Vibrio cholerae O395 tcpA pilin gene sequence and comparison of predicted protein structural features to those of type 4 pilins.

Vibrio cholerae O1 expresses a pilus that is coordinately regulated with cholera toxin production and hence termed TCP, for toxin-coregulated pilus. Insertion of Tn5 IS50L::phoA (TnphoA) into the major pilin subunit gene, tcpA, has previously been shown to render the strain avirulent as a result of its inability to colonize. One such insertion was isolated and used as a probe to screen for clones containing the intact tcpA gene. The DNA sequence of tcpA was determined by using the intact gene and several tcpA-phoA gene fusions. The deduced protein sequence agreed completely with that previously determined for the TcpA N terminus and with the size of the mature pilin protein. The reported homology with N-methylphenylalanine (type 4) pilins near the N terminus was extended and shown to include components of the atypical leader peptide as well as overall predicted structural similarities in other regions of the pilins. In contrast to the modified N-terminal phenylalanine residue found in all characterized type 4 pilins, the corresponding position in tcpA contains a Met codon, thus implying that the previously uncharacterized amino acid corresponding to the N-terminal position of the mature TcpA pilin is a modified form of methionine. Except for this difference, mature TcpA has the overall predicted structural motifs shared among type 4 pilins.

Salmonellae of serotypes gallinarum and pullorum grouped by biotyping and fimbrial-gene probing.

When salmonellae of serotypes Gallinarum (50 isolates) and Pullorum (36 isolates), that produce non-adhesive (type-2) fimbriae, were tested for their reactions in biochemical tests, 81 (94%) were found to belong to three distinct biochemical groups, I-III. Interaction of HinfI-digested DNA of both Gallinarum and Pullorum with a probe of accessory genes of type-1 fimbriation in serotype Typhimurium gave one type of Southern hybridisation pattern that was readily distinguished from that of Typhimurium strains. With a probe of the Typhimurium fimbrial subunit gene, Pullorum isolates were separated into strongly and weakly probe-reactive groups which showed restriction fragment-length polymorphism; these latter groups corresponded to biochemical groups II and III, respectively.

Pilins from the B serogroup of Bacteroides nodosus: characterization, expression, and cross-protection.

Sequences of pilin genes from four strains of serogroup B of the ovine pathogen Bacteroides nodosus have been determined. These sequences permit comparisons of amino acid sequence between pilins from different subtypes (B1, B2, B3, B4) of the B serogroup and assessment of intraserogroup variation. Pili of B. nodosus strains 234 (B1) and 183 (B2) were produced by Pseudomonas aeruginosa harboring a plasmid-borne B. nodosus pilin gene, and these pili were used in sheep vaccination trials. Pili from strain 183 (B2) were found to be a senior antigen to pili from strains of other B subtypes, providing protection against footrot infection caused by strains of the other B subtypes. Pili of this strain are therefore the most suitable candidate for inclusion in a pilus-based vaccine. Pili of strain 234 from subtype B1, the reference strain of the B serogroup, provided poor protection against infection with other subtypes.

Pili of Vibrio cholerae non-O1.

Pili of Vibrio cholerae non-O1 strain S7 were purified and characterized. The pili of S7 were morphologically, electrophoretically, and immunologically (as far as polyclonal antibody was used) indistinguishable from the 16-kilodalton pili of V. cholerae O1 strain 82P7. The purified pili and organisms had D-mannose- and L-fucose-resistant hemagglutinin. The hemagglutinating activity of the purified pili was inhibited by the Fab fraction of antipilus antibody, but the hemagglutinating activity of live organisms was not inhibited completely. The purified pili or Fab fraction of antipilus antibody did not inhibit the adhesion of V. cholerae non-O1 to rabbit intestines. Therefore, the pili were not regarded as a colonization factor of V. cholerae non-O1. A total of 148 V. cholerae non-O1 and O1 clinical isolates were screened for the presence of S7 pili by using an agglutination test with anti-S7 pilus serum; 12 of 49 V. cholerae non-O1 strains and 25 of 99 V. cholerae O1 strains were positive for agglutination. These agglutination reactions were not correlated with adhesion of the organisms to intestines.

Location of the three major agglutinogens of Bordetella pertussis by immuno-electronmicroscopy.

When the three serotypes of Bordetella pertussis (types 1,2,3; 1,2 and 1,3) were labelled with agglutinins and protein-A gold, agglutinogen 1 was found on fimbriae and on the cell surface of types 1,2,3 and 1,2 but on the cell surface only of non-fimbriate type 1,3 organisms. In contrast, agglutinogen 2 was located on fimbriae only. Agglutinogen 3 was not labelled. When protein-A gold was replaced by immunoglobulin-G gold, agglutinogen 3 was found on the cell surface only, even of fimbriate bacteria of type 1,2,3. The implications of these findings for acellular vaccines are discussed.

Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans.

Three colonial variants of Actinobacillus actinomycetemcomitans, which formed transparent rough (TR)-, transparent smooth (TS)-, and opaque smooth (OS)-surfaced colonies, were described in relation to their fimbriation. TR- and TS-cells were adhesive to agar and glass surfaces but not the OS-cells. The examination by electron microscopy revealed that TR-cells were highly fimbriated but not TS- and OS-cells. Thus, TS-cells seemed to be an intermediate type. The fimbriae were isolated from TR-cells by suspending in 0.15 M ethanolamine-HCl buffer (pH 10.5) and purified by dissolving non-fimbrial components in 0.5% deoxycholate and 0.7% n-octyl-beta-D-glucopyranoside. The relative molecular mass of the fimbrial subunit protein was 54,000.

Characteristics of binding of Escherichia coli serotype O157:H7 strain CL-49 to purified intestinal mucin.

Purified rat intestinal mucin was used as a model mucin to study the binding of Escherichia coli serotype O157:H7, a human pathogen associated with outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. Of six O157:H7 strains, only one strain (designated CL-49) bound to rat (and other) intestinal mucins by a specific and saturable process. Binding was observed only after the bacteria were serially passaged to promote the expression of type 1 pili (fimbriae). Several other type 1-piliated E. coli strains, however, did not bind to mucin. Binding of E. coli CL-49 was inhibited by D-mannose and short oligomannosyl derivatives, particularly Man-alpha-1,3-Man, Man-alpha-1,2-Man, and Man-alpha-1,3-Man-beta-1,4-N-acetylglucosamine. Other inhibitors of binding included p-nitrophenol (10(-4) M), heating at 60 degrees C (to remove pili), an antibody to type 1 pili, and purified type 1 pili of E. coli CL-49 used as hapten inhibitors. A comparison of the hydrophobicity of piliated E. coli CL-49 with other type 1-piliated E. coli strains indicated that the former strain was much more hydrophobic than the others. These findings indicate that highly purified intestinal mucins possess specific mannosyl receptor sites for bacterial type 1 pili on E. coli CL-49, but that strong hydrophobic interactions between the mucin and the pili stabilize the mannose-dependent binding process. We speculate that the mucin receptors for type 1 pili reside in oligosaccharides of the 118-kilodalton "link" glycopeptide, since this is the only mucin component known to contain mannose.

Characterization of a new putative colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype O114:H21 which produces only heat-labile enterotoxin.

Enterotoxigenic Escherichia coli (ETEC) of serotype O114:H21, which produced only heat-labile enterotoxin (LT), gave mannose-resistant hemagglutination (MRHA) with bovine erythrocytes. One strain, E20738A, was shown to possess fimbriae of approximately 7.5 nm diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular masses 17.5 and 15.5 kDa were seen; the 17.5-kDa band was the most prominent. Loss of LT and MRHA together from strain E20738A was associated with loss of a 100-MDa plasmid. An absorbed anti-strain E20738A serum reacted specifically with the 17.5- and 15.5-kDa polypeptides and bound to the intact fimbriae. This antiserum reacted positively in an ELISA with LT-positive E. coli strains of serogroups O8, O15, O48, O114, and O146. The antiserum did not react with ETEC carrying known colonization factors. The term coli-surface-associated antigen (CS) 17 has been used to describe the fimbriae.

Cloning and expression of a pili gene of Corynebacterium renale in Escherichia coli.

A plasmid gene library of Corynebacterium renale piliated strain No. 109P+ was prepared in Escherichia coli in order to study the chemical structure of the pili of C. renale. Of 3,000 recombinant clones tested, 5 reacted with anti-pili anti-serum. The gene products of these clones reacted with anti-pili monoclonal antibodies 8/4, 5/2 and B20/3 but lacked the reactivity with 13/4. SDS-PAGE analysis revealed that the expressed protein had a molecular mass of 48 kilodalton and deletion analysis showed that the encoding region for this protein was localized within a 1.4 kilobase gene including a promoter sequence. Immunoelectron microscopy showed that mouse antibodies raised to the expressed protein bound to the entire surface of the pili of C. renale. These results indicate that the cloned gene encodes a major structural protein of C. renale pili.

The complete primary structure of pilin from Haemophilus influenzae type b strain Eagan.

Adherence of Haemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an Mr of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.

Neutrophil killing of two type 1 fimbria-bearing Escherichia coli strains: dependence on respiratory burst activation.

