Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.     

Antigenic structures of Salmonella flagella. I. Presence of an antigenic determinant exposed at one end of flagellar fragments.

Salmonella flagellin, which is a constitutional subunit of the flagellum, was shown to have antigenic determinants distinct from its own serotypic ones. These antigenic determinants were found to be common to flagellins from the so-called g-complex serotypes, such as fg, mt, gm, gt, gp and gmptu, but not to those from other serotypes, such as a, i or enx. Rabbits immunized with flagellin of serotype "fg" produced anti-"fg" flagellin antibodies. Only about 20 percent of these corresponded to the serotype determinants of the "fg" on the surface of the flagella, and the remaining 80 percent reacted with the flagellin of the unrelated serotype "mt", and corresponded to the distinct determinants common to the flagellin molecules. These antigenic determinants were detected by the immunoferritin technique at only one, not both, terminals of the flagellar fragments, suggesting that a unidirectional arrangement of flagellin subunits in the flagella may expose the inherent conformation of the subunits at only one end of the flagellum.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.     

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.     

Molecular analyses of the Salmonella g. . . flagellar antigen complex.

Salmonella flagellar filaments are polymers of a highly antigenic protein, termed flagellin. Eight main subfactors have been identified in the Salmonella phase-1 g. . . series flagellar antigen. To determine the molecular basis for expression of the epitopes by which the g. . . family subfactors are distinguished, 10 members of this series were selected and their fliC (the structural gene for phase-1 flagellin) genes were sequenced. Comparative analyses of the inferred primary structures of these flagellins did not allow the identification of linear epitopes responsible for the antigen subfactors. This suggests that conformational aspects are involved in determining the antigenic specificity in these cases. A phylogenetic analysis of the flagellin sequences showed that members of the g. . . series do not form a single coherent unit.     

Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA.

In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.     

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.     

Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds.

Campylobacter coli VC167 has been shown to undergo a reversible flagellar antigenic variation between antigenic type 1 (T1) and antigenic type 2 (T2). VC167 contains two flagellin genes, and the products of both genes are incorporated into a complex flagellar filament in both antigenic types. Although there are only minor amino acid changes in the flagellins expressed by T1 and T2 cells, the two antigenic types of flagellins can be distinguished by differences in apparent M(r) on sodium dodecyl sulfate-polyacrylamide gels and by immunoreactivity with T1-specific (LAH1) or T2-specific (LAH2) antiserum. The isolation of stable variants of T1 and T2 has allowed for the transfer via natural transformation of the flagellin structural genes from the T1 background into the T2 background and from the T2 background into the T1 background. In addition, the flagellin genes from VC167 T1 and T2 have been transferred into strains of Campylobacter jejuni. The results indicate that the observed antigenic variations of VC167 flagellins are dependent on the host genetic background and independent of the primary amino acid sequence. These data provide evidence that posttranslational modifications are responsible for the antigenic variation seen in VC167 flagellins.     

Isolation and characterization of bacterial flagellar hook proteins from salmonellae and Escherichia coli.

Flagellar hook proteins from Salmonella and Escherichia coli were dissociated in acid and purified by diethylamino-ethyl-cellulose column chromatography. These two proteins had the same electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. However, analytical electrofocusing patterns showed that these proteins had different isoelectric points (4.7 for Salmonella typhimurium and 4.4 for E. coli). Immunodiffusion and immuno-electron microscopy carried out with antisera prepared against purified hook proteins from S. typhimurium and E. coli showed that these antisera reacted with both hooks. Affinity chromatography allowed separation of antibodies specific for hook proteins from each bacterial species. These results indicate that the hook proteins share common antigenic determinants as well as specific antigens, although the specificity is not quantitatively resolved. From comparisons of the amino acid composition of the hook proteins and flagellins, it was concluded that the differences between flagellins from S. typhimurium and E. coli were larger than those between hook proteins from these species.     

The flagellin gene and protein from the brewing spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis

Flagellin genes from the anaerobic Gram-negative beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were sequenced and the flagellin proteins initially characterized. Protein microsequencing led to the design of two degenerate PCR primers that allowed the P. cerevisiiphilus flagellin gene to be partially sequenced. A combination of PCR and Bubble PCR was then used to sequence the flagellin genes of three isolates from each species. Cloning and gene expression, followed by immunoblotting, confirmed the gene identities as flagellin. Analysis of the gene sequences revealed proteins similar to other bacterial flagellins, including lengths of 446 or 448 amino acids, putative sigma 28 promoters, and a termination loop. Antibody binding studies with isolated flagella correlated with gene sequence comparisons, with both indicating that the P. cerevisiiphilus isolates studied are very similar but that the P. frisingensis isolates show greater variation. Purified flagellins were found to be glycosylated, probably through an O linkage. Phylogenetic analysis revealed greater diversity within the flagellin sequences than within the 16S rRNA genes. Despite the Gram-negative morphology of Pectinatus, this genus proved most closely related to Gram-positive Firmicutes.     

Characterization of a post-translational modification of Campylobacter flagellin: identification of a sero-specific glycosyl moiety

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.     

