Identification and characterization of Haemophilus parainfluenzae antigens associated with IgA nephropahty

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Although IgA nephropathy is the most frequent nephritis that has poor prognosis in Japan, etiology of this disease is still unknown. Since the early report describing the association of IgA nephropathy and Haemophilus influenzae, we have started investigation to identify the antigen (s) of H.parainfluenzae. In previous studies, we successfully isolated the novel gene, named hpnA, as a candidate that cause IgA nephropathy. Those studies also indicated the existence of antgens other than hpnA. Therefore, we designed current experiments to find those antigens. Six strains of H.parainfluenzae isolated irom patients with IgA nephropathy were utilized. Outer membrane fractions were prepared by differential centrifugation, and separated by SDS-PAGE. For immunoblot analysis, sera from patients or healthy volunteers were used for the first antibodies, and bands only visible with patients' sera were excised from membrane. After trypsinization, samples were applied onto mass spectrometer. Proteins in samples were determined by comparison towards protein database. Results of these experiments indicated that OMP and P6 were newly identified In H.parainfluenzae, nucleotide sequence for P6 has been determined, we synthesized recombinant OMP that has histidine-tag at N-terminus, and applied it to immunoblot analysis. We found the existence of IgA antibodies against P6 in many patients with IgA nephropathy, indicating the possible involvement of P6 in the pathogenesis of this disorder.