Biochemical and antigenic properties of the Campylobacter flagellar hook protein.

The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.     

Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.     

Protection against Campylobacter jejuni infection in suckling mice by anti-flagellar antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.     

Topologic mapping of protective and nonprotective epitopes on the variant surface glycoprotein of the WRATat 1 clone of Trypanosoma brucei rhodesiense

Monoclonal antibodies were used in competitive antibody binding assays to define and map epitopes on the variant surface glycoprotein of the WRATat 1 clone of T. b. rhodesiense. By using a panel of 30 WRATat 1-specific monoclonal antibodies, 16 epitopes were defined that fall into four clusters, having 1, 1, 3, and 11 distinct epitopes respectively. All epitopes were easily classified as being 1) exposed uniformly on the surface of the trypanosome, 2) exposed only in the region of the flagellar pocket, or 3) "buried", based on the ability or inability of the monoclonal antibodies to bind living trypanosomes in a fluid phase immunofluorescence assay. Monoclonal antibodies that bind exposed surface epitopes are protective, whereas only three of seven that bind exclusively to flagellar pocket epitopes are protective. None of the nine monoclonal antibodies that recognize buried epitopes are protective. Also, antibody-mediated immunity to WRATat 1 trypanosomes is not associated with any particular subclass of antibody. The IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA subclasses each contain examples of protective monoclonal antibodies.     

Purification and antigenic analysis of flagella of Campylobacter jejuni

The flagella of Campylobacter jejuni strain FUM158432 were purified and a flagellin preparation consisting of only a single peptide of 63,000 daltons was obtained. The peptide of 92,000 daltons usually associated with a flagellar preparation was shown to be a peptide derived from the hook region. Antiserum was prepared by immunizing a rabbit with the flagellin preparation. The reaction of the antiserum was found to be highly specific for the flagellar filament by immunoelectron microscopy and for flagellin peptide by the immunoblotting method. Seventeen of 23 clinically isolated strains of C. jejuni reacted with this antiserum but the other six strains did not, indicating the existence of antigenic variation of the flagella of C. jejuni. The flagella of a few strains of C. coli also reacted with this antiserum.     

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.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

The flgE gene of Campylobacter coli is under the control of the alternative sigma factor sigma54.

The flgE gene encoding the flagellar hook protein of Campylobacter coli VC167-T1 was cloned by immunoscreening of a genomic library constructed in lambdaZAP Express. The flgE DNA sequence was 2,553 bp in length and encoded a protein with a deduced molecular mass of 90,639 Da. The sequence had significant homology to the 5' and 3' sequences of the flgE genes of Helicobacter pylori, Treponema phagedenis, and Salmonella typhimurium. Primer extension analysis indicated that the VC167 flgE gene is controlled by a sigma54 promoter. PCR analysis showed that the flgE gene size and the 5' and 3' DNA sequences were conserved among C. coli and C. jejuni strains. Southern hybridization analyses confirmed that there is considerable sequence identity among the hook genes of C. coli and C. jejuni but that there are also regions within the genes which differ. Mutants of C. coli defective in hook production were generated by allele replacement. These mutants were nonmotile and lacked flagellar filaments. Analyses of flgE mutants indicated that the carboxy terminus of FlgE is necessary for assembly of the hook structure but not for secretion of FlgE and that, unlike salmonellae, the lack of flgE expression does not result in repression of flagellin expression.     

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.     

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60- kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.     

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.     

Motility as an intestinal colonization factor for Campylobacter jejuni

The colonization of the intestinal tract of suckling mice by Campylobacter jejuni was examined by orally challenging the mice with a wild-type strain and several nonmotile mutant strains which were isolated after treating the wild-type strain with mutagens. The wild-type strain had colonized the lower portion of the small intestine, the caecum and the colon 2 d after inoculation. Two nonmotile strains, one of which (M8) had lost all the flagellar structure including the filament, the hook and the basal structure, and the other (M1) which had lost only the filament region, were both cleared from the intestinal tract 2 d after challenge. Another nonmotile strain (M14), which had a complete flagellar structure like that of the wild-type strain, did not colonize and was cleared from the intestinal tract like the other nonmotile and nonflagellated strains. One atypically motile strain (M5), which had a shorter flagellar filament than that of the wild-type strain, colonized the intestinal tract only when mice were challenged with a large inoculum. None of the mice challenged with either the wild-type or any of the mutant strains showed signs of illness. We concluded that motility is an important factor in the colonization of the intestinal tract of suckling mice by C. jejuni.     

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold–protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.     

Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase

We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).     

Information essential for cell-cycle-dependent secretion of the 591-residue Caulobacter hook protein is confined to a 21-amino-acid sequence near the N-terminus

Recent findings suggest that axial flagellar proteins and virulence proteins of Gram-negative bacteria are exported from the cytoplasm via conserved trans-location systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591-residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38–58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21-residue sequence is not conserved in other axial proteins and does not correspond to the SGL-, ANN LAN- and heptad repeat motifs that are located Just upstream of the essential secretion information in the hook protein and are conserved near the N-termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell-cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.