DNA-based protein phylogram of <i>S. enterica</i> FimH, derived from ZP analysis.

<p>The tree was built based on the 50 <i>fimH</i> sequences of <i>S. enterica</i> subsp. I. Each circle represents a unique structural variant, and the size of the circle is proportional to the number of representative sequences. The dashed line separates the long-term (green) from the recently emerged variants (black). Branches marked in blue indicate branches containing synonymous mutations. The length of each branch is proportional to the number of non-synonymous mutations that were acquired. The strain tags of systemically invasive serovars are in red and the non-invasive serovars in black.</p

Amino acid variation in <i>S. enterica</i> subspecies I FimH.

<p>Residues identical to the amino acid sequence of <i>S.</i> Typhimurium SL1344 (Thm1) FimH are indicated by dots. Systemically invasive serovars are shown in red and non-invasive serovars are shown in black. Position -10 represents the position in the signal peptide of unprocessed FimH upstream of the cleavage site.</p

List of bacterial strains used in this study.

1<p>Harborview Medical Center, Seattle, WA, USA.</p>2<p>Institute for Environmental Health, Lake Forest Park, WA, USA.</p>3<p>University of Iowa, Iowa City, IA, USA.</p>4<p>Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland.</p>5<p>Ohio State University, Columbus, OH, USA.</p>6<p>University of Illinois, Urbana, IL, USA.</p

FimH-mediated bacterial interaction with epithelial and macrophage cell lines.

<p>Bacterial adhesion to (A and C) and invasion of (B and D) Hep-2 cells (A and B) and RAW264.7 cells (C and D). Different variants of FimH were expressed in <i>S.</i> Typhimurium SL1344H3 and bacterial binding was tested in the absence and presence of 50 mM methyl-D-mannopyranoside (α-mm). Data are the means ± SD of triplicates from one representative experiment of three experiments that were performed.</p

Schematic representation of evolutionary changes in the FimH binding phenotype of selected <i>S. enterica</i> serovars.

<p>The evolutionary changes in the FimH of <i>S.</i> Enteritidis, <i>S.</i> Pullorum, <i>S.</i> Gallinarum and <i>S.</i> Dublin (A), <i>S.</i> Indiana, <i>S.</i> Paratyphi C and <i>S.</i> Choleraesuis (B), <i>S.</i> Paratyphi B (C) and <i>S.</i> Paratyphi A, <i>S.</i> Sendai and <i>S.</i> Typhi (D). Low (green)-FimH with low-binding phenotype; High (blue)-FimH with high-binding phenotype; None (orange)-inactive variant of FimH. The strain tags of systemically non-invasive serovars are in black and the systemically invasive serovars in red. Structural mutations are given along each arrow. Structural hot-spot mutations are underlined. The activating mutations are in blue and the inactivating mutations are in orange. The FimH variants from strains with phylogenetic relatedness supported by MLST are shown in tan boxes.</p

Binding phenotypes of natural <i>S. enterica</i> subspecies I FimH variants.

<p>Static adhesion of <i>S.</i> Typhimurium LBH4 transformed with plasmids encoding different variants of FimH or plasmids carrying <i>fimH</i> deletion (fimHΔ) to Man-BSA (red bars) and anti-FimH^SE antibody (grey bars). The binding of ³H- labeled bacteria was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002733#s4" target="_blank">Material and Methods</a>. Data are the means ± SD of triplicates from one representative experiment of three experiments that were performed. The strain tags of systemically invasive serovars are in red and the non-invasive ones in black. Bacterial binding was >95% inhibitable in the presence of 50 mM methyl-D-mannopyranoside (not shown). * The non-binding FimH variants of <i>S.</i> Gallinarum were not tested in this study.</p

Maximum-likelihood DNA phylograms of <i>S. enterica fimH</i> and concatenated MLST loci (<i>aroC</i>, <i>hisD</i> and <i>thrA</i>).

<p>The <i>fimH</i> tree (A) was built based on an alignment of <i>fimH</i> sequences amplified from 55 isolates including 45 different strains of subspecies I and 10 strains of subspecies II–VI (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002733#ppat-1002733-t001" target="_blank">Table 1</a>). Five additional alleles of <i>fimH</i> obtained from GenBank (<i>S.</i> Typhimurium AJB3 (Thm3), <i>S.</i> Typhimurium LB5010 (Thm4), <i>S.</i> Gallinarum 287/91 (Gal1) and 589/02 (Gal2), and <i>S.</i> Paratyphi C 49 [RKS4594] (PaC1) were included. The MLST loci tree (B) was built on an alignment of concatenated sequences of three genes (<i>aroC</i>, <i>hisD</i> and <i>thrA</i>) obtained for 57 study strains. The trees shown were rooted using <i>S. enterica</i> subsp. II (2993). The italicized values along the branches denote % bootstrap values based on 1000 runs (the bootstrap proportions along the terminal branches separating isolates within single serovars as well as the ones below 50% are not shown). Systemically invasive serovars are shown in red and non-invasive serovars are shown in black. Strain tags are as used in the text.</p