen (B), BSA (C), or CA (D). (E) Examination of uninoculated fibrinogen-supplemented urine by negative staining with 1% uranyl acetate and electron microscopy revealed lattice-like structures that were consumed after 24 hours of culture with E. faecalis. Scale bar, 500 nm. (F) Biofilm formation in vitro on 1-cm silicon catheters in urine supplemented with fibrinogen and BSA. Scale bar, 500 nm. (G to I) Biofilm formation in a standard 96-well polystyrene plate assay comparing wild-type (OG1RF) and MIDAS mutant (EbpA^AWAGA) strains in fibrinogen-supplemented (H) or either BSA- or CA-supplemented urine (I). Biofilm formation in a standard growth medium (TSGB) was included for comparison. Values represent means ± SEM derived from at least three independent experiments with differences between mean values evaluated for significance using a paired t test: *P < 0.05; **P < 0.005; ***P < 0.0005; ns, P > 0.05. Human urine was pooled from three healthy female donors, clarified by centrifugation, and adjusted to pH 6.5 before use.

Fig. 5. EbpA adhesin-based vaccine protects mice from E. faecalis CAUTIs

Mice were immunized and received two booster immunizations with the indicated doses of the various Ebp proteins (EbpB, EbpC, EbpA^Full, EbpA^NTD, and EbpA^CTD). Four weeks after the final immunization, mice were implanted with catheters and challenged with 1 × 10⁷ CFU of E. faecalis OG1RF. After 24 hours of infection, bacterial burdens in bladder tissue (A and D) or recovered catheters (B and E) were quantitated as the number of CFU recovered. (C) The presence and distribution of bacteria and fibrinogen were assessed in catheters recovered from three mice taken randomly in the indicated treatment groups by immunofluorescence staining using antibody staining to detect fibrinogen (anti-Fg) and E. faecalis (anti–group D). (F) Ability of sera recovered from immunized mice to block binding of purified EbpA proteins to a fibrinogen-coated surface was evaluated by ELISA. Mouse anti-EbpA^Full was used to detect all species of EbpA. Rabbit anti-EbpA^CTD was used as a negative control. Rabbit anti-fibrinogen was used to block fibrinogen before adding the proteins. Values represent means ± SEM. Mann-Whitney U test was used for mouse experiments and paired t test for binding assays. P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.005; ***P < 0.0005; ns, values were not statistically different. The horizontal bar represents the median value. The horizontal broken line represents the limit of detection of viable bacteria. Animals that lost the catheter were not included in this work.