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

Abstract

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (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 EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), 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.

Keywords: Enterococcus faecium; UTI; biofilm; ebp; pathogenesis; pilus; virulence.

Figure 1

Schematic representation of the deleted ebpABC_fm locus of TX82 and transcriptional analysis of its effect on bps_fm, the downstream class C sortase gene. (A) A genetic map of the ebp-bps_fm region, showing the segment deleted from TX82ΔebpABC_fm by allelic replacement with a cat gene. The previously predicted transcriptional terminator in the intergenic region between ebpC_fm and bps_fm is indicated with a lollypop and the lengths of the previously determined mRNA transcripts are marked by arrows above the ebp-bps_fm region. Arrowheads indicate locations of each primer pair for RT-PCR (see B below). (B) RT-PCR analysis of bps gene expression of WT TX82 and its isogenic ebpABC_fm deletion mutant. Gel on left, RT-PCR of total RNA (20 ng), isolated from mid-exponential cells and treated with DNase; middle gel, control reaction of the same RNA sample amplified without RT; gel on right, control reaction with genomic TX82 DNA as template. An intragenic region of gyrase (gyrA) was included as an internal control. Lane numbers correspond to the primer pairs shown in (A). M, molecular weight marker.

Figure 2

Characterization of EbpC_fm surface expression by TX82 and its ebpABC_fm deletion and complementation derivates. (A) Surface expression of EbpC_fm in different growth stages and growth media, using whole-cell ELISA. Cells were washed and adjusted to OD_{600 nm} = 0.5 before coating on wells. EbpC_fm expression was detected using an affinity-purified anti-EbpC_fm antibody. Bars represent the means of absorbance at 590 nm ± SD from four independent assays, each performed in triplicate. (B) Flow cytometry analysis. Labeling by the anti-EbpC_fm antibody is shown for each strain and by a pre-immune antibody (PI) for TX82, and indicated as log fluorescence intensity on the X-axis. Bacteria were analyzed using side scatter as the threshold for detection. Each histogram represents 50,000 events of bacterium-sized particles. (C) Western blot of mutanolysin extracts. Five µg of mutanolysin cell wall extracts were electrophoresed and blotted onto the membrane and probed with affinity-purified anti-EbpC_fm antibodies or pre-immune antibodies. Lane 1, TX82; lane 2, TX82ΔebpABC_fm; lane 3, TX82ΔebpABC_fm (pAT392); lane 4, TX82ΔebpABC_fm (pAT392::ebpABC_fm), lane 5, rec-EbpC_fm.

Figure 3

Immuno-EM analysis of pilus production by WT TX82 and its ebpABC_fm deletion mutant and complementation derivates. (A) TX82 grown on BHI plate; (B) higher magnification of the pilus seen in (A); (C) TX82 grown in BHI broth, showing two cells possibly connected by a pilus; (D) TX82 grown in TSBG; (E) TX82 grown on a blood agar plate; (F–I) Complemented ebpABC_fm deletion mutant (TX82ΔebpABC_fm (pAT392::ebpABC_fm)) grown in BHI broth. Note the pili apparently linking several cells in (I); (J) TX82ΔebpABC_fm grown in BHI broth; (K) TX82ΔebpABC_fm (pAT392) grown in BHI broth; (L) TX82 grown in BHI broth. (A–K) were stained with affinity-purified EbpC_fm-specific antibodies and (L) with pre-immune antibodies. Scale bars 200 nm.

Figure 4

Comparison of biofilm formation of TX82 (WT) versus its ebpABC_fm deletion and complementation derivates. (A) Biofilm formation without added gentamicin. (B) Biofilm formation with overnight inoculum grown in the presence of gentamicin, but cultures in biofilm plate in its absence. (C) Biofilm growth with both inoculum and plate incubated in the presence of gentamicin. Median OD_{570 nm} values and interquartile ranges, with the minimum and maximum values marked by whiskers, represent combined data from at least three independent assays and 48 wells for each construct. Statistical analyses were performed by the Mann-Whitney test and p values are shown in the figure.

Figure 5

Effect of the ebpABC_fm deletion on primary attachment to polystyrene. (A) Adherence of cells grown without gentamicin. (B) Adherence of cells grown with gentamicin. Median values and interquartile ranges show data combined from four independent assays. Adhering bacteria were counted microscopically from a total of 64 fields (each ∼2,900 µm²) for each construct. Statistical analyses were performed by the Mann-Whitney test and p values are shown in the figure.

Figure 6

Effect of deletion of ebpABC_fm in a murine model of UTI, using a mixed inoculum. (A) Percentage of bacteria recovered from kidneys. (B) Percentage of bacteria recovered from bladders. Two independent experiments are shown in both panels. Exp. 1, mice infected with equal CFUs of TX82 and TX82ΔebpABC_fm (2.8 × 10⁶ CFU/mouse); Exp. 2, mice infected with 10-fold overdose of TX82ΔebpABC_fm (2.2 × 10⁷ CFU/mouse) versus TX82 (2.2 × 10⁶ CFU/mouse). Horizontal bars represent the means of percentages of total bacteria in kidneys and bladders Statistical analyses were performed by the paired t test and p values are shown in the figure. Empty circles and empty triangles represent percentages of TX82 and TX82ΔebpABC_fm in inocula, respectively, while solid circles and solid triangles represent percentages of TX82 and TX82ΔebpABC_fm recovered from kidneys and bladders 48 h post infection, respectively.