Characterization of the ebp_fm pilus-encoding operon of enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection

We recently identified 15 genes encoding putative surface proteins with features of MSC RAMMs and/or pili in the Enterococcus faecium TX16 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC_fm', is transcribed as an operon, that its putative major pilus subunit, EbpC_fm (also called PilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC_fm operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC_fm expression and eliminated EbpCfm-containing pili from the cell surface. However, transcription of the downstream sortase, bpsfm, was not affected. Importantly, the ebpABC_fm deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC_fm in trans, which also restored cell surface expression of EbpC_fm and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and <0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC_fm locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.