Abstract
The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.
Highlights
Enterococcus faecalis is a gram-positive coccus that is a common commensal of the human gastrointestinal tract
E. faecalis strains were routinely grown in/on brain heart infusion (BHI) (Difco Laboratories, Detroit, MI) broth/agar or, for some experiments, in tryptic soy broth supplemented with 0.25% glucose (TSBG) and, for in vivo experiments, in BHI supplemented with 40% horse serum (BHIS)
Subsequent Reverse transcriptase (RT)-PCR analysis found that deletion of any one of the ebp genes had no appreciable effect on expression of the remaining ebp genes or bps at the transcriptional level (Figure S2)
Summary
Enterococcus faecalis is a gram-positive coccus that is a common commensal of the human gastrointestinal tract. Our previous studies demonstrated that a pilus-deficient disruption mutant of E. faecalis OG1RF was attenuated in a rat endocarditis model and in the ability to form biofilm, leading to the name “endocarditis and biofilm-associated pili” or Ebp [4]. Pilus-deficient mutants were found to be attenuated in catheter-associated and non-catheter associated UTI models [5,6]. Ebp pili are involved in adherence of OG1RF to host fibrinogen and collagen [6] as well as to human platelets [7], suggesting that they exhibit multiple functions during the infection process. Gene expression studies demonstrated that the three ebp genes and bps produce a single polycistronic transcript and that bps is independently expressed from a second promoter. Bee (biofilm enhancer in Enterococcus) [9], carried on a plasmid, has been identified and it is only found in ~1-2% of E. faecalis isolates [7]
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