Bacterial Infections

Relative Contributions of Ebp Pili and the Collagen Adhesin Ace to Host Extracellular Matrix Protein Adherence and Experimental Urinary Tract Infection by Enterococcus faecalis OG1RF

Sreedhar R. Nallapareddy, Kavindra V. Singh, Jouko Sillanpää, Meng Zhao, Barbara E. Murray
A. Camilli, Editor
DOI: 10.1128/IAI.00038-11

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    Analysis of cell surface expression of EbpC and Ace by E. faecalis OG1RF, its isogenic ΔebpABC and ΔaceΔebpABC deletion mutants, and their complementation derivatives using whole-cell ELISA. (A) Surface expression of EbpC. (B) Surface expression of Ace. BHI-S-grown cells were coated onto wells of microtiter plates, and affinity-purified anti-rEbpC and anti-rAce antibodies were used for detection of cell surface expression. Bars represent the means of absorbance at 405 nm ± standard deviations (SD) from 16 wells, representing results from three independent assays.

  • Fig. 2.
    Fig. 2.

    Western blot analysis of cell wall extracts from E. faecalis OG1RF, its ΔebpABC and ΔebpABCΔace deletion mutants, and their complementation constructs. Mutanolysin cell wall extracts were separated by gradient SDS-PAGE gels, transferred onto polyvinylidene difluoride (PVDF) membranes, and probed with affinity-purified anti-rEbpC and anti-rAce antibodies. Positions of molecular mass markers are indicated on the left.

  • Fig. 3.
    Fig. 3.

    Effect of ebpABC deletion and ebpABC-ace double deletion on the adherence of OG1RF to immobilized ECM proteins. (A) Adherence to collagen type I by ebp and ace mutants; (B) adherence to fibronectin by the ebp mutant; (C) adherence to collagen type I by complementation constructs. Bars represent mean percentages ± SD of 35S-labeled cells adhering to 16 ECM protein-coated wells from four independent experiments. BSA was used as a negative control. Means of adherence percentages of different strains were compared using ANOVA and Bonferroni's posttest. Means of complemented strains were compared with appropriate vector-only control strains using t test or ANOVA and Bonferroni's posttest. *, P < 0.05 versus the wild type by post hoc test; **, P < 0.001 versus the wild type and ΔebpABC mutant by post hoc test; ***, P < 0.001 versus the wild type and ΔebpABC mutant and P < 0.05 versus the Δace mutant by post hoc test; ****, P < 0.001 versus vector-only control by post hoc test.

  • Fig. 4.
    Fig. 4.

    Binding of native Ebp pilus-enriched high-molecular-weight (HMW) surface extracts to collagen types I, IV, and V and fibronectin. (A) Western blot analysis. An HMW surface extract fraction was obtained by ultrafiltration and dialysis of mutanolysin surface extracts. (B) Binding of pili from the HMW surface extract detected with an affinity-purified anti-rEbpC antibody. Bars represent means ± SD for 10 ECM protein-coated wells from up to four independent experiments. BSA was used as a negative control. Similar results obtained with anti-rEbpA antibody are not shown. Mean OD405 values from surface extracts of the wild type and the ΔebpABC mutant were compared using unpaired t test. *, P < 0.0001 versus ΔebpABC HMW surface extracts; CI, collagen type I; CIV, collagen type IV; CV, collagen type V; Fn, fibronectin; EbpAM, EbpA monomer.

  • Fig. 5.
    Fig. 5.

    Adherence of OG1RF and its Δace, ΔebpABC, and ΔaceΔebpABC deletion mutants to the collagen-secreting cell line 3T6. Each bar represents mean percentages ± SD of bacteria adhering to cells coating 9 wells from three independent experiments. Data for each strain were normalized with respective data of OG1RF, which was set to 100%. Mean adherence percentages of different strains were compared using ANOVA and Bonferroni's post test. *, P < 0.001 versus the wild type by post hoc test; **, P < 0.001 versus the wild type and P < 0.01 versus the Δace mutant by post hoc test.

  • Fig. 6.
    Fig. 6.

    Effect of ebpABC deletion on the adherence of OG1RF to immobilized fibrinogen. Bars represent mean percentages ± SD of 35S-labeled cells adhering to 16 fibrinogen-coated wells from four independent experiments. BSA was used as a negative control. Mean adherence percentages of the wild type and the ΔebpABC mutant were compared using unpaired t test. *, P < 0.0001 versus wild type; **, P < 0.001 versus vector-only control.

  • Fig. 7.
    Fig. 7.

    Effect of anti-rEbp antibodies on adherence of OG1RF to fibrinogen. (A) Effect of anti-rEbp antibody mix. (B) Effect of individual anti-rEbpA, anti-rEbpB, and anti-rEbpC antibodies. 35S-labeled OG1RF cells were preincubated with different concentrations of individual anti-rEbp antibodies or an equal mixture of affinity-purified anti-rEbpA, anti-rEbpB, and anti-rEbpC antibodies. Data were normalized with respective data of bacteria adhering in the absence of immunoglobulins and expressed as percentages. Data points represent the means ± standard deviations (results are from two to three independent experiments). Ig, immunoglobulin.

  • Fig. 8.
    Fig. 8.

    Effect of deletion of ace and double deletion of ace and ebpABC in a murine model of UTI (monoinfection using 106- and 105-CFU inocula). Data are expressed as log10 CFU/g of bacteria recovered from kidney homogenates 48 h after transurethral challenge. The log10 CFU from both kidneys were combined and averaged. Horizontal bars represent geometric means. Mean difference in CFU is given as log10 ± SD for the respective inocula. Mean log10 CFU of bacteria were analyzed for significance by unpaired t test. Results for the wild type versus ebp allelic replacement mutant TX5475 were reported in reference 54, and the CFU differences (of comparable inocula) between the ebp allelic replacement mutant and the ΔaceΔebpABC mutant were nonsignificant.

Additional Files

  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Fig. S1. Effect of different polar ebp mutations and a nonpolar bps mutation on the adherence of OG1RF to immobilized ECM proteins.
      Fig. S2. Analysis of cell surface expression of EbpA by E. faecalis OG1RF, its ΔebpABC and ΔebpABC Δace deletion mutants, and their complementation derivatives using whole-cell ELISA.
      Fig. S3. Western blot analysis of cell wall extracts from E. faecalis OG1RF, its ΔebpABC and ΔebpABC Δace deletion mutants, and their complementation constructs.
      Fig. S4. Comparison of biofilm formation by OG1RF versus its ebpABC deletion and complementation constructs.
      Fig. S5. Effect of affinity-purified anti-rEbpA, anti-rEbpB, and anti-rEbpC antibody mix on adherence of OG1RF to ECM proteins.
      Fig. S6. Regression analysis of fibrinogen adherence data from a previously published study and surface display of Ebp pilins by 22 E. faecalis strains.
      Fig. S7. Effect of deletion of ace and double deletion of ace and ebpABC on bladder CFU in a murine model of UTI.
      PDF file, 216K.
    • Supplemental file 2 - Table S1. Bacterial strains and plasmids used in this study.
      Table S2. Primers used in this study.
      Zipped MS Word document, 24K.
    • Supplemental file 3 - List of references cited in supplemental material.
      MS Word document, 41K.
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