Isolation and characterisation of dog uropathogenic Proteus mirabilis strains.

Proteus mirabilis strains isolated from the urine of dogs with urinary tract infections, were characterised with respect to the production of haemolysin and fimbriae. In contrast to healthy dogs, P. mirabilis was also isolated in high numbers from the faeces of dogs suffering from recurrent urinary tract infections. Production of fimbriae was demonstrated by electron microscopy and the presence of genes for two different types of major fimbrial subunits (MR/P-like or UCA-like) was demonstrated by Southern hybridisation. These genes were absent in the Proteus vulgaris, Providentia rettgeri and Morganella morganii strains tested. All but one P. mirabilis strains were haemolytic and most strains produced fimbriae albeit in different amounts. The UCA fimbrial subunits from dog and human isolates have identical molecular weights and N-terminal sequences and are immunologically cross reactive. It was concluded that dog uropathogenic P. mirabilis strains are very similar to human uropathogenic P. mirabilis strains.

P-fimbriae of Escherichia coli as carriers for gonadotropin releasing hormone: development of a recombinant contraceptive vaccine.

The demand for an effective and low cost means of fertility control of domestic animals has raised interest in the development of contraceptive vaccines. A promising candidate for a vaccine component is the brain peptide gonadotropin releasing hormone (GnRH), which plays a central role in the regulation of reproductive functions in vertebrates. Neutralization of GnRH by vaccine-induced antibodies is expected to prevent the reproductive activity in a wide range of species. A GnRH-protein conjugate was prepared by means of recombinant DNA technology. The oligonucleotides encoding GnRH were inserted in hypervariable regions of the subunit gene of P-fimbriae of Escherichia coli. Hybrid fimbriae encoded by the manipulated fimbrial gene clusters were expressed efficiently on the cell surface of Escherichia coli. Vaccination of female rats and bull calves with purified hybrid GnRH-fimbriae strongly affected the reproductive characteristics. We conclude that P-fimbriae represent a very attractive carrier system for application of GnRH in a new type of vaccine.

Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities.

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.

Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli.

An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined. The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids. The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids. The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins. As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al. (1987). The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al. (1983). FtwA contains two deletions found in comparison to the other fimbrial subunits.

Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and fsoF influence fimbrial binding to renal tubuli and immobilized fibronectin.

P fimbriae of the F7(1) serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Gal alpha(1-4)Gal-specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Gal alpha(1-4)Gal-containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin-coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F7(2), F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue-binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.

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.

Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin.

Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure. It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E. These complexes are commonly found at the tip of the fimbrial structure. In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae. Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.

Expression of foreign epitopes in P-fimbriae of Escherichia coli.

Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.

Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae.

Type 1 fimbriae of Escherichia coli are surface organelles which mediate binding to D-mannose-containing structures. By direct binding of FimH to D-mannose attached to a carrier protein, we demonstrated that this protein was uniquely responsible for the receptor specificity. Furthermore, we show by receptor immunoelectron microscopy that the FimH protein is located laterally in the structure of the type 1 fimbriae.

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.

Functional analysis of the fsoC gene product of the F7(1) (fso) fimbrial gene cluster.

Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.

Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae.

The influence of genetic manipulation of the structural genes coding for major P-fimbrial subunits on the formation of fimbriae in Escherichia coli was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein. The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.

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 crossreaction 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.

Biogenesis of F7(1) and F7(2) fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein.

The F7(1) and F7(2) P-fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985). With the immunocytochemical gold-labelling technique it was demonstrated that both the FsoE and FstE proteins are non-adhesive minor fimbrial subunits located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.

Biogenesis of F71 and F72 fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein.

The F71 and F71 P-fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985). With the immunocytochemical gold-labelling technique it was demonstrated that both the FsoE and FstE proteins are non-adhesive minor fimbrial sub-units located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.

Dot-blot hybridization method.

Nucleic acid hybridization is a very potent technique that can be used for the identification of DNA and RNA species with varying degree of homology and for the estimation of relative amounts of nucleic acid with known homolgy. In most cases, single-stranded (ss) (denatured) DNA or RNA is bound to a filter support (e.g., nitrocellulose) and incubated with a radioactively labeled ss DNA or RNA fragment ("probe") complementary to the nucleic acids of interest. In subsequent washing steps the nonhybridized probe is removed. Hybridized probe will be retained, but may be washed off in further washing steps, depending on the ion concentration and temperature of the wash.

Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli.

The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally. The results show that these gene clusters are closely related.

The role of fimbriae of uropathogenic Escherichia coli as carriers of the adhesin involved in mannose-resistant hemagglutination.

The gene clusters encoding various P-fimbriae (F7(1), F7(2), F9 and F11) were compared. Deletion plasmids that lack the gene encoding the fimbrillin were derived from these gene clusters. Introduction of these deletion plasmids into an E. coli K12 strain resulted in non-fimbriated cells that still showed mannose-resistant hemagglutination (MRHA). However when introduced into wild type E. coli strains no MRHA was observed. Derivatives of the wild type E. coli strains with reduced amounts of O-antigen on the other hand showed MRHA when harbouring these plasmids. These results indicate that adhesion and presence of fimbriae are not necessarily linked. P-fimbriae could function as a carrier for the adhesin and thus endow adhesive capacity to cells with a complete O-antigen.

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.