Positions 73, 158 and 166 in the crystal structure of FimH in complex with FimG (pdb id: 3JWN).

<p>Carbon atoms are shown in green for FimH and in violet for FimG. Oxygen atoms are shown in red and nitrogen atoms in blue. The hydrogen bonds are shown in red dashed lines, and close contact in blue dashed lines.</p

FimH protein Zonal Phylogeny and MLST tree of AIEC used in this study.

<p>(A) Each circle represents a FimH variant coded by phylogenetically distinct <i>fimH</i> allele(s). Circle size reflects the number of strains (from 1 to 12) carrying the corresponding FimH variant. Consensus, the most common and evolutionarily primary FimH variant. All amino acid changes indicated are derivatives from the consensus variant. Circles inside the black rings represent evolutionarily fixed FimH variants coded by multiple phylogenetically linked <i>fimH</i> alleles with silent variations only. The rest are evolutionarily recent FimH variants coded by a single <i>fimH</i> allele. In red – the proportion of AIEC strains carrying the corresponding FimH variant. In green – the proportion of non-AIEC strains. (B) Minimum spanning tree based on the MLST allelic profiles portraying the clonal distribution of 45 AIEC strains and 3 reference strains. Each dot represents a given sequence type (ST) and the size of the circle is proportional to the number of strains analyzed. Connecting lines of increasing length and the numbers on these lines demonstrate the number of different alleles between two STs. The color of the dots represents the different AIEC clades and the reference strains.</p

Construction of <i>fimH</i> chromosomal mutants.

<p>(A) Construction of <i>fimH</i> chromosomal mutants. (B) Electron microscopy examination of AIEC LF82 bacteria, LF82-Δ<i>fimH</i> isogenic mutant, LF82-Δ<i>fimH/fimH_LF82</i>, LF82-Δ<i>fimH/fimH_K12</i>, LF82-Δ<i>fimH/fimH₇₀₈₂</i>, LF82-Δ<i>fimH/fimH_LF28</i>, LF82-Δ<i>fimH/fimH_LF16</i>, LF82-Δ<i>fimH/fimH₇₀₄₉</i>, LF82-Δ<i>fimH/fimH_LF31</i>, LF82-Δ<i>fimH/fimH_7136;</i> LF82-Δ<i>fimH/fimH_LF73</i> showing flagella and type 1 pili expression (magnification ×40 000). (C) Yeast agglutination titer of AIEC LF82 bacteria, LF82-Δ <i>fimH</i> isogenic mutant and <i>fimH</i> chromosomal mutants.</p

Phylotype of <i>E. coli</i> according to <i>fimH</i> clades.

<p>Phylotype of <i>E. coli</i> according to <i>fimH</i> clades.</p

Primers used in this study.

<p>Primers used in this study.</p

Evolution and hotspot mutations in <i>fimH</i> from AIEC and non-AIEC strains.

*<p> <b>χ² P<0.05.</b></p>**<p> <b>χ² P<0.01.</b></p>***<p> <b>2×2 χ² P<0.001 for 3 hotspot mutations in FimH (specific to AIEC) compared to the rest.</b></p

Bacterial colonization, colonic mucosa inflammation and translocation in CEABAC10 mice infected with LF82<i>ΔfimH/fimH_LF82</i> or LF82<i>ΔfimH/fimH_K12</i> mutants.

<p>(A) Quantification of LF82-Δ<i>fimH/fimH_LF82</i> (black square) or LF82-Δ<i>fimH/fimH_K12</i> (white square) bacteria in the feces of CEABAC10 mice receiving 0.25% DSS in drinking water after oral infection with 10⁹ bacteria on day 0. (B) Quantification of colonic mucosal-associated LF82-Δ<i>fimH/fimH_LF82</i> or LF82-Δ<i>fimH/fimH_K12</i> bacteria on day of sacrifice. (C) Quantification of bacteria on day of sacrifice in the liver and spleen. (D) DAI was performed for CEABAC10 transgenic mice infected with (black square) LF82<i>ΔfimH/fimH_LF82</i> or with (white square) LF82<i>ΔfimH/fimH_K12</i>. (E) Il-1β secretion by colonic mucosa. (F) Histopathological scoring for several parameters of colonic inflammation was performed for CEABAC10 transgenic mice infected with (black square) LF82<i>ΔfimH/fimH_LF82</i> or with (white square) LF82<i>ΔfimH/fimH_K12</i>. * P<0.05; ** P<0.01; and *** P<0.001.</p

FimH variants of the AIEC isolates studied.

a<p>Variants are defined as combinations of amino acid mutations.</p>b<p>Blank entries indicate identity with the wild type. Mutations are indicated.</p

Bacterial persistence and IL-1β secretion by colonic mucosa of CEABAC10 mice according to FimH sequence.

<p>(A) Quantification of LF82-Δ<i>fimH/fimH_LF82</i> (black square), LF82-Δ<i>fimH/fimH₇₀₈₂</i> (white circle), LF82-Δ<i>fimH/fimH_K12</i> (white square) and LF82-Δ<i>fimH/fimH_LF28</i> (white triangle) bacteria in the feces of CEABAC10 mice infected with 10⁹ bacteria on day 3 post-infection. (B) Quantification of IL-1β release by colonic loops infected with LF82 bacteria expressing <i>fimH</i> from LF82 or 7082 (S70/N78 clade) and from K12 or LF28 (consensus clade). * P<0.05; ** P<0.01.</p

Adhesion ability of AIEC and non-AIEC strains with regard to <i>fimH</i> clade and CEACAM6 expression.

<p>(A) Cell-associated bacteria were quantified using non differentiated T84 cells after a 3 H infection period, and results were analyzed with regard to <i>fimH</i> clade. (B) Cell-associated AIEC bacteria belonging to the <i>fimH</i> S70/N78 clade were quantified using undifferentiated and differentiated T84 cells after a 3 H infection period. (C) Western blot analysis of whole protein extracts from non-differentiated and differentiated T84 cells using anti-CEACAM6 and anti-GAPDH antibodies. (D) Cell-associated bacteria (AIEC and non-AIEC bacteria belonging to the <i>fimH</i> S70/N78 clade) were quantified using differentiated T84 cells after a 3 H infection period. (E) Yeast agglutination titer of AIEC and non-AIEC strains belonging to the <i>fimH</i> S70/N78 clade. (F) Transmission electron micrograph of negatively stained AIEC and non-AIEC bacteria belonging to the <i>fimH</i> S70/N78 clade, magnification ×25 000.</p