Anti-flagellin Antibodies Research Articles

Cloning and sequence analysis of the flagellin gene ofCampylobacter jejuni TGH9011

Flagella are essential for motility and have been implicated to be one of the pathogenic determinants. The flagellum ofCampylobacter jejuni is a polymeric structure of a 62-kd protein. Using a high-affinity flagellin antibody to screen a lambda gt 11 phage genomic expression library ofC. jejuni strain TGH9011 (Serotype LIO36), a recombinant phage clone lambda gt 11RK that expresses theC. jejuni flagellin protein was isolated. The recombinant lambda gt 11 RK produced a 56-kd protein upon induction with isopropylthiogalactoside, which reacted specifically with anti-flagellin antibody. The flagellin gene was sequenced, and comparative analysis of the nucleotide and amino acid sequence identified a region of the flagellin that shows hypervariability among differentCampylobacter species and strains.

Local immune responses to the Campylobacter flagellin in acute Campylobacter gastrointestinal infection.

Intestinal immunoglobulin A (IgA) anti-campylobacter flagellin antibodies were measured by an enzyme-linked immunosorbent assay in patients with and without campylobacter infections. The level of fecal IgA antiflagellin antibodies was significantly higher in patients than in controls, but only 12 of 29 patients (41.4%) with a campylobacter infection had detectable IgA antibodies against flagellin (optical density, greater than or equal to 0.100). Further testing of fecal IgA antibodies from 10 patients with homologous and heterologous purified flagellins showed strain specificity in 1 patient. Cross-reactivity of fecal IgA antibodies with heterologous flagellins did not correlate with the Lior serotype of the isolate.

High levels of anti- Campylobacter-flagellin IgA antibodies in breast milk

Several studies have shown the beneficial role of immunity against enteric Campylobacter sp. Circulating antibodies against flagellin and possibly maternal antibodies seem to protect against intestinal colonization by Campylobacter. Milk from women in Central Africa (n = 120) and in France (n = 31) were investigated for the presence of anti-flagellin antibodies. Results showed that most French and all African milk possessed anti-flagellin IgA but not IgG. The titres of anti-flagellin IgA were elevated in colostrum and decreased in later milk. Specific activities were constant during lactation, suggesting constant antigenic stimulation. The fact that IgA but not IgG are elicited against flagellin could be due to an infection limited to the mucosal surface.

Expression of two Rhizobium meliloti flagellin genes and their contribution to the complex filament structure

The complex flagellar filaments of Rhizobium meliloti are composed of two related (87% identical) flagellins that are encoded by closely linked, separately transcribed genes, flaA and flaB (E. Pleier and R. Schmitt, J. Bacteriol. 171:1467-1475, 1989). To elucidate the role of the subunits, A and B, in assembling the complex filament, the wild-type alleles were replaced with defective ones containing a 2,249-bp deletion (accompanied by substitution of a kanamycin resistance cartridge), which eliminates 74% of flaA (3' end) and 85% of flaB (5' end). The resulting nonmotile, filamentless mutant, RU11011, was tested for complementation with wild-type flaA, flaB, and flaA flaB genes provided on the multiple-copy vector pRK290. Whereas flaA alone did not restore motility and filament production, both flaB and flaA flaB restored 20 to 30% of wild-type motility. Apparent causes of this reduced motility were fewer flagella per cell and/or shortened filaments sometimes ending in unusually thin, fragile structures. Tests with enzyme-linked antiflagellin antibodies indicated that flaA is expressed at higher levels than flaB and that multiple copies of flaA lead to reduced flagellin export. We conclude that the proximal portion of the complex filament is assembled from B subunits (not produced sufficiently to form full-length flagella) and that the distal portion is made from A subunits. Multiple copies of the strong flaA promoter may offset transcriptional controls that regulate the synthesis of flagellar structures required for flagellin export.

Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi

A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease. Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins. The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266). However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein. The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease. The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.

Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals

Borrelia burgdorferi expresses a conserved, species-specific 39-kDa protein (P39) that can stimulate antibodies during human infection. To confirm that anti-P39 antibodies are produced consistently in animals exposed to infectious spirochetes, white-footed mice, Peromyscus leucopus, and laboratory white mice, Mus musculus (strain BALB/c), were experimentally inoculated with either infectious or noninfectious B. burgdorferi and the antibody response to P39 was determined by immunoblot at 21 days postinoculation. All mice inoculated with approximately 10(7) infectious B. burgdorferi produced anti-P39 antibodies and were cultured positive for this spirochete. Mice inoculated with similar numbers of inactivated or viable noninfectious B. burgdorferi still producing P39 did not induce anti-P39 antibodies. By contrast, putative antiflagellin antibodies were detected in less than 18% of the infected animals, which supports the notion that antibody reactive with flagellin may not be reliable as a marker for B. burgdorferi exposure as was originally thought. Mice infected with B. burgdorferi following exposure to ticks (Ixodes dammini) produced anti-P39 antibodies no later than 7 days postinfection, indicating that P39 is an effective immunogen in natural infections. Notably, anti-P39 antibodies were the predominant B. burgdorferi reactive antibodies detected early in the infection. Our results indicate that anti-P39 antibodies are produced in response to an active infection and are therefore reliable markers for infection in experimentally and naturally inoculated animals.

Infection of adult Syrian hamsters with flagellar variants of Campylobacter jejuni

Two variants of Campylobacter jejuni IN1 differing in the expression of flagella, IN1 (Fla+ Mot+, wild type) and IN1-NM (Fla- Mot-), were tested for their ability to establish infection in adult hamsters. Animals were challenged intracecally with 2 X 10(9) to 5 X 10(10) CFU and monitored for evidence of infection. None of the challenged animals developed illness. There was a significant difference, however, in the ability of IN1 to infect hamsters (35 of 43) compared with that of IN1-NM (1 of 42) (P less than 0.01). Additionally, eight animals challenged with IN1-NM excreted only the campylobacters of Fla+ phenotype, which were shown to be identical with the parental Fla+ Mot+ type. Both groups of animals developed serum immunoglobulin G antibodies to outer membrane proteins of IN1 as measured by an enzyme-linked immunosorbent assay; however, only animals that excreted Fla+ organisms developed antiflagellin antibodies. In vitro reversibility from Fla+ to Fla- occurred with a frequency of 9.2 X 10(-6) per cell per generation; however, reversion from Fla- to Fla+ could not be detected in vitro. Further characterization of IN1-NM showed that it produced a cytoplasmic 62K flagellin subunit protein, but this protein lacked six epitopes detected in IN1 and also differed in its two-dimensional gel electrophoresis pattern. The results of these studies support the concept that an intact flagellum is necessary for intestinal infection with C. jejuni and that this model may be useful for studying the immune response to C. jejuni.

Human antibody response to Campylobacter jejuni flagellin protein and a synthetic N-terminal flagellin peptide.

We measured isotype-specific human antibodies directed against Campylobacter jejuni native flagellin and a synthetic peptide derived from the N-terminal amino acid sequence of the protein by using a microdilution enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with gastrointestinal infection caused by C. jejuni (n = 20) and control samples (number from normal subjects = 20; number from patients with diarrhea other than campylobacter = 20) were tested in this assay. Serum specimens from patients with campylobacter infection showed statistically significant higher isotype-specific antiflagellin antibody titers than control samples did. Detection of immunoglobulin G (IgG) antibodies was less specific (70%) than detection of either IgA or IgM antibodies in infected patients (95%). The sensitivity of testing for any of the isotypes ranged from 64 to 100% in acute-phase serum specimens and 85 to 95% in convalescent-phase serum specimens. An ELISA with an N-terminal synthetic peptide derived from the flagellin protein as antigen was not sensitive (60%) for detecting campylobacter infection but was very specific (97.5%). In conclusion, detection of serum IgA or IgM against C. jejuni flagellin may be a useful marker of infection. Although the N-terminal synthetic peptide was antigenic in a few patients with infection and showed good specificity in the ELISA, additional amino acid sequences with better sensitivity for detecting infection need to be identified.

