Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections.

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an O7:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic O6:K+ and meningitis O18:K1 and O83:K1 strains. The sfa determinant was also detected on the chromosome of K1 isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against F1C-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.     

S-Fimbria-encoding determinant sfa(I) is located on pathogenicity island III(536) of uropathogenic Escherichia coli strain 536

The sfa(I) determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III(536), is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III(536) also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III(536). The presence of known PAI III(536) sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.     

Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesion determinant: nucleotide sequence of the genes sfa B, C, D, E, F

The S fimbrial adhesin (sfa) determinant of E. coli comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfa C situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation studies indicated that the genes sfa B and sfa C play a role in regulation of the sfa determinant. In addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfa A and sfa G responsible for S subunit proteins, were determined. It is suggested that these genes are involved in transport and assembly of fimbrial subunits. Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1C fimbriae (foc) and type I fimbriae (fim). The evolutionary relationship of fimbrial adhesion determinants is discussed.     

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.     

Novel molecular variants of allele I of the Escherichia coli P fimbrial adhesin gene papG

P fimbriae of extraintestinal pathogenic Escherichia coli mediate digalactoside-specific adherence via the tip adhesin molecule PapG, which occurs in three known variants (I to III), which are encoded by the corresponding three alleles of papG. In the present study, newly discovered variants of papG allele I and the respective wild-type source strains were characterized. One of the new papG allele I variants conferred a unique agglutination phenotype that combined the phenotypes associated with papG alleles I, II, and III. Comparative hydrophilicity analysis of predicted PapG peptides revealed regions that might explain the observed phenotypic similarities and differences between the PapG variants. The new papG allele I variants occurred either as the sole papG allele or together with both papG alleles II and III, rather than with only papG allele III, as in archetypal strains J96 and CP9. They also occurred in the absence of the usual F13 papA allele. One of the new papG allele I variants occurred in a serogroup O6 strain that, according to random amplified polymorphic DNA analysis, was phylogenetically distant from the "J96-like" clonal group of E. coli O4:H5, which includes all previously identified examples of papG allele I. Cluster analysis of nucleotide and predicted peptide sequences suggested that papG allele I represents the earliest evolutionary branch from a common papG ancestor. These results demonstrate unexpected diversity within papG allele I and, together with previous findings, suggest that the J96-like clonal group of E. coli O4:H5 may represent the original source of papG within the species.     

Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins.

We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins.     

Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin

The erythrocyte receptor for Escherichia coli K99 fimbrial adhesin was isolated from equine erythrocytes and characterized as Neu5Gc-alpha(2----3)-Galp-beta(1----4)-GLcp-beta(1----1)-Ceramide. This glycolipid acted as the receptor for K99 by four different experimental approaches: inhibition of equine erythrocyte hemagglutination by preincubation of K99-positive bacteria or purified K99 fimbriae with the isolated glycolipid; inhibition of attachment of K99-positive bacteria to porcine intestinal epithelial cells in the presence of the isolated glycolipid; induction of binding of K99-positive bacteria or purified K99 fimbriae to normally unreactive guinea pig erythrocytes by coating these cells with the isolated glycolipid; and isolation of the receptor by affinity chromatography with K99 coupled to CNBr-activated Sepharose 4B, indicating a strong interaction between K99 and the isolated glycolipid.     

The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds to beta 1-linked galactosyl residues in glycosphingolipids.

The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine. In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis. The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 +/- 21, 117 +/- 21, and 73 +/- 22 pM, respectively. Thin-layer chromatography overlay binding analysis demonstrated that serotype O8:K87:K88ab:H19 E. coli bound to hydroxylated galactosylceramide, gangliotriaocylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides. The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GD1a, GD2, GD3, GQ1b, or GT1b. The binding pattern observed with the K88 adhesin suggests that beta 1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly. It is suggested that beta 1-linked galactose residues may form the molecular basis of both glycoprotein and glycolipid receptors for the K88 fimbrial adhesin in the porcine small intestine.     

Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli.

The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).     

Comparison of Escherichia coli fimbrial antigen F7 with type 1 fimbriae.

Two Escherichia coli O6:K2:H1 strains, C1212 and C1214, isolated from urinary tract infections, were compared for their capacity to adhere to various cells. After growth on solid medium, only C1212 bacteria agglutinate human erythrocytes and attach to urinary epithelial cells. Both of these reactions are mannose resistant. In contrast, C1214 bacteria cause a mannose-sensitive agglutination of guinea pig erythrocytes, show a mannose-sensitive attachment to buccal epithelial cells, and attach to urinary mucus. Immunoelectron microscopy revealed that C1214 bacteria possess type 1 fimbriae (mannose sensitive), which are not present in C1212 bacteria when this strain is grown on solid medium. The fimbriae of C1212 (mannose resistant) were also demonstrated by immunoelectron microscopy. We call these fimbriae demonstrated in C1212 the E. coli F7 antigen. Urinary mucus, and probably mucous material elsewhere, may function as a trap for Enterobacteriaceae with type 1 fimbriae by the specific adherence of such bacteria. We consider this a nonimmune resistance mechanism against disease caused by Enterobacteriaceae.     