The production of reactive oxygen metabolites by neutrophils is thought to play a key role in host defense against invading microorganisms. In this study, the generation of oxygen metabolites induced by two uropathogenic Escherichia coli strains, ABU2 and PN7, and their subsequent killing in neutrophils were investigated. Both strains were grown to promote type 1 (mannose-sensitive) fimbria formation, but they differ with respect to other surface structures. When interacting with human neutrophils, the ABU2 bacteria adhered to and were phagocytized by the neutrophils, whereas PN7 bacteria adhered to the neutrophils but resisted phagocytosis. Both strains induced a pronounced neutrophil chemiluminescence response. However, when the intracellular and extracellular parts of the oxidative response were separated, we found that the predominant part of the response was of intracellular origin with the ABU2 bacteria as prey, whereas a large fraction of the response induced by the PN7 bacteria was extracellular. The general opinion is that production of reactive oxygen metabolites should be intraphagosomal to minimize the tissue-damaging effects of the metabolites and to optimize their bactericidal effects. However, since the surface-adherent bacteria (the PN7 cells) are killed in an aerobic but not an anaerobic milieu, whereas the ingested bacteria (the ABU2 cells) are killed in both aerobic and anaerobic milieu, we propose that extracellularly generated oxygen metabolites are of importance in killing E. coli strains that can resist neutrophil engulfment.

Plasmid content of piliated and nonpiliated forms of Moraxella bovis.

Plasmid profiles were compared between nonpiliated and piliated forms of Moraxella bovis isolates. The piliated form of M bovis isolate IBH64 contained 1 fewer plasmid than did the nonpiliated form. Piliated and nonpiliated cells of IBH64 contained plasmids having molecular size of 45, 32.8, 4.9, and 4.6 kilobases (kb). Single- and double-restriction endonuclease digestion by Ava I and Nde I indicated that the size of the additional plasmid carried by the nonpiliated form of IBH64 was approximately 43.6 kb. The M bovis isolates, Newport and GRS, contained the same number of plasmids in either their piliated or nonpiliated form.

The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae.

The fimD gene of Escherichia coli K12 was shown to be necessary for surface localization of type 1 fimbriae, since deletion of the gene resulted in a virtually bald phenotype. The FimD protein was found to be located in the outer membrane. Expressed alone, this protein had a very deleterious effect on cell growth. The DNA sequence of the fimD gene was determined; the corresponding amino acid sequence of the FimD protein was compared with those of the PapC and FaeD proteins. A deletion derivative of FimD gave clues as to which parts of the protein were necessary for outer membrane integration.

Purification and analysis of colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae from enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli fimbriae are immunogenic and play a key role in intestinal colonization. Native colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae were purified by a common method involving shearing, differential centrifugation, gel filtration, and density gradient ultracentrifugation. The compositions and N-terminal sequences were determined. Coli surface antigen 3 possesses two N-terminal isoforms, one of which matches the published DNA sequence, except for the previously proposed signal sequence cleavage point.

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium.

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial antiserum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.

Binding of Escherichia coli S fimbriae to cultured human endothelial cells.

The binding of Escherichia coli adhesins to human umbilical vein endothelial cells was studied by a cell monolayer enzyme-linked immunosorbent assay. S fimbriae displayed a concentration-dependent and saturable binding to the endothelial cells which was mediated by their sialylgalactoside-specific lectin activity. P fimbriae exhibited only low binding, and type 1 fimbriae exhibited no binding to these cells.

Variable expression of P fimbriae in Escherichia coli urinary tract infection.

Fresh urinary isolates were examined by immunofluorescence with polyclonal rabbit antibodies against type 1 and P fimbriae. This procedure showed P-fimbriate Escherichia coli in 22 of 24 samples from patients with asymptomatic bacteriuria, 24 of 26 samples from patients with cystitis, and 6 of 6 samples from patients with pyelonephritis. Type 1 fimbriae were expressed by less than 40% of isolates in all three groups. There was no relation between the presence of symptoms or the site of infection and fimbrial expression, of P or type 1, by bacteria adherent to freshly isolated uroepithelial cells.

In vivo expression and variation of Escherichia coli type 1 and P pili in the urine of adults with acute urinary tract infections.

In vivo expression of pili by Escherichia coli in the urine of 41 adults with lower urinary tract infections was analyzed by immunostaining with polyclonal antiserum to type 1 and P pili. Type 1 pili were detected in 31 of 41 urine specimens, while P pili were detected in 6 of 18 specimens. The piliation status of bacterial populations in urine was heterogeneous, varying from predominantly piliated to a mixture of piliated and nonpiliated cells. Bacteria frequently adhered to exfoliated uroepithelial cells and leukocytes in urine. Expression of pili in vivo did not always correlate with the hemagglutination phenotype after growth in vitro. Strains isolated from different sites in the urogenital tract of two individuals showed phenotypic variation in the state of piliation. The results demonstrate that E. coli type 1 and P pili are expressed and are subject to variation in vivo during acute urinary tract infections in adults.