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.

Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants

Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.

Impairment of Jones-Mote hypersensitivity and specific antibody response against depolymerized flagellin in lepromatous leprosy.

Cutaneous hypersensitivity and antibody-producing capacity were assessed in patients with lepromatous leprosy with defective immunity, by immunizing them with monomeric flagellin from Salmonella adelaide. Results were compared with those of controls, matched for age and sex, derived from similar socioeconomic stratum, but without any defect of the immunological system. In contrast to the normal individuals, who showed Jones-Mote type of hypersensitivity, no lepromatous patient could mount any 'delayed-in-time' cutaneous hypersensivivity reaction against an intradermal challenge of monomeric flagellin. However, when immunized through the subcutaneous route, both groups could produce adequate amounts of specific serum antibody. In addition to this unique split tolerance found in all lepromatous patients, some patients showed low levels of 'natural' IgM antibody, reduced formation of specific antibody when immunized through the subcutaneous route, and incomplete maturation of IgG class of anti-flagellin antibody. When immunized by the intradermal route, however, production of both anti-flagellin antibody and maturation of IgG antibody was significantly inhibited in normal adults but not in lepromatous patients. Thus, contrary to the earlier concept of hyperactivity of the humoral immune apparatus in lepromatous leprosy, the present study detected B-cell hypofunction in some patients.

Classification of Bacillus subtilis flagellins

Purified flagellins derived from 16 strains of Bacillus subtilis were classified into at least five distinct groups on the basis of their reaction with antiflagellar filament antibody and antiflagellin antibody. This classification was in good accord with that derived independently on the basis of amino acid analyses of the flagellins. Flagellar antigenicity appears to provide a useful typological character in classifying B. subtilis strains.

Conditional surface structure mutants of Caulobacter crescentus temperature-sensitive flagella formation due to an altered flagellin monomer

Two classes of temperature-sensitive surface structure mutants of Caulobacter crescentus have been isolated. Mutants in one class were unable to form flagella at the restrictive temperature (37°C) and mutants in the second class were unable to form a stalk structure at 37°C. All of the mutants appeared normal at the permissive temperature (23°C). Analysis of the conditional flagella mutant revealed that the temperature-sensitive lesion resided in the structural flagellin monomer, since (1) extracts of mutant cells grown at the restrictive temperature contained material cross-reacting with anti-flagellin antibody although flagella were not assembled. (2) Mutant flagellin prepared from cells grown at the permissive temperature was heat-denatured to a different conformation than wild-type flagellin, as demonstrated by lateral shifts in microcomplement fixation assays. (3) Amino acid analysis showed the presence of tyrosine in the mutant flagellin, whereas none was detected in the wild-type control. (4) Chymotryptic peptide maps of mutant and wild-type flagellin showed the appearance of two new peptides in the former case. (5) A spontaneous revertant regained both motility and wild-type flagellin amino acid composition and chymotryptic peptide maps. An alteration in the primary structure of the mutant flagellin therefore results in a temperature-dependent alteration in flagellin conformation, perhaps accounting for the inability to assemble a flagellar structure at the cell surface. Comparison of immunoprecipitates of intracellular and extracellular mutant flagellin, as well as flagellin concentrations, further showed that the inability to assemble a flagellar structure interfered with the regulatory processes controlling both the amount and structural integrity of the intracellular flagellin at the restrictive temperature.

