Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Absence of autoantibodies to peptides shared by HLA-B27.5 and Klebsiella pneumoniae nitrogenase in serum samples from HLA-B27 positive patients with ankylosing spondylitis and Reiter's syndrome.

Some microorganisms which are pathogenic in humans share amino acid sequences with human proteins (molecular mimicry). It has been suggested that molecular mimicry might be a reason for autoimmunity as a result of immunological cross reactivity. A homologous sequence of six amino acids has been found in both Klebsiella pneumoniae nitrogenase and the HLA-B27.5 molecule. In addition, (auto)antibodies to a synthetic peptide that contained the HLA-B27.5/klebsiella mimicking epitope have been detected in serum samples from HLA-B27 positive patients with ankylosing spondylitis and Reiter's syndrome. Confirmation of these data is important, because ankylosing spondylitis and Reiter's syndrome have so far been assumed to be 'seronegative' rheumatic diseases. It was, however, not possible to confirm the presence of autoantibodies against the mimicking peptide in serum samples from patients with ankylosing spondylitis and Reiter's syndrome. Serum samples from 81 patients with ankylosing spondylitis, 38 patients with Reiter's syndrome, and 81 healthy blood donors were tested against the 'mimicking peptide' in an enzyme linked immunosorbent assay (ELISA). Some of the serum samples from patients showed high but non-specific binding to the mimicking peptide. A highly significant correlation between binding to plastic coated with the mimicking peptide, to plastic coated with an irrelevant peptide, and even to non-coated plastic was observed. The nature of the serum component(s) in these patient serum samples (and some control serum samples) responsible for the high non-specific binding to plastic remains unclear. It was also shown that antibodies to the HLA-B27 peptide (containing the mimicking epitope) induced in rabbits do not cross react with the klebsiella peptide and vice versa.

Antibody activity in ankylosing spondylitis sera to two sites on HLA B27.1 at the MHC groove region (within sequence 65-85), and to a Klebsiella pneumoniae nitrogenase reductase peptide (within sequence 181-199).

74 overlapping peptides of varying lengths from Klebsiella pneumoniae nitrogenase reductase (residues 181-199) and from the HLA B27.1 molecule (residues 65-85) were synthesized and tested by ELISA against sera from HLA B27+ ankylosing spondylitis (AS) patients, and sera from HLA B27+ and HLA B27- healthy first-degree relatives. Antibody activity in AS sera to Klebsiella peptides of four to eight amino acids was maximal with the peptide NSRQTDR. Activity to HLA B27 peptides was maximal with the peptide KAKAQTDR (named epitope I). These peptides overlap with, but are proximal to the NH2 terminus from QTDRED, which is homologous in HLA B27.1 and K. pneumoniae nitrogenase reductase. A second weaker reactive site was noted in the HLA B27.1 peptides, proximal to the COOH terminus from the homologous sequence, namely peptide REDLRTLL (named epitope II). Little activity was seen against peptides that included the entire homologous sequence. Sera from 50 AS patients showed higher total Ig activity against peptides KAKAQTDR (p less than 0.001) and NSRQTDR (p less than 0.02) than did sera from 22 B27+ and 22 B27- healthy controls. These data indicate that AS patient sera contain antibodies that bind to K. pneumoniae nitrogenase peptides and HLA B27.1 peptides, and that there are at least two epitopes on HLA B27.1 in the alpha 1 domain, at the MHC groove region, that are autoantigenic in AS patients. Epitope I may be a site for crossreactivity between HLA B27 and Klebsiella.

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.

Studies of humoral and cell-mediated immunity to peptides shared by HLA-27.1 and Klebsiella pneumoniae nitrogenase in ankylosing spondylitis.

One-hundred and twenty-four patients with spondylarthropathies were studied for antibodies to the peptides from HLA-B27.1 and Klebsiella pneumoniae nitrogenase which share a QTDRED hexamer sequence. Of 60 male Norwegian ankylosing spondylitis (AS) patients 23.3% showed positive ELISA reactivity for B27.1 peptide compared with 4% of Norwegian male controls (P less than 0.10). This difference was not observed among patients and controls from New Mexico. All patients with anti-B27.1 antibody were HLA-B27+. Antibody to B27.1 peptide was present in 20% of normal female controls with at least one previous pregnancy. No female control without previous pregnancy showed positive anti-B27.1 peptide reactivity. Anti-Klebsiella peptide antibody was neither significantly elevated in AS nor correlated with anti-B27.1 peptide antibody. Significant migration inhibition by these peptides was not observed in AS or normal controls. The possible influence of epitope conformation, rather than sequence homology, in potentially cross-reacting determinants shared by bacterial antigens and human Class I molecules requires further study.

Cross-reactive epitope with Klebsiella pneumoniae nitrogenase in articular tissue of HLA-B27+ patients with ankylosing spondylitis.

Synovial tissues from patients with ankylosing spondylitis or reactive arthritis were examined by an immunoperoxidase technique, using antisera to synthetic peptides representing antigens shared between HLA-B27.1 and Klebsiella pneumoniae nitrogenase. With either antiserum, all HLA-B27+ patients with synovial inflammation showed strong immunoperoxidase staining in synovial lining cells, vascular endothelium, and infiltrating inflammatory cells. These findings indicate that antigens showing cross-reactivity between HLA-B27.1 and Klebsiella nitrogenase epitopes are strongly expressed within inflamed synovial tissues of HLA-B27+ patients.

Molecular mimicry between human leukocyte antigen B27 and Klebsiella. Consequences for spondyloarthropathies.

Ankylosing spondylitis and Reiter's syndrome are the two major spondyloarthropathies highly associated with human leukocyte antigen (HLA) B27. Although the development of spondylitis is unclear, it has been hypothesized that HLA-B27 may predispose to spondyloarthropathies via the phenomenon of molecular mimicry. A computer search for homologies between HLA-B27 and microbes revealed a sequence of six consecutive amino acids (glutamine-threonine-aspartic acid-arginine-glutamic acid-aspartic acid) shared by HLA-B27.1 (residues 72 to 77), and Klebsiella pneumoniae nitrogenase (residues 188 to 193). Antibodies raised against a peptide derived from HLA-B27 containing this six-amino-acid sequence cross-reacted with the peptide derived from Klebsiella that contained these six amino acids, and vice-versa. These antibodies also reacted with articular tissues from HLA-B27-positive patients with ankylosing spondylitis. Sera from 53 percent of Reiter's patients and 27 percent of patients with ankylosing spondylitis showed binding to these same peptides. These results suggest that molecular mimicry may have a role in disease development.

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli.

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.

Comparison of active and inactive forms of iron protein from Rhodospirillum rubrum.

The Fe protein of nitrogenase from Rhodospirillum rubrum was purified in its active and inactive forms. It is shown that the inactive form exists as a two-subunit modified form of the enzyme as previously reported [Ludden & Burris (1978) Biochem. J. 175, 251--259]. In contrast, the active form exists as a single-subunit unmodified form of the enzyme. The upper subunit (on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) of the inactive form was shown to contain at least the phosphate group of the covalently bound modifying group. The active and inactive forms of the enzyme were shown to be identical proteins on the basis of amino-acid composition, tryptic-digest pattern and immunological cross-reactivity.

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component.

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the Mo-Fe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-reaction of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified Mo-Fe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the Mo-Fe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in 270-fold enhancement of activity indication the presence of active Mo-Fe protein in these extracts. Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the Mo-Fe component of nitrogenase is carried by Rhizobium.