Science Translational Medicine. 2014 Sep 17;6(254):254ra127

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

 

Ana L. Flores-Mireles, Jerome S. Pinkner, Michael G. Caparon*, & Scott J. Hultgren*

Department of Molecular Microbiology and Center for Women’s Infectious Disease Research Washington University School of Medicine, Saint Louis, MO 63110-1093, USA.

*Corresponding authors. E-mail: hultgren@borcim.wustl.edu (S.J.H.); caparon@borcim.wustl.edu (M.G.C.)

 

ABSTRACT

Enterococci are a frequent cause of catheter-associated urinary tract infections (CAUTIs), the most common type of hospital-acquired infection. Treatment has become increasingly challenging due to emergence of multi-antibiotic resistant enterococcal strains and their ability to form biofilms on catheters. In this study, we identified and targeted a critical step in biofilm formation to develop a vaccine that prevents CAUTI. In a murine model of CAUTI, formation of catheter-associated biofilms by E. faecalis depends on EbpA, which is the minor subunit that tips a heteropolymeric surface fiber known as the endocarditis and biofilm-associated (Ebp) pilus. Here we show that EbpA is an adhesin that mediates bacterial attachment to host fibrinogen, which is released and deposited on catheters following introduction of the catheter into the bladder. Fibrinogen-binding activity resides in the N-terminal domain of EbpA (EbpANTD) and vaccination with EbpA and EbpANTD, but not its C-terminal domain or other Ebp subunits, inhibited biofilm formation in vivo and significantly protected against CAUTI. Analysis in multiple in vitro assays 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 catheter material. This approach may provide a new strategy for the prevention of CAUTI and other catheter-associated diseases.

 