The effect of chemical modification of flagellin on its subsequent immunogenicity

Substitution of flagellin with a variety of reagents caused a reduction in antigenicity relative to unmodified flagellin. This reduction resulted in a decreased capacity (1) to elicit a primary anti-flagellin antibody response and (2) to prime for a secondary response. However compared with acetoacetylated flagellin, there was less correlation between the loss of antigenicity and the capacity of the antigen to prime for delayed-type hypersensitivity. For example, when flagellin was lightly succinylated or iodinated, the resulting antigen induced enhanced DTH. Further substitution depressed the DTH response induced. Injection of extensively iodinated flagellin into rats induced a state of partial antibody tolerance similar to that found earlier with acetoacetylated flagellin. However, iodinated flagellin which induced antibody tolerance did not prime for enhanced DTH.

Cleavage of bacterial flagellin with proteolytic enzymes: II. Induction of enhanced delayed-type hypersensitivity to flagellin by peptic fragments of the protein

Digestion of Salmonella adelaide flagellin (mol wt 40,000) with pepsin at suboptimal pH results in the release of 3–4 large, “pepsin-resistant peptides” (mol wt 13,000–15,000), which carry most of the antigenic determinants of the protein. These large peptides are slowly degraded by prolonged pepsin treatment into low molecular weight peptic peptides (mol wt < 4,000) which have little or no antigenic activity. A single injection into rats of the peptic digests of flagellin failed to produce detectable anti-flagellin antibodies, but was still capable of priming rats for a secondary antibody response to flagellin. This priming effect was mediated by the “pepsin-resistant peptides” present in the peptic digests, but not by the low molecular weight peptic peptides. In contrast to the antibody response, preparations of flagellin that had been subjected to prolonged peptic digestion induced higher levels of flagellin-specific delayed-type hypersensitivity than undigested flagellin. Furthermore, on fractionation of such digests, it was found that both the “pepsin-resistant peptides” and the low molecular weight peptic peptides induced delayed-type hypersensitivity to flagellin. A partial purification of the small peptic peptides which provoked delayed hypersensitivity was achieved by high voltage paper electrophoresis. The hypersensitivity inducing peptides were shown to be derived from regions of the flagellin molecule which carried antibody forming determinants. From the observation that degraded antigen preferentially induces delayed hypersensitivity, it is proposed that macrophage-degraded antigen plays an important role in regulating humoral and cell-mediated immunity in vivo. It is also suggested that degraded antigen favours the induction of delayed hypersensitivity due to its inability to promote cell-cell interaction between immunocompetent cells.

Relation of the Human Antibody Response to Flagellin to Gm Genotype

Abstract The incidence and distribution of the Gm allotypic markers Gm (a), (x), (g), (b) and (f) were studied in 113 Caucasian subjects who were classified on the basis of the serum titer of “natural” anti-flagellin antibodies and their response to injection with 5µg flagellin from Salmonella adelaide. There were 59 subjects with a low titer (&amp;lt; 100) of “natural” anti-flagellin antibodies (LNA) and of these, 43 had a low primary response (LPR, peak titer &amp;lt; 100) to injected flagellin; these 59 subjects were classified as “low responders.” There were 18 subjects with a high titer of “natural” antibodies (HNA, titer τ; 600) and a separate group of 36 with a high primary response (HPR, titer τ; 10,000); these 54 subjects were classified as “high responders.”. As expected in Caucasians, all subjects positive for Gm (a) were also positive for Gm (g) (64.6%) and all subjects positive for Gm (b) were also positive for Gm (f) (89.4%). The number of Gm (a,g) subjects was significantly higher in group HNA than in group LNA (P &amp;lt; 0.05) and significantly higher in “high responders” than in “low responders” (P &amp;lt; 0.05). Otherwise stated, the absence of the Gma,g gene group was associated with a lower response to flagellin. The results indicate a significant association in these subjects between the magnitude of the immune response to flagellin (indicated by antibody titers) and the Gm genotype.

Immune response to chemically modified flagellin. I. Induction of antibody tolerance to flagellin by acetoacetylated derivatives of the protein.

