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.

Adhesins, Bacterial↗

PubMed1995

Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.

DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.

Animals↗

PubMed1994

Passive protection of mice against group A streptococcal pharyngeal infection by lipoteichoic acid.

Previous studies have shown that lipoteichoic acid (LTA) of group A streptococci plays a central role in the adherence of these organisms to epithelial cells. In this study, intranasal instillation of purified LTA but not deacylated LTA in mice blocked colonization and prevented death after intranasal challenge infection with group A streptococci. Bacteria pretreated with rabbit antisera against LTA also failed to colonize or infect mice after intranasal challenge. In vitro studies showed that LTA and M protein inhibited adherence of type 24 streptococci to mouse pharyngeal cells. Passive intranasal administration of purified type 24 M protein protected mice from death after challenge infection with type 24 streptococci but had no significant effect on pharyngeal colonization. Surface LTA and M protein may mediate adherence of streptococci to mouse pharyngeal cells, and blocking adherence with LTA prevents colonization and infection in this animal model.

Animals↗

PubMed1994

Analysis of the role of M24 protein in group A streptococcal adhesion and colonization by use of omega-interposon mutagenesis.

We recently concluded that M protein mediates adherence of group A streptococci to HEp-2 tissue culture cells, because the N-terminal half of M protein blocked adherence and M+ strains attached in greater numbers than M- streptococci. To further assess the role of M protein in adhesion, an M-, isogenic mutant of M type M-, isogenic mutant of M type 24 group A streptococci was constructed by insertional inactivation of the emm24 gene with the omega-interposon flanked by emm24 gene sequences. Southern blot analysis confirmed that the omega-element inserted only into emm24. The M- isogenic mutant M24-omega 3 did not react with antiserum to M24 protein, not did it survive in whole human blood. Electron micrographs of M24-omega 3 showed a diminution of surface fibrillae and reduced binding of plasma components compared with the parent strain. The adhesion of the M+ parent to HEp-2 cells and to mouse oral epithelial cells was dramatically greater than the adhesion of the M24-omega 3 mutant, although there was no difference between the two in adhesion to human buccal cells. In addition, the parent strain was dramatically more effective than the M24-omega 3 mutant in colonizing the oral cavity of mice. These results indicate that the M24 protein can serve as an adhesin in streptococcal attachment to human cells in tissue culture and is important in the colonization of mouse mucosal surfaces.

Adhesins, Bacterial↗

PubMed1994

Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci.

Lipoteichoic acid and several streptococcal proteins have been reported to bind fibronectin (Fn) or fibrinogen (Fgn), which may serve as host receptors. We searched for such proteins by screening a library of genes from M type 5 group A streptococci cloned into Escherichia coli. Lysates of clones were probed with biotinylated Fn and biotinylated Fgn. One clone expressed a 54-kDa protein that reacted with Fn and Fgn. The protein, termed FBP54, was purified and used to immunize rabbits. Anti-FBP54 serum reacted with purified, recombinant FBP54 and with a protein of similar electrophoretic mobility in extracts of M type 5, 6, and 24 streptococci. Anti-FBP54 serum also reacted with 5 of 15 strains of intact, live streptococci, suggesting that FBP54 may be a surface antigen. Southern blot analysis confirmed that the gene is found in group A streptococci but not in Staphylococcus aureus or E. coli. The cloned gene was sequenced and contained an open reading frame encoding a protein with a calculated molecular weight of 54,186. Partial amino acid sequencing of purified FBP54 confirmed that this open reading frame encoded the protein. As determined by utilizing fusion proteins containing truncated forms of FBP54, the primary Fn/Fgn-binding domain appears to be contained in residues 1 to 89. These data suggest that FBP54 may be a surface protein of streptococci that reacts with both Fn and Fgn and therefore may participate in the adhesion of group A streptococci to host cells.

Adhesins, Bacterial↗

PubMed1994

FimH family of type 1 fimbrial adhesins: functional heterogeneity due to minor sequence variations among fimH genes.