SUPPLEMENT

The incidence of health care-associated infections (HAI) has increased over the last several years to become a major health problem, particularly in hospital settings1. Catheter-associated urinary tract infections (CAUTI) (Fig. 1) are the most common cause of HAI and according to the Centers for Disease Control (CDC), have increased over 6% between 2009 and 2013. The risk of developing a CAUTI increases from 3% to 10% for each day the catheter is in place and CAUTI can lead to serious complications, such as bloodstream infections and death2. Several species in the genus Enterococcus, most prominently E. faecalis and E. faecium, are major causes of bacterial CAUTI3. The treatment of enterococcal CAUTI is difficult due to their intrinsic resistance to multiple antibiotics and aseptic solutions and because the organisms are members of the normal gut microbiota. Of particular concern, they have recently acquired resistance to antibiotics considered to be the “last line of defense,” including vancomycin, daptomycin, linezolid, and tigercycline4. Current treatment is now based on antibiotic combinations, such as penicillin with vancomycin/ciprofloxacin; or novobiocin with doxycycline. Unfortunately, combinatorial therapy has proven to be unpredictable5. There are promising new antibiotics, such as fluoroquinolones, streptogramins, oxazolidinones, semisynthetic glycopeptides, and glycylcyclines4,5; however, enterococci rapidly develop resistance, so these antibiotics may also become obsolete. Furthermore, even effective antibiotic therapy will alter the patient’s gut microflora, leading to unwanted outcomes, such as development of Clostridium difficile colitis3. An ideal alternative would target enterococci without altering the gut microbiota and would be recalcitrant to the development of resistance. However, this goal requires a detailed understanding of the molecular basis of enterococcal pathogenesis, which has been problematical due to the lack of representative experimental models of CAUTI. To meet this challenge, our group developed a murine model of E. faecalis CAUTI by trans-urethral implantation of a silicone tube into the bladder lumen that faithfully reproduces features of the human disease6. We found that catheter biofilm formation is critical for CAUTI pathogenesis facilitating E. faecalis persistence in the bladder. Furthermore, we found that the endocarditis and biofilm-associated (Ebp) pilus, a hair-like extracellular fiber that is attached to the cell wall, plays a critical role in CAUTI pathogenesis7,8. The Ebp fiber is a heteropolymer composed of multiple copies of a major shaft subunit (EbpC) and single copies of two minor subunits, EbpB at the base attaching the fiber to the cell wall and EbpA located at the fiber’s distal tip. Analysis of mutants constructed to lack pilus subunits revealed that the EbpA tip subunit was critical for biofilm formation and CAUTI pathogenesis. Using various in vitro binding assays, we found that EbpA binds to the host protein fibrinogen (Fg). Molecular dissection of EbpA revealed that its Fg-binding activity was located in the N-terminal half of protein (EbpANTD). If EbpA’s function during infection is to bind to Fg, this raised an important question: Is Fg present in a catheterized bladder? Fg is a prominent host protein associated with the inflammatory response and indwelling urinary catheters are known to induce inflammation, a phenomenon reproduced in the murine CAUTI model. Thus, we hypothesized that catheter-induced inflammation would lead to accumulation of Fg in the bladder. When examined, we found that not only was Fg released subsequent to catheter implantation, but that it was deposited on the catheters themselves and accumulated at levels correlated with the duration of indwelling time (Fig. 2). Moreover, while E. faecalis cannot bind to non-implanted catheters, it readily bound to Fg-coated catheters in vitro and in vivo and binding was dependent on EbpA (Fig. 3). Binding alone was not sufficient to explain pathogenesis, as when bound to a catheter, the major nutrition source for the bacteria will come from the urine stream. However, the E. faecalis strain we used (strain OG1RF) grows poorly in urine and does not form a biofilm, making it unclear how it grows and forms a biofilm during CAUTI. This paradox was resolved by the discovery that Fg was also consumed as a nutrient to promote enterococcal growth and biofilm formation in the presence of urine9. Now that we had identified a critical step in enterococcal CAUTI biofilm formation, the next question was: Can we target this step to prevent infection? Using a vaccination strategy, our initial results were disappointing, as immunization with a C-terminal domain of EbpA (EbpACTD) was not protective. We then vaccinated mice with individual Ebp subunits, including EbpC, EbpB, EbpA and EbpANTD and found that vaccination with EbpA and EbpANTD inhibited biofilm formation in vivo and significantly protected against CAUTI, while similar to EbpACTD, the other subunits were not protective. Furthermore, protection was correlated with the development of serum antibody that could block EbpA-Fg interaction (Fig. 4)9. Comparison of Ebp amino acid sequences across multiple enterococcal species, such as E. faecalis, E. faecium, E. gallinarum and E. casseliflavus, revealed that EbpANTD is highly conserved (approx. 95% identical) and that it is the most conserved Ebp domain overall. This suggests that structural constraints imposed by its Fg-binding requirement restricts variation in the EbpANTD sequence, making it immunologically vulnerable. Furthermore that the heteropolymeric structure of Ebp contributes to immune evasion by restricting its vulnerable Fg-binding site to a very minor component of the entire structure so that the majority of the antibodies made against the pilus will not be protective. This also suggests that the EbpANTD-based vaccine is effective because it uniquely targets the vulnerable domain of the pilus and that it will be effective against a broad range of enterococci, including multi-drug resistant isolates. Furthermore, it will be of interest to determine whether high-titer therapeutic antibodies can be developed by specifically targeting the Fg-binding subdomain within EbpNTD and whether passive administration of these antibodies would be efficacious against an established CAUTI. This study has unveiled a new approach that may provide a new strategy for the prevention of CAUTI and for other enterococcal infections where Fg is present such as infective endocarditis, bacteremia, and intra-abdominal and surgical-site infections.

 

Figures:

CM fig1Figure 1. Bacterial biofilm in a human urinary catheter from a patient with CAUTI.

CM fig2Figure 2. Fibrinogen deposition on catheters

CM fig3

Figure 3. Biofilm formation on the catheter

 

CM fig4 Figure 4. Model of anti-EbpANTD antibodies mechanism of protection by neutralizing EbpA-Fg interaction during E. faecalis CAUTI.

 

Acknowledgments:

This work was supported by 2012 Berg-Morse Postdoctoral Fellowship to A.L.F.-M. and National Institute of Diabetes and Digestive and Kidney Diseases grants R01-DK051406, R01-AI108749-01, and P50-DK0645400 from the NIH.

 

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  9. Flores-Mireles, A.L., Pinkner, J.S., Caparon, M.G. & Hultgren, S.J. EbpA vaccine antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-associated bladder infection in mice. Sci Transl Med 6, 254ra127 (2014).

 

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