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.     

Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi.

Four mutants of Escherichia coli that are resistant to the flagellotropic phage chi, but are motile, were isolated. When they were observed in liquid culture bylight microscopy, one mutant exhibited circular movement and another tumbled at high frequency on the surface of a glass slide. The remaining two mutants moved normally. None of these mutants adsorbed the wild-type strain of chi. P1 transduction revealed that the mutation sites of these four mutants were more than 97% contransducible with a site in hag, the structural gene for flagellin. When flagellins of these mutants were chromatographed on a diethylaminoethyl-cellulose column, two eluted slower and one eluted slightly faster than the flagellin of the parental strain. The other flagellin eluted at the same position as that of the parent. Host range mutants of phage chi, which could infect these bacterial mutants, were isolated.     

Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement.

Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.     

Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin

The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.     

The amino acid composition of Rhizobium meliloti flagellin: Occurrence of a single tryptophan residue

Flagella were isolated from cultures of $\text{Rhizobium meliloti}$ strain RM2011 and purified to homogeneity as determined by the presence of a single band of the monomeric flagellin subunit following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Classical hydrochloric acid hydrolysis showed that the amino acid composition of RM2011 flagellin was generally similar to that of other bacterial flagellins. Hydrolysis with methanesulfonic acid indicated that, unlike other flagellins, RM2011 flagellin contained a single tryptophan residue per molecule. Analysis of the products obtained by the reaction of the flagellin with N-bromosuccinimide under conditions specific for cleavage at tryptophan residues confirmed the presence of a single tryptophan residue.     

Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.     

Identification of proinflammatory flagellin proteins in supernatants of Vibrio cholerae O1 by proteomics analysis

The genome of Vibrio cholerae contains five flagellin genes that encode proteins (FlaA-E) of 39-41 kDa with 61-82% identity among them. Although the existing live oral attenuated vaccine strains against cholera are protective in humans, there is an intrinsic residual cytotoxic and inflammatory component associated with these candidate vaccine strains. Bacterial flagellins are known to be potent inducers of proinflammatory molecules via activation of Toll-like receptor 5. Here we found that purified flagella from wild type V. cholerae 395 induced significant release of interleukin (IL)-8 from cultured HT-29 human colonic epithelial cells. Furthermore we found that filtered supernatants of KKV90, a DeltaflaA isogenic strain unable to produce flagella, were still able to activate production of IL-8 albeit to significantly lower levels than the wild type, suggesting that other activators of proinflammatory molecules were still present in these supernatants. A comparative proteomics analysis of secreted proteins of V. cholerae 395 and KKV90 identified additional proteins with potential to induce IL-8 release in HT-29 cells. Secreted proteins in the range of 30-45 kDa identified by two-dimensional electrophoresis and mass spectrometry revealed the presence of two additional flagellins, FlaC and FlaD, that appeared to be secreted 3- and 6-fold more, respectively, in the mutant compared with the wild type. Double isogenic mutants flaAC and flaAD were unable to trigger IL-8 release from HT-29 cells. In sum, we have shown that purified flagella and secreted flagellin proteins (FlaC and FlaD) are inducers of IL-8 release from epithelial cells via Toll-like receptor 5. This observation may explain, in part, the observed reactogenicity of cholera vaccine strains in humans.     

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.     

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.     

Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion.

Immunological methods were used to examine the flagellin production of Salmonella typhimurium strains that carried a mutation in one of the two possible genes for flagellin (H1 or H2) and also were incapable of expressing the other gene. Some mutants produced flagellin that was excreted into the culture medium; others accumulated flagellin intracellularly. These two phenotypes were detected in both H1 and H2 mutants. The mutation sites were mapped on the corresponding deletion map (consisting of 21 segments in the case of H1 and 31 segments in the case of H2). H1 and H2 mutations causing excretion of flagellin were clustered mainly in segment 12 and segment 6 from the proximal end, respectively, suggesting that the corresponding segments of the flagellins play a role in polymerization. Mutations causing accumulation in the cytoplasm were clustered in segments 19 to 21 of the H1 map and in segments 25 to 29 of the H2 map, suggesting that an essential region for flagellin transport exists toward the C terminus of flagellin.     

Overexpression of Methanococcus voltae Flagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.     

Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis

Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.     

Mechanism of flagellin polymerization in Bacillus brevis

The optical properties of acid-dissociated and tetranitromethane-modified flagellin has been studied by circular dichroism (CD) techniques. The flagellins have the same CD spectra which do not change over the 2.9-10.0 range. The spectra character depends on pH in the presence of polyethylene glycol (PEG) and ammonium sulphate which accelerate polymerization. As the content of PEG goes up to 20% and ammonium sulphate to 1 M, the value of the molecular ellipticity at 222 nm ([O]222) of the both flagellins considerably increases at pH 4.3-10.0, however [O]222 does not achieve the value for bacterial flagella. PEG and ammonium sulphate addition at pH 2.9-3.9 gives less dramatic increase of the [O]222 value. It has been concluded that the changes in the CD spectra at pH 4.3-10.0 is a result of conformational rearrangements in flagellin before it incorporates into the flagella.