Protein sequence alignments for the BcfD (A) and StfH (B) allelic groups from S. Newport. Alignments were made using ClustalW (MegAlign; DNAStar, Madison, WI). Identical amino acids are shadowed in black with white text, while different amino acids appear as black text with no shadowing. Allelic sequence labels are indicated at left and amino acid positions at right. Arrows indicate predicted signal sequence cleavage sites.
Association of S. Newport strain sources with adhesin alleles. The percentages of S. Newport strains for which the source of isolation was associated with a specific adhesin allele (group A or B) are displayed as colors, based on a gradient from blue (0% association) to black (50% association) and yellow (100% association). The exact numbers of strains are indicated in each cell, and total numbers for each source of isolation are shown at the bottom of the heat map.
Combinations of adhesin alleles for each isolation source. (a) Relative frequencies of S. Newport strains, with each detected combination of adhesin alleles grouped by isolation source. The detected allele combinations of FimH/BcfD/StfH for each source are listed on the right of the corresponding pie charts. The following colors were used for the different combinations: dark blue (A/A/A1), medium blue (A/A/B1), light blue (A/B/B1), green (A/A/B2), pink (B/B/B1), red (B/B/B2), and purple (B/A/A1). Six strains with an StfH group A2 allele (four bovine strains, one porcine strain, and one avian strain) were excluded from the charts. (b) Numbers of each combination of alleles are shown with stacked columns, with each color representing the corresponding number for one source of isolates (human, black; environment, brown; avian, orange; equine, light blue; porcine, blue; bovine, red).
Transmission electron microscopy of Bcf- and Stf-fimbriated bacteria. (A) E. coli AAEC189 pLDBAD-Stf, after Stf induction. The inset is a close-up image of fimbriae and fimbrial aggregates at the bacterial cell surface. (B) Immunogold-labeled Stf fimbriae and fimbrial aggregates on E. coli SE5000 containing pLDBAD-Stf using adsorbed rabbit anti-Stf antisera, as described in Materials and Methods, followed by anti-rabbit antibodies conjugated to 10-nm-diameter gold particles. The inset is a close-up image showing numerous gold particles attached to fimbrial aggregates. (C) E. coli AAEC189 pBAD33 (empty vector control). The white arrow denotes a flagellum (flagella were absent from SE5000). (D) Immunogold labeling of E. coli SE5000 pBAD33, as described for panel B. (E) E. coli AEEC189 pLDHSG-Bcf-S after Bcf induction. (F) Immunogold-labeled strain AJB4ΔbcfC pLDHSG-Bcf-L processed with adsorbed rabbit anti-Bcf antisera followed by anti-rabbit antibodies conjugated to 20-nm-diameter gold particles, showing entangled fimbriae with numerous gold particles attached. (G) E. coli SE5000 pHSG576 (empty vector control). (H) Immunogold labeling of strain AJB4ΔbcfC pHSG576, as described for panel F.
Bacterial adhesion to host-specific intestinal epithelial cells. The binding of recombinant E. coli AAEC189 expressing S. Newport Fim, Bcf, or Stf fimbriae with their different adhesin alleles, respectively, FimH (a to e), BcfD (f to j), or StfH (k to o), was determined with porcine (IPEC-J2), bovine (CMS and J8), and human (RKO and Caco-2) intestinal epithelial cell lines. The histograms indicate the percentages of adherent bacteria (CFU of the cell-associated bacteria divided by CFU of the inoculum). Results are the means of the results of three independent assays done in triplicate, with error bars representing standard errors. Asterisks above each bar represent significant differences (unpaired t tests) between percentages of bound bacteria with allele A or A1 (red bars), allele A2 (pink bars), allele B or B2 (black bars) or allele B1 (gray bars) for the respective adhesins, as shown on the right side (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Nonfimbriated E. coli AAEC189 pAZ37 (Fim), pBAD33 (Stf), or pHSG-576 (Bcf) was used as a background binding control (open bars) to determine statistically significant adhesion, as mentioned in Results. The FimH data are from Yue et al. (19).
Association of S. Newport strain sources with adhesin alleles. The percentages of S. Newport strains for which the source of isolation was associated with a specific adhesin allele (group A or B for FimH and BcfD; subgroup A1, A2, B1, or B2 for StfH) are displayed as colors, based on a gradient from blue (0% association) to black (50% association) and yellow (100% association). The exact numbers of strains are indicated in each cell, and total numbers for each source of isolation are shown at the bottom of the heat map. Download FIG S1, PDF file, 0.3 MB.
Numbers of S. Newport isolates for each adhesin allele and for each strain source. (Top left panel) Strains with allele A or B of FimH. (Top right panel) Strains with allele A or B of BcfD. (Bottom left panel) Strains with allele A or B of StfH. (Bottom right panel) Strains with allele A1, A2, B1, or B2 of StfH. Significant different distributions of allelic groups between two sources of strains were determined by the Fisher’s exact test (see Tables S3 to S5); levels of statistical significance are shown as stars (*, P < 0.05; **, P < 00.1; ***, P < 0.001) below brackets, with an open end indicating a given source and a closed end indicating a compared source. Download FIG S2, PDF file, 0.3 MB.
Statistical data (*, P < 0.05; **, P < 0.01; ***, P < 0.001 [unpaired t tests]) for the numbers of adherent strains with different FimH (A), BcfD (B), or StfH (C) alleles that bind to each intestinal epithelial cell line (Ø, nonfimbriated empty vector control strain). Download TABLE S3, PDF file, 0.04 MB.
Bacterial adhesion inhibition for Fim, Bcf, or Stf fimbriated E. coli and IPEC-J2 or RKO cells. Binding of recombinant E. coli AAEC189 expressing S. Newport Fim, Bcf, or Stf fimbriae with their different adhesin alleles, including FimH A and B alleles, BcfD A and B alleles, and StfH A1 and B1 alleles. ΔFimH, ΔBcfD, and ΔStfH are corresponding empty vector controls. White columns indicate percentages of bacteria adhering to the corresponding intestinal epithelial cells; black columns indicate percentages of bacteria adhering in the presence of 50 mM methyl α-d-mannopyranoside. Data are means of results of triplicate assays (± standard errors of the means [SEM]), repeated independently three times. P values were calculated by using a one-sided unpaired t test (*, P < 0.05; ns, P > 0.05). Download FIG S3, PDF file, 0.1 MB.