We recently reported that the type 1-fimbriated Escherichia coli strains CSH-50 and HB101(pPKL4), both K-12 derivatives, have different patterns of adhesion to yeast mannan, human plasma fibronectin, and fibronectin derivatives, suggesting functional heterogeneity of type 1 fimbriae. In this report, we provide evidence that this functional heterogeneity is due to variations in the fimH genes. We also investigated functional heterogeneity among clinical isolates and whether variation in fimH genes accounts for differences in receptor specificity. Twelve isolates obtained from human urine were tested for their ability to adhere to mannan, fibronectin, periodate-treated fibronectin, and a synthetic peptide copying the 30 amino-terminal residues of fibronectin. CSH-50 and HB101(pPKL4) were tested for comparison. Selected isolates were also tested for adhesion to purified fragments spanning the entire fibronectin molecule. Three distinct functional classes, designated M, MF, and MFP, were observed. The fimH genes were amplified by PCR from chromosomal DNA obtained from representative strains and expressed in a delta fim strain (AAEC191A) transformed with a recombinant plasmid containing the entire fim gene cluster but with a translational stop-linker inserted into the fimH gene (pPKL114). Cloned fimH genes conferred on AAEC191A(pPKL114) receptor specificities mimicking those of the parent strains from which the fimH genes were obtained, demonstrating that the FimH subunits are responsible for the functional heterogeneity. Representative fimH genes were sequenced, and the deduced amino acid sequences were compared with the previously published FimH sequence. Allelic variants exhibiting >98% homology and encoding proteins differing by as little as a single amino acid substitution confer distinct adhesive phenotypes. This unexpected adhesive diversity within the FimH family broadens the scope of potential receptors for enterobacterial adhesion and may lead to a fundamental change in our understanding of the role(s) that type 1 fimbriae may play in enterobacterial ecology or pathogenesis.

Adhesins, Escherichia coli↗

PubMed1993

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a.

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

A 28-kilodalton fibronectin-binding protein of group A streptococci.

Lipoteichoic acid (LTA) has been implicated as a major adhesin of group A streptococci that interacts with fibronectin (Fn). It has been suggested that protein adhesins may also be involved in the binding of Fn to streptococci. We searched for such a protein by transblotting membrane preparations from M types 5, 19, and 24 group A streptococci to nitrocellulose and reacting the blot with 125I-Fn. The Fn reacted with a 28-kDa polypeptide from all three serotypes of streptococci. Using affinity-purified antibodies to the 28-kDa protein in immunoblots of membrane preparations from various streptococci, we demonstrated that the 28-kDa protein is present in all 17 strains tested. Affinity-purified antibodies to the 28-kDa protein also reacted in varying degrees with intact streptococci, demonstrating that the antigen is exposed on the surface of intact organisms. Our results suggest that, in addition to LTA, group A streptococci contain a common Fn-binding moiety that is expressed as a major component of membrane preparations and that is accessible on the surface of streptococci for interactions with Fn.

Adhesins, Bacterial↗

PubMed1992

Lipoteichoic acid and M protein: dual adhesins of group A streptococci.

The roles of lipoteichoic acid (LTA) and M protein in the adherence of group A streptococci to human cells were investigated. Both M+ and M- streptococci bound to pharyngeal and buccal epithelial cells in similar numbers. Streptococcal attachment was inhibited by LTA, but not by the pepsin-extracted, amino-terminal half of M protein (pep M), suggesting that M protein does not mediate attachment to these cells. However, a purified, recombinant, intact M protein did block attachment of streptococci to buccal cells. Using synthetic peptides, the inhibitory domain was localized to a region of intact M protein that is within or near the bacterial cell wall. Evidence is presented to suggest that on the surface of streptococci this region of the M protein is probably not accessible for interactions with host cell receptors and that M protein does not mediate attachment to buccal or pharyngeal cells. In contrast, approximately 10-times more M+ streptococci bound to Hep-2 cells than did M- streptococci and pep M protein blocked binding of streptococci to Hep-2 cells. The data suggest that at least two streptococcal adhesins, LTA and M protein, are involved in the adherence of streptococci to certain cells and that the relative contributions of these adhesins to the attachment process depends on the type of host cells used to study adherence.

Amino Acid Sequence↗

PubMed1992

Functional heterogeneity of type 1 fimbriae of Escherichia coli.

