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.     

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 position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy

Flagellin contains conserved N/C domains for TLR5 binding to activate innate immunity and a middle hypervariable domain harboring the major antigenic epitopes. However, conflict results existed in the previous studies as to whether the hypervariable domain was involved in the cytokine production and adjuvancy of flagellin. Here we constructed three flagellin variants (designated as FliCΔ190-278, FliCΔ220-320, and FliCΔ180-400) with deletions in the hypervariable domain. Our data demonstrated that all deletion variants lost substantial antigenicity but not mucosal adjuvancy. Surprisingly, the variant with deletion of amino acids 220-320 (FliCΔ220-320) induced higher production of IL-8, MCP-1, and TNF-α, and showed higher mucosal adjuvancy than full-length FliC flagellin. Our data supported the notion that the hypervariable domain was involved in the cytokine production by flagellin and more importantly demonstrated that the hypervariable domain was important for the mucosal adjuvancy of flagellin.     

The archaeal flagellum: a different kind of prokaryotic motility structure

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.     

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.     

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.     

Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni

The organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c.0.2kb, similar to that reported for Campylobacter coli. The 5' flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N-terminal 170 amino acids and C-terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface-exposed regions and may represent areas in which surface epitopes are located.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

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.     

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.     

A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is composed primarily of many subunits of a flagellin protein. The highly conserved N and C termini of flagellin are important in its export and self-assembly, whereas the middle sequence region varies greatly in size and composition in different species and is known to be deletion-tolerant. In Salmonella typhimurium phase 1 flagellin, this "hypervariable" region encodes two solvent-exposed domains, D2 and D3, that form a knob-like feature on flagella fibers. The functional role of this structural feature in motility remains unclear. We investigated the structural and physiological role of the hypervariable region in flagella assembly, stability and cellular motility. A library of random internal deletion variants of S. typhimurium flagellin was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative variants were determined in semi-solid and liquid medium using colony swarming motility assays, video microscopy and optical trapping of single cells. All ten variants exhibited diminished motility, with varying extents of motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. The mechanical stability of the variant flagella fibers also decreased with increasing size of deletion. Comparison of the variant sequences with the wild-type sequence and structure indicated that all deletions involved loss of hydrophobic core residues, and removal of both partial and complete segments of secondary structure in the D2 and D3 domains. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region D2 and D3 domains appear to stabilize the folded conformation of the flagellin protein and contribute to the mechanical stability and propulsive force of the flagella fibers.     

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.     

Antigenic Difference Between Polar Monotrichous and Peritrichous Flagella of Vibrio parahaemolyticus

Polar monotrichous and peritrichous flagella of Vibrio parahaemolyticus were isolated and purified separately. On hydroxylapatite column chromatography, the flagellins of polar monotrichous flagella were eluted with a higher concentration of phosphate than those of peritrichous flagella. Gel diffusion tests showed an antigenic difference between the flagellins of polar monotrichous and peritrichous flagella. Electron microscope observations on cells stained with ferritin-conjugated antibodies demonstrated that polar monotrichous and peritrichous flagella reacted specifically with antimonotrichous flagellin and antiperitrichous flagellin antisera, respectively.     

Recent advances in the structure and assembly of the archaeal flagellum

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.     

Identification of genes involved in the assembly and attachment of a novel flagellin N-linked tetrasaccharide important for motility in the archaeon Methanococcus maripaludis

Recently, the flagellin proteins of Methanococcus maripaludis were found to harbour an N -linked tetrasaccharide composed of N -acetylgalactosamine, di-acetylated glucuronic acid, an acetylated and acetamidino-modified mannuronic acid linked to threonine, and a novel terminal sugar [( 5S )-2-acetamido-2,4-dideoxy-5-O-methyl-α-L- erythro -hexos-5-ulo-1,5-pyranose]. To identify genes involved in the assembly and attachment of this glycan, in-frame deletions were constructed in putative glycan assembly genes. Successful deletion of genes encoding three glycosyltransferases and an oligosaccharyltransferase (Stt3p homologue) resulted in flagellins of decreased molecular masses as evidenced by immunoblotting, indicating partial or completely absent glycan structures. Deletion of the oligosaccharyltransferase or the glycosyltransferase responsible for the transfer of the second sugar in the chain resulted in flagellins that were not assembled into flagella filaments, as evidenced by electron microscopy. Deletions of the glycosyltransferases responsible for the addition of the third and terminal sugars in the glycan were confirmed by mass spectrometry analysis of purified flagellins from these mutants. Although flagellated, these mutants had decreased motility as evidenced by semi-swarm plate analysis with the presence of each additional sugar improving movement capabilities.     

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins.The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.     

Unique sequences in region VI of the flagellin gene of Salmonella typhi

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.     

Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants

Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.