Flagellin (mol. wt. 40,000) from S. adelaide organisms was acetoacetylated to varying extents with diketene (acetoacetic anhydride). Chemical studies demonstrated that the amino groups of flagellin were more readily acetoacetylated than the hydroxyl groups. Several antigenic tests revealed that as flagellin was acetoacetylated to increasing extents there was a steady decline in the affinity of the molecule for anti-flagellin antibodies. Loss in antigenic activity following acetoacetylation was not related to the number of acetoacetyl groups attached but was determined by the type of residue substituted. Reactive lysine residues were much less important anti-genically than easily substituted hydroxyl groups. Acetoacetylation very readily destroyed the antibody-forming capacity of flagellin in rats. This fall in immunogenicity was related to the antigenic activity of the preparations. In fact, only a 40% reduction in the antigenic activity of flagellin produced a 90-95% reduction in primary antibody formation. The more heavily acetoacetylated flagellins produced no detectable antibody and, in fact, rendered adult rats tolerant (in terms of antibody formation) to a subsequent challenge of flagellin. Tolerance was induced by acetoacetylated flagellins which had drastically reduced affinities for anti-flagellin antibodies. These results were interpreted as indicating that the affinity of antigen for receptors on cells appears to be of crucial importance in determining whether antibody formation or immunological tolerance (antibody suppression) occurs.

Immune response to chemically modified flagellin. II. Evidence for a fundamental relationship between humoral and cell-mediated immunity.

Flagellin (mol.wt. 40,000) from S. adelaide organisms and a series of acetoacetyl derivatives of flagellin were tested for their ability to induce humoral and cell-mediated immunity in adult rats. It was found that unmodified flagellin was an excellent inducer of antibody formation but a poor inducer of delayed-type hypersensitivity. In contrast, increasing acetoacetylation steadily destroyed the ability of flagellin to initiate antibody formation but enhanced the capacity of the molecule to induce flagellin-specific cell-mediated immunity and antibody tolerance. In fact, it appeared that in adult rats antibody formation and cell-mediated immunity may well be opposing immunological processes. Furthermore, the affinity of the acetoacetyl flagellins for anti-flagellin antibodies appeared to determine the type of immune response which predominated. High affinity antigen produced antibody formation whereas low affinity antigen induced cell-mediated immunity and antibody tolerance. The importance of affinity was further evidenced by the fact that a CNBr digest of flagellin induced humoral and cellular immune responses identical to an acetoacetylated flagellin of comparable antigenic activity. From these studies it was proposed that both humoral and cell-mediated immunity can be directed against the same antigenic determinants but that the specificity requirements for delayed hypersensitivity (and antibody tolerance) are less than those required for antibody formation. Some remarkable immunological features of the flagellin system were revealed. Flagellin induced comparable delayed-type hypersensitivity when injected in either saline or FCA. Furthermore, FCA only slightly enhanced the delayed responses induced by the acetoacetyl flagellins and in fact these preparations produced antibody tolerance whether injected in saline or adjuvant. Finally, in contrast to the adult tolerance induced by the acetoacetylated flagellins, which existed only at the antibody level, tolerance in neonatal rats existed at the level of both humoral and cell-mediated immunity. This finding is the first indication of a fundamental difference between neonatal and adult tolerance. The significance of these findings is discussed in the light of current immunological concepts and a hypothesis proposed to explain these phenomena.

Variation in the primary structure of Bacillus subtilis flagellins.

The flagella derived from 18 strains Bacillus subtilis were tested for their reaction with antiflagellar filament antibody and antiflagellin antibody. On the basis of their reactivity, at least five serologically distinct classes could be identified. Peptide map analysis of tryptic digests of the subunit proteins were consistent with the immunochemical analysis. Large differences in sequence existed among proteins of the different classes; proteins within an antigenic group differed by only a few peptides. Furthermore, 9 of the 27 tryptic peptides resolved were common to flagellin proteins from all the classes examined. The relationship between antigenic specificity, variability in peptide pattern, and the conformation of the flagellin protein are discussed.