Comparison of the nucleotide sequences of the genes encoding the KS71A and F7(1) fimbrial antigens of uropathogenic Escherichia coli

DNA fragments encompassing the genes for the KS71A and F7(1) fimbrial subunits of Escherichia coli strains KS71 (O4:K12) and AD110 (O6:K2), respectively, have been subjected to DNA sequencing. The nucleotide sequences of the two fimbrillin genes were identical and they encode a polypeptide of 187 amino acids of which 21 amino acids probably will constitute the signal sequence. The primary structure of these fimbrillins showed significant homology with the primary structure of other E. coli fimbrillins.     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Nucleotide sequence of the gene encoding the F72 fimbrial subunit of a uropathogenic Escherichia coli strain

The cloned DNA fragment encoding the F72 fimbrial subunit from the uropathogenic Escherichia coli strain AD110 has been identified. The nucleotide sequence of the structural gene and of 196 bp of the noncoding region preceding the gene was determined. The structural gene codes for a polypeptide of 188 amino acid residues, including a 21-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the F72 gene were compared with the reported sequences of the papA gene (B?ga et al., 1984). Both genes code for subunits of fimbriae that are involved in mannose-resistant hemagglutination (MRHA) of human erythrocytes. The available data show that there is absolute homology between the noncoding regions preceding both genes over 129 bp. The two proteins are homologous at the N terminus and C terminus; there is less, but significant, homology in the region between the N and C termini.     

Immunofluorescence study of fimbrial phase variation in Escherichia coli KS71

An immunofluorescence assay was developed to study fimbrial phase variation in a pyelonephritogenic Escherichia coli strain, KS71. By using fluorochrome-labeled antibodies specific for either P, type-1C, or type-1 fimbriae of strain KS71, it was shown that in a broth culture of strain KS71 the fimbrial types mostly occurred on different cells. Only 9% of the cells carried more than one fimbrial type. The KS71 cell population was fractionated into subpopulations expressing only one of the fimbrial types or lacking fimbriae. Immunofluorescence assay of the subpopulations revealed a rapid phase variation in fimbrial synthesis. Kinetic analyses of a nonfimbriated cell population suggested that a change from one fimbrial phase to another was not totally random.     

Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His⁴⁴, Met⁹⁰, His⁹⁷, and His¹²⁷, and CuB, a second degenerate octahedral geometry with the addition of Glu⁴⁶. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.     

Molecular cloning and characterization of F9 fimbriae from a uropathogenic Escherichia coli

Abstract The genes responsible for the formation of F9 fimbriae of the uropathogenic Escherichia coli strain C1018 were cloned by a cosmid cloning procedure. A positive clone was further subcloned by removing two Bam HI fragments and the remaining plasmid pPIL288-10 had a size of 25 kb. This clone still produced fimbriae as judged by electron microscopy and mannose-resistant haemagglutination (MRHA). Antisera were raised against the clone and against fimbriae purified from the clone. The first antiserum was used in a Western blot to prove the purity of the F9 fimbriae. The antiserum raised against purified fimbriae was used in inhibition tests of MRHA and adherence of cloned bacteria to human uroepithelial cells.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

Molecular cloning of F11 fimbriae from a uropathogenic Escherichia coli and characterization of fimbriae with polyclonal and monoclonal antibodies

Abstract The genes coding for F11 fimbriae from the uropathogenic Escherichia coli C1976 were cloned by a cosmid cloning procedure. Two cosmid clones expressed F11 fimbriae and these clones possessed an identical DNA fragment of 8.9 kb. This fragment was subcloned into pBR322 and this plasmid still produced fimbriae and caused a mannose-resistant haemagglutination (MRHA). Polyclonal and monoclonal antibodies were produced against purified cloned F11 fimbriae. Both types of antibodies were used in inhibition tests of MRHA and adherence of bacteria to the uroepithelial cell line T24. After preincubation of bacteria with polyclonal antiserum the MRHA and the MR adherence were totally inhibited. Preincubation of bacteria with monoclonal antibodies did not inhibit MRHA and MR adherence.     

Genome of multidrug-resistant uropathogenic Escherichia coli strain NA114 from India

Uropathogenic Escherichia coli (UPEC) causes serious infections in people at risk and has a significant environmental prevalence due to contamination by human and animal excreta. In developing countries, UPEC assumes importance in certain dwellings because of poor community/personal hygiene and exposure to contaminated water or soil. We report the complete genome sequence of E. coli strain NA114 from India, a UPEC strain with a multidrug resistance phenotype and the capacity to produce extended-spectrum beta-lactamase. The genome sequence and comparative genomics emanating from it will be significant in under-standing the genetic makeup of diverse UPEC strains and in boosting the development of new diagnostics/vaccines.     

The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce 'attachment pili'. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21.5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

P fimbriae on uropathogenic Escherichia coli O16:K1 and O18 strains

Abstract The fimbrial composition of 12 P-fimbriate uropathogenic Escherichia coli O16 and O18 strains was analysed by immunoprecipitation with 14 fimbria-specific antisera. All the O16 strains possessed a P fimbrial serovariant with an apparent M _r of 17500. One strain had an additional, serologically closely related P fimbria with an apparent M _r of 19 800. Two groups were found among the O18 strains; one possessing a type 1C fimbria and a 19800-Da P fimbria, the other lacking type 1C fimbriae and possessing a P-fimbrial variant with an apparent M _r of 17 800. Fimbriae on strains within the groups were serologically similar by immunoprecipitation assays. Also, the fimbriae on the O16 and O18 strains were mutually cross-reactive. The grouping of the O18 strains by fimbrial serology corresponded to the previous clonal grouping based on other phenotypic characters.     

Proteomic analysis of uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common of bacterial infections in humans. Although a number of Gram-negative bacteria can cause UTIs, most cases are due to infection by uropathogenic E. coli (UPEC). Genomic studies have shown that UPEC encode a number of specialized activities that allow the bacteria to initiate and maintain infections in the environment of the urinary tract. Proteomic analyses have complemented the genomic data and have documented differential patterns of protein synthesis for bacteria growing ex vivo in human urine or recovered directly from the urinary tracts of infected mice. These studies provide valuable insights into the molecular basis of UPEC pathogenesis and have aided the identification of putative vaccine targets. Despite the substantial progress that has been achieved, many future challenges remain in the application of proteomics to provide a comprehensive view of bacterial pathogenesis in both acute and chronic UTIs.