The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins

In order to circumvent problems associated with direct chemical analysis of the phase-1 flagellar filament protein (flagellin) of Salmonella typhimurium, the covalent structure was determined by recombinant DNA procedures. The corresponding structural gene (H-1i) was cloned into plasmid pBR322 in a 4.3-kilobase fragment produced by EcoRI digestion of chromosomal DNA, and the nucleotide sequence of the region specifying the flagellar protein was determined. Comparison of the data obtained with the limited information available for other salmonellar flagellins supported the concept that both ends of the molecule are conserved in this genus. Additionally, a conservation of base sequence in the region of H-1 genes coding for the N-terminal end of flagellins was apparent, suggesting that this area may have an additional regulatory role. The i flagellin was found to be unrelated to proteins in the NBRF data base with the exception of other flagellins. The three flagellins which have been sequenced to date (those produced by Bacillus subtilis, Caulobacter crescentis, and phase-1 S. typhimurium) show homologies in amino acid sequence at both the N-terminal and C-terminal ends despite large differences in their total molecular weight, and comparison suggests that B. subtilis and Salmonella are more closely related to each other than either is to Caulobacter.     

Structural study of binding of flagellin by Toll-like receptor 5

In order to predict the binding regions within the complex formed by Toll-like receptor 5 (TLR-5) and flagellin, a complementary hydropathy between the two proteins was sought. A region common to the flagellins of Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Listeria monocytogenes was shown to be hydropathically complementary to the 552-to-561 fragment of TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profile of this region is shared with flagellins of 377 bacterial species out of a total of 723 publicly available sequences. A conformational analysis of the predicted binding site of TLR-5, whose structure is still unknown, was carried out with a methodology already applied to similar problems. To sample the conformations available to the peptide chain, a plot of the number of conformations per unit energy interval (density of states) versus energy was built. Following a theoretical argument, conformations belonging to maxima in this plot were selected. The most stable structure obtained in this search, an alpha-helical conformation, was shown to form the electrostatic interactions Glu552-Gln89, Asp555-Arg92, and Arg558-Glu93 with the predicted binding site of the flagellin of S. enterica serovar Typhimurium, formed by the 88-to-97 chain fragment (LQRVRELAVQ), which is likewise alpha helical.     

Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

Tyrosine phosphate in a- and b-type flagellins of Pseudomonas aeruginosa.

Both a- and b-type purified flagellins from a number of Pseudomonas aeruginosa strains grown in radiolabeled phosphate were shown to be phosphorylated. Analysis of partial acid-hydrolyzed flagellar filaments revealed that 32Pi was in phosphotyrosine. Three 32P-phosphopeptides apparently are common to a- and b-type flagellins, but a fourth peptide was found only in b-type hydrolysates. P. aeruginosa PAK flagellin, containing only two tyrosines, both in the variable region, was readily labeled and gave the same peptide pattern as flagellins containing additional tyrosines. Data showing that a- and b-type flagellins gave positive immunoblots with antiphosphotyrosine monoclonal antibody and that release of P(i) by alkaline phosphatase occurred indicated that unmodified tyrosine phosphate exists in flagellin.     

Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.     

Flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking part of the outer domain of flagellin.

We have isolated spontaneous mutants of Salmonella typhimurium which can swim in the presence of antifilament antibodies. The molecular masses of flagellins isolated from these mutants were smaller than that (52 kDa) of wild-type flagellin. Two mutants which produced the smallest flagellins (42 and 41 kDa) were selected, and the domain structures of the flagellins were analyzed by trypsin digestion and then subjected to amino acid sequencing. The two flagellins have deletions at Ala-204 to Lys-292 and Thr-183 to Lys-279, respectively. These deleted parts belong to the outer domain (D3) of flagellin, which is believed to be at the surface of the filament. These mutant filaments aggregated side by side in the presence of salt, resulting in disordered motility.     

Location of epitopes on Campylobacter jejuni flagella.

Flagella were isolated from strains of Campylobacter jejuni belonging to different heat-labile serogroups and from a strain of Campylobacter fetus, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the flagellin molecular weights (Mr) were approximately 62,000. The flagellins were cleaved by hydrolysis with cyanogen bromide, and sodium dodecyl sulfate-urea peptide gel electrophoresis showed that the C. jejuni flagellins were structurally similar, and differed from C. fetus flagellin. Immunochemical analysis by Western blotting, enzyme-linked immunosorbent assay, immune electron microscopy, and immunoprecipitation with polyclonal and monoclonal antibodies revealed the presence of both internal and surface-exposed epitopes. The internal epitopes were antigenically cross-reactive and linear, and in the case of C. jejuni flagellin were located on cyanogen bromide peptides of apparent Mr 22,400 and 11,000. Antigenically cross-reactive epitopes were also present on an Mr 43,000 cyanogen bromide peptide of C. fetus flagellin. The Mr 22,400 peptide of C. jejuni VC74 flagellin also carried closely positioned internal linear epitopes for two monoclonal antibodies. One epitope was strain specific, while the other was shared by some but not all Campylobacter flagellins. The flagella of C. jejuni VC74 also displayed both surface-exposed antigenically cross-reactive and surface-exposed serospecific epitopes. Both linear and conformational epitopes contributed to the serospecificity of C. jejuni VC74 flagella, and a linear serospecific epitope was located on a cyanogen bromide peptide of apparent Mr 4,000.