Flagellin Fragment Research Articles

Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes.

Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Enzyme-linked immunosorbent assay using recombinant OspC and the internal 14-kDa flagellin fragment for serodiagnosis of early Lyme disease.

The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen. According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto.

The intrathecal synthesis of borrelial-specific IgM- and IgG-antibodies was studied in 67 patients with neuroborreliosis and in 14 patients with neurosyphilis (controls). Antibody concentrations in serum and in the cerebrospinal fluid were determined by an enzyme immunoassay (EIA) using, as antigens, a sonicate of Borrelia burgdorferi, the recombinant 14 kDa flagellin fragment, the outer surface protein C (22 kDa), and the high molecular mass protein p83 (83 kDa). In the sonicate EIA, IgG- and/or IgM-antibodies to Borrelia burgdorferi in serum were detected in all patients with neuroborreliosis and in 71% of patients with neurosyphilis. Intrathecal synthesis of borrelial-specific IgG- and/or IgM-antibodies was demonstrated in 82% of patients with neuroborreliosis and in 71% of patients with neurosyphilis. Immunoglobulin G- and/or IgM-antibodies in serum against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in none of those with neurosyphilis. Intrathecal synthesis of IgG- and/or IgM-antibodies to individual recombinant antigens was demonstrated in 67% of patients with neuroborreliosis and in none of those with neurosyphilis. The sensitivity of the recombinant antigens in serum was almost equal to that of the sonicate EIA, whereas the recombinant antigens were clearly less sensitive in the estimation of the intrathecal specific immune response. It was concluded that in suspected cases of neuroborreliosis, the estimation of high specific antibodies in the recombinant EIA will be helpful in confirming the diagnosis.

Radial Mass Analysis of the Flagellar Filament of Salmonella: Implications for the Subunit Folding

X-ray fiber diffraction patterns of the R-type straight flagellar filament of Salmonella typhimuriumSJW1655 strain showed layer-lines with an axial spacing of 1/437 Å, which could be resolved only due to very small disorientation angles (<2 °) of the filaments in oriented sol specimens. Although the equatorial layer-line was situated between the relatively strong first layer-lines right above and below it, these small disorientation angles and a new method of two dimensional angular deconvolution allowed us to determine the equatorial layer-line intensities quite accurately. The equatorial data were phased by using the amplitude difference between the native flagellar filament and its heavy atom derivatives. One of the heavy-atom derivatives was prepared by introducing a cysteine residue by site-directed mutagenesis and applying a mercury compound. From the equatorial structure factors, the radial density distribution of the filament was calculated at 11 Å resolution. A prominent feature was two pairs of high density peaks at radii of around 25 and 45 Å and a deep density trough between them, which corresponds to the concentric double tubular structure in the core region that has been found in the density map recently deduced by helical image reconstruction from electron micrographs of frozen hydrated filaments. The molecular masses were estimated for four radial segments that correspond to the morphological domains identified in the map of helical image reconstruction. Then the domains were assigned to sequence positions by correlating the estimated masses with those of proteolytic fragments of flagellin. The assignment is consistent with the distributions of secondary structures and in particular α-helical coiled-coils that were predicted from the sequence. It also helps to understand how the polymerization behaviour is affected by truncation of the disordered terminal regions of flagellin and why mutations in a specific region are responsible for changes in the polymorphic shape of the filament.>

Isotype, Specificity, and Kinetics of Systemic and Mucosal Antibodies to Campylobacter jejuni Antigens, Including Flagellin, during Experimental Oral Infections of Chickens

The immune response of chickens to Campylobacter jejuni infection was studied as a step in the search for vaccine candidates. One-day-old chicks orally challenged with C. jejuni strain 81116 showed significant increases in specific IgG, IgA, and IgM circulating antibodies, as detected by enzyme-linked immunosorbent assay (ELISA). These levels peaked at 9, 5, and 7 weeks postinfection, respectively. Maternal IgG antibodies were also detected over the first 2 weeks. Specific mucosal IgG and IgA antibody levels also increased significantly. All of the birds demonstrated a major response to the 62-kDa flagellin protein by Western blotting techniques. The immunodominance of flagellin was confirmed by ELISA using an antigen preparation from an aflagellate mutant. When overlapping recombinant polypeptide fragments of flagellin were used, epitopes detected by chicken antibodies were observed in region IV, between residues 95-340 of the protein. Thus flagellin may be suitable candidate for a vaccine, although its role in protection must first be established.

Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato.