Escherichia coli and other members of the family Enterobacteriaceae express surface fibrillar structures, fimbriae, that promote bacterial adhesion to host receptors. Type 1 fimbriae possess a lectinlike component, FimH, that is commonly thought to cause binding to mannose-containing oligosaccharides of host receptors. Since adhesion of type 1 fimbriated organisms are inhibited by mannose, the reactions are described as mannose sensitive (MS). We have studied the adhesion of the type 1 fimbriated CSH-50 strain of E. coli (which expresses only type 1 fimbriae) to fibronectin (FN). E. coli CSH-50 does not bind detectable amounts of soluble FN but adheres well to immobilized plasma or cellular FN. This adhesion was inhibited by mannose-containing saccharides. By using purified domains of FN, it was found that E. coli CSH-50 adheres primarily to the amino-terminal and gelatin-binding domains, only one of which is glycosylated, in an MS fashion. Binding of the mannose-specific lectin concanavalin A to FN and ovalbumin was eliminated or reduced, respectively, by incubation with periodate or endoglycosidase. Adhesion of E. coli CSH-50 to ovalbumin was reduced by these treatments, but adhesion to FN was unaffected. E. coli CSH-50 also adheres to a synthetic peptide copying a portion of the amino-terminal FN domain (FNsp1) in an MS fashion. Purified CSH-50 fimbriae bound to immobilized FN and FNsp1 in an MS fashion and inhibited adhesion of intact organisms. However, fimbriae purified from HB101 (pPKL4), a recombinant strain harboring the entire type 1 fim gene locus and expressing functional type 1 fimbriae, neither bound to FN or FNsp1 nor inhibited E. coli adhesion to immobilized FN or FNsp1. These novel findings suggest that there are two forms of type 1 MS fimbriae. One form exhibits only the well-known MS lectinlike activity that requires a substratum of mannose-containing glycoproteins. The other form exhibits not only the MS lectinlike activity but also binds to nonglycosylated regions of proteins in an MS manner.

Bacterial Adhesion↗

PubMed1992

Multiple adhesins of streptococci.

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Adhesins, Bacterial↗

PubMed1992

Glycerol-induced unraveling of the tight helical conformation of Escherichia coli type 1 fimbriae.

Glycerol was found to unravel the helical conformation of Escherichia coli type 1 fimbriae without appreciable depolymerization. The linearized fimbrial polymers have a diameter of 2 nm, react strongly with a monoclonal antibody directed at an inaccessible epitope on native fimbriae, and display greater mannose-binding activity and trypsin sensitivity than native fimbriae. Removal of glycerol by dialysis results in spontaneous reassembly of the linear polymers into structures morphologically, antigenically, and functionally indistinguishable from native fimbriae.

Escherichia coli↗

PubMed1991

Aggregation of group A streptococci by human saliva and effect of saliva on streptococcal adherence to host cells.

The aggregation of group A streptococci by whole, stimulated human saliva (WHS) and the effect of saliva on streptococcal adherence to host cells was investigated. WHS samples from 11 individuals were found to aggregate both M+ and M- group A streptococci to various degrees. The aggregating activity was sensitive to heat, EDTA, EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], sodium dodecyl sulfate, and lipoteichoic acid. None of the simple sugars tested, mercaptoethanol, albumin, or nonionic detergents had any effect on aggregation. The aggregating activity of EDTA-treated saliva was restored by 0.1 mM Ca2+ and 1.0 mM Mn2+ but not by up to 5 mM Mg2+. Only streptococci from the stationary phase were aggregated. Hyaluronidase treatment of streptococci from the exponential phase of growth restored their ability to be aggregated, suggesting that the hyaluronic acid capsule interferes with agglutination. Adsorption of WHS by one strain of Streptococcus pyogenes removed aggregating activity for other strains of S. pyogenes and Streptococcus sanguis but not agglutinins for Escherichia coli, suggesting that the agglutinin is specific for certain gram-positive bacteria. Molecular sieve chromatography of WHS and identification of streptococcus-binding components of saliva suggest that either a glycoprotein of approximately 360 kDa or a mucin of saliva of greater than 1,000 kDa mediates aggregation of streptococci. WHS also inhibited adherence of S. pyogenes to buccal epithelial cells.

Adsorption↗

PubMed1991

Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.

Bacterial Adhesion↗