Nucleotide sequence of the genes coding for minor fimbrial subunits of the F1C fimbriae of Escherichia coli.

F1C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces. This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae. The foc gene cluster encoding F1C fimbriae has been cloned, as described previously. Here we present the nucleotide sequence (2081 bp) coding for the F1C minor fimbrial subunits. The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids. The deduced amino acids of the F1C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae. The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower.     

Monoclonal antibodies against the nonhemagglutinating fimbrial antigen 1C (pseudotype 1) of Escherichia coli.

Hybridoma-derived monoclonal antibodies were produced with fimbrial preparations from Escherichia coli 20025 (04:K12:H-) with fimbrial (F) antigens 1C, 13, one related to 12, and one preliminarily termed y and from E. coli 2980 (018ac:K5:H-) with F antigens 1C and 8. Two clones of subclonal hybrid cells were studied which produced monoclonal antibodies (mc-20025-F2b, immunoglobulin G2b [IgG2b]; mc-2980-F2, IgG1) that were reactive with E. coli 20025, 2980, and a number of additional strains which exhibited the F1C antigen. Results of enzyme-linked immunosorbent assay and Western blot analysis indicated that the antibodies had F1C specificity, and competitive enzyme-linked immunosorbent assay with 125I-labeled antibodies showed that they recognized different epitopes on the fimbrial subunit. Neither of the antibodies agglutinated F1C-fimbriated E. coli but bound to the bacteria. There was no binding to E. coli without F1C fimbriae.     

Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate.

S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Like the SfaI complex, SfaII consists of the major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding the subunit proteins of SfaII were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with those of the SfaI complex subunits. Although the sequences of the two major SfaA subunits differed markedly, the sequences of the minor subunits showed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of the meninigitis isolate. These data were confirmed by the isolation and characterization of the SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of SfaI and SfaII revealed that differences between the two Sfa complexes with respect to their capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other than SfaS.     

The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin.

Escherichia coli IHE11088(pRR-5) expressing the G (F17) fimbria adhered to immobilized laminin as well as to reconstituted basement membranes. No adhesion was seen with the plasmidless strain IHE11088 or with the deletion derivative IHE11088(pHUB110), which expresses the G-fimbrial filament with a defective GafD lectin and lacks N-acetyl-D-glucosamine-specific binding. Adhesion of IHE11088(pRR-5) to laminin and to reconstituted basement membranes was specifically inhibited by N-acetyl-D-glucosamine, and adhesion was abolished after N-glycosidase F treatment of laminin. The results show that the GafD lectin binds to laminin carbohydrate and suggest a novel function for the F17 fimbria in binding to mammalian basement membranes.     

Molecular cloning of the Escherichia coli O75X adhesin.

The uropathogenic strain Escherichia coli IH11128 (O75:K5:H-) exhibits a mannose-resistant O75X adhesin. The genes encoding the O75X adhesin were cloned from a clinical strain and transferred to E. coli K-12 derivatives. The recombinant plasmids were found to express a 15-kilodalton fimbrial subunit protein, a fimbrialike extracellular structure, and mannose-resistant hemagglutination. An indirect immunofluorescence assay was used to study attachment of the clone and purified adhesin to frozen sections of human kidney. The clone bound selectively to the interstitial areas and notably to Bowman's capsule. The purified adhesin bound to the basement membrane of the tubules and to Bowman's capsule.     

Evaluation of receptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesin variants using porcine serum transferrin and glycosphingolipids as model receptors

Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 microM), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a beta-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a beta-linked HexNAc. In addition, the presence of a terminal galactose beta-linked to this HexNAc residue enhances K88 adhesin binding.     

Prediction of antigenic determinants and secondary structures of the K88 and CFA1 fimbrial proteins from enteropathogenic Escherichia coli.

Recently the amino acid sequences of the K88ab and the CFA1 fimbrial proteins from enteropathogenic Escherichia coli have become available. To elucidate the immunological interrelationships among the K88 family of antigenic variants, and to estimate the nature and position of the antigenic determinants in the K88 and CFA1 proteins, the positions of these features in the amino acid sequences have been predicted. This has been done by computerized algorithms, incorporating such factors as hydrophilicity and secondary structural potential. Some of the predicted determinants of K88 show good correlation with the known sequence differences between the antigenic variants of this protein. The presented results point to a possible use of synthetic vaccines against fimbriated pathogenic E. coli strains.     

Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation

Isolates of Klebsiella pneumoniae are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains of K. pneumoniae was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of K. pneumoniae expressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intact mrk gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.     

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.