Major Fimbrial Subunit Research Articles

Serological conservation and location of the adhesin of avian Escherichia coli type 1 fimbriae

By inoculation of mice with purified type 1 -like fimbriae isolated from an avian Escherichia coli strain, a monoclonal antibody (mAb G5) was obtained. mAb G5 reacted in an enzyme-linked immunosorbent assay (ELISA) with type 1-like and type 1A fimbriae differing in the molecular masses of their major fimbrial subunit and isolated from several avian E. coli strains. The specificity of mAb G5 for type 1 fimbriae was assessed in a whole bacteria ELISA with 16 reference E. coli strains expressing different types of fimbriae. Immunoblotting experiments showed that mAb G5 recognized the 29 kDa minor component of reference type 1A fimbriae which has been identified as the adhesin. 14mAb G5 also recognized the 29 kDa component of type 1-like and type 1A fimbriae expressed by avian E. coli strains, suggesting that the adhesin is antigenically conserved among these fimbriae. Immunoelectron microscopic studies gave evidence that the adhesin could be located mainly at the tip or both at the tip and along the fimbriae, depending on the strain.

Evaluation of a small-scale method for the extraction of the K88 antigen from enterotoxigenic Escherichia coli

A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed. Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile. There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released. Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells. We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.

Evaluation of a small-scale method for the extraction of the K88 antigen from enterotoxigenicEscherichia coli

A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed. Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile. There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released. Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells. We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.

Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens

The nucleotide sequence of a DNA fragment that contains the fimA gene, encoding the major fimbrial subunit, of Serratia marcescens IA506 and associated flanking sequences has been elucidated. In addition, the origin of transcription has been identified and is located 120 base pairs upstream of the fimA initiation codon. The predicted amino acid sequence of the FimA polypeptide exhibits some degree of sequence homology with the fimbrial subunits encoded by the fimA determinants of Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli and also to Smf2, the major structural component of mannose-resistant (MR) fimbriae of S. marcescens. The Serratia adhesin that facilitates haemagglutination mediated by type 1 fimbriae is less susceptible to inhibition by D-mannose than has been observed to be the case in other type 1 fimbrial adhesins. The molecule conferring this adherence specificity has been shown to be distinct from the fimA gene product and, therefore, is analogous to the fimbrial systems reported in other species.

Epitope analysis of the F4 (K88) fimbrial antigen complex of enterotoxigenic Escherichia coli by using monoclonal antibodies

So far, three subtypes of the F4 (K88) fimbrial antigen of porcine enterotoxigenic Escherichia coli, F4ab, F4ac, and F4ad, have been distinguished by using polyclonal antisera in agglutination and precipitation tests. The a factor represents one or more common epitopes, whereas each of the b, c, and d factors represents one or more subtype-specific epitopes. We further characterized the F4 antigen complex by using a panel of 40 F4-specific monoclonal antibodies (MAbs). The specificity of all MAbs was proven by enzyme-linked immunosorbent assays, agglutination and radioimmunoprecipitation tests, and immunoelectron microscopy. The MAbs either reacted with all F4 subtypes, reacted with two subtypes, or were subtype specific. Epitope analysis by competition enzyme-linked immunosorbent assays revealed at least 11 epitope clusters on the F4 antigen complex, designated a1 to a7, b1, b2, c, and d. The following antigenic formulas were found for the F4 subtypes: F4ab, a1a2a3a4a5a6b1b2; F4ac, a1a2a3(a4)a5a6a7c; and F4ad, a1a2a3a4a7d. All MAbs were directed against conformational epitopes located on the 27,500-dalton major fimbrial subunits. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed.

F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits

The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified. focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA. Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.

Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.

Expression of type 1 fimbriae and mannose-sensitive hemagglutinin by recombinant plasmids

Deletions within the cloned genes (fimA) encoding the type 1 major fimbrial subunits of two isolates of Klebsiella pneumoniae resulted in a nonfimbriate but hemagglutinating phenotype after transformation of Escherichia coli HB101 or ORN103. Phenotypic expression of type 1 fimbriae could be restored by transformation with plasmids containing the fimA genes of the fimbrial gene clusters from different strains. The surface fimbriae expressed were serologically identical to those of the polymerized product of the introduced fimA gene. The fimA gene products of Salmonella typhimurium and Serratia marcescens could utilize the accessory fimbrial genes of K. pneumoniae to produce surface-associated, hemagglutinating fimbriae. The relatedness of the type 1 fimbrial gene clusters from multiple isolates of members of the family Enterobacteriaceae was examined by DNA hybridization techniques. These analyses demonstrated little nucleotide sequence agreement among distinct genera of the enteric bacteria.

Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.

Molecular characterization of the type 3 (MR/K) fimbriae of Klebsiella pneumoniae.

The expression of type 3 (MR/K) fimbriae by Klebsiella pneumoniae requires the production of at least four polypeptides with molecular masses of 20.5, 25, 34, and 78 kilodaltons. The genes encoding these polypeptides are located on a gene cluster 5,500 base pairs in length. The nucleotide sequence of the major fimbrial structural gene was determined, and its transcription was shown to initiate in vitro 157 base pairs upstream of the translational start site. The predicted amino acid sequence for the fimbrial subunit comprised 202 residues, including a signal peptide of 22 amino acids. Despite similarities in the organization of the gene cluster, the nucleotide and the amino acid sequence of the major fimbrial subunit revealed little agreement with other known fimbrial subunit sequences.

Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

A uropathogenic isolate of Klebsiella pneumoniae was shown to exhibit a mannose-sensitive hemagglutinating phenotype and to produce type-1 fimbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of fimbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major fimbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniae fimbrial genes revealed a nucleotide sequence agreement of 73%, and amino acid sequence agreement of 84% for the mature fimbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both fimbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).

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

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.