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.     

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.     

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.     

Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors

The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.     

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.     

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.     

The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannose-containing structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannose-coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesion is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.     

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.     

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.     

The bacterial fimbrial tip acts as a mechanical force sensor

There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.     

Fimbriae in Escherichia Coli Isolated from the Small Intestine of Piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively. Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated. Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

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.     

Differential binding to and biofilm formation on,HEp-2 cells by Salmonella enterica serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster

Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.     

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.     

Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae

Summary Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both the major fimbrial subunit gene, fimA, and the fimH gene in combination with either the fimF or the fimG gene were required for mannose-specific adhesion. The fimF, fimG and fimH gene products were likewise shown to play a major role in the fimbrial morphology as longitudinal modulators. The amount of FimF, FimG and FimH proteins appeared to control the length and number of the fimbriae. The DNA sequence of a 2050 bp region containing the three genes was determined. The corresponding protein sequences all exhibited homology with the fimbrial subunit protein, FimA.     

Glycosylation changes as important factors for the susceptibility to urinary tract infection

FimH is the type?1 fimbrial tip adhesin and invasin of Escherichia coli. Its ligands are the glycans on specific proteins enriched in membrane microdomains. FimH binding shows high-affinity recognition of paucimannosidic glycans, which are shortened high-mannose glycans such as oligomannose-3 and -5. FimH can recognize equally the (single) high-mannose glycan on uroplakin Ia, on the urinary defence protein uromodulin or Tamm-Horsfall glycoprotein and on the intestinal GP2 glycoprotein present in Peyer's patches. E. coli bacteria may attach to epithelial cells via hundreds of fimbriae in a multivalent fashion. This binding is considered to provoke conformational changes in the glycoprotein receptor that translate into signalling in the cytoplasm of the infected epithelial cell. Bladder cell invasion by the uropathogenic bacterium is the prelude to recurrent and persistent urinary tract infections in humans. Patients suffering from diabetes mellitus are more prone to contract urinary tract infections. In a study of women, despite longer treatments with a more potent antibiotic, these patients also have more often recurrences of urinary tract infections compared with women without diabetes. Type?1 fimbriae are the most important virulence factors used not only for adhesion of E. coli in the urinary tract, but also for the colonization by E. coli in patients with Crohn's disease or ulcerative colitis. It appears that the increased prevalence of urinary tract infections in diabetic women is not the result of a difference in the bacteria, but is due to changes in the uroepithelial cells leading to an increased adherence of E. coli expressing type?1 fimbriae. Hypothetically, these changes are in the glycosylation of the infected cells. The present article focuses on possible underlying mechanisms for glycosylation changes in the uroepithelial cell receptors for FimH. Like diabetes, bacterial adhesion induces apoptosis that may bring the endoplasmic reticulum membrane with immature mannosylated glycoproteins to the surface. Indicatively, clathrin-mediated vesicle trafficking of glucose transporters is disturbed in diabetics, which would interfere further with the biosynthesis and localization of complex N-linked glycans.     

Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis.

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.     

Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases

A total of 78 E. coli strains isolated from adults with different types of urinary tract infections were screened by polymerase chain reaction for prevalence of genetic regions coding for virulence factors. The targeted genetic determinants were those coding for type 1 fimbriae ( fimH ), pili associated with pyelonephritis ( pap ), S and F1C fimbriae ( sfa and foc ), afimbrial adhesins ( afa ), hemolysin ( hly ), cytotoxic necrotizing factor ( cnf ), aerobactin ( aer ). Among the studied strains, the prevalence of genes coding for fimbrial adhesive systems was 86 %, 36%, and 23% for fimH, pap , and sfa/foc , respectively. The operons coding for Afa afimbrial adhesins were identified in 14% of strains. The hly and cnf genes coding for toxins were amplified in 23% and 13% of strains, respectively. A prevalence of 54% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from the hospitalized patients displayed a greater number of virulence genes and a diversity of gene associations compared to the strains isolated from the ambulatory subjects. A rapid assessment of the bacterial pathogenicity characteristics may contribute to a better medical approach of the patients with urinary tract infections.