Immunodominant proteins are variable in molecular and antigenic structure among different genospecies of Borrelia burgdorferi sensu lato. We have recently developed an immunoblot using five recombinant antigens: the chromosomal-encoded B. burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof, and the plasmid-encoded outersurface proteins A (OspA) and C (OspC). In the present study the same antigens (derived from strain PKo, genospecies B. afzelii) were compared with the homologous recombinant proteins from strain B31 (genospecies B. burgdorferi sensu stricto) and with OspA, OspC and the internal flagellin fragment from strain PBi (genospecies B. garinii). Patients with neuroborreliosis (n = 28) and patients with acrodermatitis chronica atrophicans (n = 20) were investigated in the IgG immunoblot; the IgM immunoblot was performed only in patients with neuroborreliosis. There was a small increase in the detection rate of OspA-specific IgG or IgM antibodies using the different variants of recombinant OspA; however, OspA remained an insensitive antigen for antibody detection in Lyme borreliosis. The same was true to OspC-specific IgG antibodies. The sensitivity of OspC, which is the immunodominant antigen for IgM antibody detection, could not be increased using recombinant antigens derived from different strains. However, some sera which were negative in the recombinant immunoblot reacted with OspC in the conventional immunoblot using B. burgdorferi whole cell lysate as antigen. The most unexpected finding was the high degree of immunological heterogeneity of the internal flagellin fragments: IgG antibodies were detected in 18 of 48 patients using B31 fragments, in 25 of 48 using PKo fragments, in 23 of 48 using PBi fragments versus 33 of 48 when the three recombinant proteins were combined. PKo-derived fragments were more sensitive for antibody detection in patients with acrodermatitis chronica atrophicans, B31- and PBi-derived fragments for antibody detection in patients with neuroborreliosis. This is in agreement with the fact that isolates from patients with neuroborreliosis are predominantly belonging to the genospecies B. burgdorferi sensu stricto and B. garinii. For detection of IgM antibodies in sera from patients with neuroborreliosis, recombinant internal fragments derived from strains B31 and PBi were more sensitive than the PKo-derived fragment. The best discrimination between neuroborreliosis sera and control sera was achieved when the IgM blot was performed using recombinant internal flagellin fragments derived from strains PKo and PBi and OspC derived from B31 or PKo.

Recombinant immunoblot in the serodiagnosis of Lyme borreliosis

A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encoded Borrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans, n = 31; neuroborreliosis state II, n = 60; Lyme arthritis, n = 24 and acrodermatitis chronica atrophicans, n = 19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.

Mobility of the terminal regions of flagellin in solution.

The mobility of the disordered terminal regions of flagellin was examined in detail based on 1H NMR chemical shifts and spin-lattice relaxation times in the rotating frame. Proteolytic fragments of flagellin with terminal deletions of different sizes were used to compare the dynamical properties of various N- and C-terminal segments. We found that dynamic properties of different terminal segments were similar to each other and were close to those of the heat-denatured state of flagellin. The main chain of these terminal segments undergoes rapid motions with effective correlation times of 1.3-4.1 x 10(-9) s. The terminal regions contain no large segments with well-defined structure. However, comparison with the random-coiled state of poly-L-lysine suggests significant structural constraints in the terminal regions (as well as in the heat-denatured flagellin) which may reflect the existence of some highly fluctuating secondary structure, as suggested by earlier CD studies.

Role of the disordered terminal regions of flagellin in filament formation and stability

Terminal regions of flagellin from Salmonella typhimurium, residues 1 to 65 and 451 to 494, have no ordered tertiary structure in solution, which makes them very susceptible to proteolytic degradation. Flagellin was subjected to mild controlled proteolytic treatment with highly specific proteases to remove terminal segments from the disordered regions. It is demonstrated here that various fragments can be readily prepared that differ from each other in 1 × 10 3 to 2 × 10 3 M r segments in their NH 2- or COOH-terminal regions. Terminally deleted fragments of flagellin were used to clarify the role of the disordered regions in the self-assembly of flagellin. The polymerization ability of the fragments was tested by inducing filament formation with ammonium sulfate. We found that fragments of flagellin containing large terminal deletions could form straight filaments, although the stability of these filaments required high salt concentrations. Even a fragment lacking the whole mobile COOH-terminal part of flagellin and 36 residues from the NH 2-terminal region could form long filaments. The fragments could be also polymerized onto native flagellar seeds, suggesting that the subunit packing of the filaments of fragments is similar to that of the native ones. The fragments could also copolymerize with native flagellin, resulting in various helical forms. Filaments of fragments were found to be straight at both pH 4.0 and pH 12.5, indicating that they might have lost their polymorphic ability. Our results show that the major part of the disordered terminal regions of flagellin is not essential for polymerization, but it does play an important role in stabilization of the filaments and in influencing their polymorphic conformation.

Molecular characterization of the humoral response to the 41-kilodalton flagellar antigen of Borrelia burgdorferi, the Lyme disease agent

The earliest humoral response in patients infected with Borrelia burgdorferi, the agent of Lyme disease, is directed against the spirochete's 41-kDa flagellar antigen. In order to map the epitopes recognized on this antigen, 11 overlapping fragments spanning the flagellin gene were cloned by polymerase chain reaction and inserted into an Escherichia coli expression vector which directed their expression as fusion proteins containing glutathione S-transferase at the N terminus and a flagellin fragment at the C terminus. Affinity-purified fusion proteins were assayed for reactivity on Western blots (immunoblots) with sera from patients with late-stage Lyme disease. The same immunodominant domain was bound by sera from 17 of 18 patients. This domain (comprising amino acids 197 to 241) does not share significant homology with other bacterial flagellins and therefore may be useful in serological testing for Lyme disease.