Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn''s disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH _LF82 (expressing FimH with an AIEC-associated mutation) with fimH _K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.     

Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes.

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics.     

FimH-mediated autoaggregation of Escherichia coli

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N-terminal receptor-binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30 degrees C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild-type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.     

Clonal analysis reveals high rate of structural mutations in fimbrial adhesins of extraintestinal pathogenic Escherichia coli

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintestinal E. coli are under strong positive selection (Dn/Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.     

Haplotype Diversity in “Source-Sink” Dynamics of <Emphasis Type="Italic">Escherichia coli</Emphasis> Urovirulence

FimH, the mannose-specific, type 1 fimbrial adhesin of Escherichia coli, acquires amino acid replacements adaptive in extraintestinal niches (the genitourinary tract) but detrimental in the main habitat (the large intestine). This microevolutionary dynamics is reminiscent of an ecological “source-sink” model of continuous species spread from a stable primary habitat (source) into transient secondary niches (sink), with eventual extinction of the sink-evolved populations. Here, we have adapted two ecological analytical tools—diversity indexes D _S and α—to compare size and frequency distributions of fimH haplotypes between evolutionarily conserved FimH variants (“source” haplotypes) and FimH variants with adaptive mutations (putative “sink” haplotypes). Both indexes show two- to threefold increased diversity of the sink fimH haplotypes relative to the source haplotypes, a pattern that ran opposite to those seen with nonstructural fimbrial genes (fimC and fimI) and housekeeping loci (adk and fumC) but similar to that seen with another fimbrial adhesin of E. coli, papG-II, also implicated in extraintestinal infections. The increased diversity of the sink pool of adhesin genes is due to the increased richness of the haplotypes (the number of unique haplotypes), rather than their evenness (the extent of similarity in relative abundances). Taken together, this pattern supports a continuous emergence and extinction of the gene alleles adaptive to virulence sink habitats of E. coli, rather than a one-time change in the habitat conditions. Thus, ecological methods of species diversity analysis can be successfully adapted to characterize the emergence of microbial virulence in bacterial pathogens subject to source-sink dynamics. [Reviewing Editor: Dr. Margaret Riley]     

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella.     

Allosteric catch bond properties of the FimH adhesin from Salmonella enterica serovar Typhimurium

Despite sharing the name and the ability to mediate mannose-sensitive adhesion, the type 1 fimbrial FimH adhesins of Salmonella Typhimurium and Escherichia coli share only 15% sequence identity. In the present study, we demonstrate that even with this limited identity in primary sequence, these two proteins share remarkable similarity of complex receptor binding and structural properties. In silico simulations suggest that, like E. coli FimH, Salmonella FimH has a two-domain tertiary structure topology, with a mannose-binding pocket located on the apex of a lectin domain. Structural analysis of mutations that enhance S. Typhimurium FimH binding to eukaryotic cells and mannose-BSA demonstrated that they are not located proximal to the predicted mannose-binding pocket but rather occur in the vicinity of the predicted interface between the lectin and pilin domains of the adhesin. This implies that the functional effect of such mutations is indirect and probably allosteric in nature. By analogy with E. coli FimH, we suggest that Salmonella FimH functions as an allosteric catch bond adhesin, where shear-induced separation of the lectin and pilin domains results in a shift from a low affinity to a high affinity binding conformation of the lectin domain. Indeed, we observed shear-enhanced binding of whole bacteria expressing S. Typhimurium type 1 fimbriae. In addition, we observed that anti-FimH antibodies activate rather than inhibit S. Typhimurium FimH mannose binding, consistent with the allosteric catch bond properties of this adhesin.     

The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts

Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.     

Convergent Molecular Evolution of Genomic Cores in Salmonella enterica and Escherichia coli

One of the strongest signals of adaptive molecular evolution of proteins is the occurrence of convergent hot spot mutations: repeated changes in the same amino acid positions. We performed a comparative genome-wide analysis of mutation-driven evolution of core (omnipresent) genes in 17 strains of Salmonella enterica subspecies I and 22 strains of Escherichia coli. More than 20% of core genes in both Salmonella and E. coli accumulated hot spot mutations, with a predominance of identical changes having recent evolutionary origin. There is a significant overlap in the functional categories of the adaptively evolving genes in both species, although mostly via separate molecular mechanisms. As a strong evidence of the link between adaptive mutations and virulence in Salmonella, two human-restricted serovars, Typhi and Paratyphi A, shared the highest number of genes with serovar-specific hot spot mutations. Many of the core genes affected by Typhi/Paratyphi A-specific mutations have known virulence functions. For each species, a list of nonrecombinant core genes (and the hot spot mutations therein) under positive selection is provided.     

Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages.The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections.     

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

Enterobacterial adhesins and the case for studying SNPs in bacteria

Single-nucleotide polymorphisms (SNPs) in structural genes can have a dramatic effect on the biology of whole organisms, from bacteria and viruses to mammals. Here, we underscore the importance of SNPs in bacterial genes that contribute to the ability of pathogens to cause disease. SNPs that confer an adaptive advantage for bacterial pathogens have been discovered in the genes encoding the FimH and Dr adhesins of Escherichia coli and, most recently, Salmonella enterica sv. Typhimurium FimH.     

Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

Weak rolling adhesion enhances bacterial surface colonization

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as Escherichia coli when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.     

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.     

Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens

Adhesion of meningitis-associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of alpha-methyl-D-mannoside. A cat cassette was introduced into the filmA gene of IHE 3034, and the resulting mutant strain IHE 3034-2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S-fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034-2. The complemented strain IHE 3034-2(pRPO-1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K-12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH-null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino-acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034-FimH were individually substituted to the corresponding amino acid in the PC31-FimH. The substitution S62-->A completely abolished collagen adhesiveness. The reverse substitution A62-->S in the PC31-FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034-FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino-acid residue Ala-62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.     

A potential role of Escherichia coli pathobionts in the pathogenesis of pediatric inflammatory bowel disease

Through genomic analysis of mucosa-associated Escherichia coli strains, we found a close genetic association among isolates from pediatric inflammatory bowel disease (IBD) patients. A specific E. coli pathovar, adherent-invasive E. coli (AIEC), was found in Crohn's disease (CD) adult patients - this pathovar has enhanced adhesive and invasive properties, mainly due to the mannose-bonding FimH protein. We aimed to characterize 52 mucosa-associated E. coli strains isolated from pediatric IBD and non-IBD patients. Eleven E. coli strains, showing a strong similarity in fimH gene sequence to that of E. coli AIEC LF82, were characterized for fimH gene sequence, genomic profiling, adhesive and invasive ability, and phylogrouping. The results were compared with E. coli strains AIEC LF82 and MG1655. The 11 E. coli isolates showed 82.4% ± 1.4% fimH sequence similarity and 80.6% ± 1.3% genomic similarity to strain AIEC LF82. All these strains harbored V27A and S78N FimH mutations, as found in LF82. Nine of them belonged to the more virulent B2 and D phylogroups. Neuraminidase treatment, mimicking inflamed mucosa, enhanced adhesion of all 11 strains by 3.5-fold, but none showed invasion ability. It could be argued that the 11 selected strains could be a branch of an E. coli subpopulation (pathobionts), that could take advantage in an inflamed context because of a suitable genomic and (or) genetic backdrop.