Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

Abstract

In patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA), synovial fluid mononuclear cells (SFMC) show proliferation to bacterial antigens that trigger ReA, i.e. Chlamydia, Yersinia, Campylobactor, Shigella and Salmonella species. We have shown previously that SFMC proliferate significantly to outer membrane proteins of S typhimurium in Salmonella induced ReA. In the present study we characterized the immunoreactive fractions of outer membrane protein (Omp) of S typhimurium in Salmonella induced ReA. Omp of Salmonella was isolated and fractionated by continuous elution sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using Prep-Cell into eight Omp fractions based on molecular weight. Twenty-three patients with ReA were screened for the bacterial trigger using the SFMC proliferative response to crude lysates of Y enterocolitica, S flexneri, C jejuni and S typhimurium using thymidine uptake assay. SFMC from patients with salmonella induced ReA were tested against eight fractions. Seven of 23 patients with ReA had S typhimurium-induced ReA. Of these seven patients, five patients SFMC had a significant stimulation index (SI) against < 22, 22-26, 25-35 and 28-40 kDa fractions of Omp. These fractions were analysed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which revealed 10 proteins. These proteins were 37 kDa OmpA, 33 kDa TsX, 28 kDa putative Omp, 28 kDa Vac J, 39 kDa OmpD, 18 kDa OmpX, 23 kDa OmpW, 43 kDa OmpS1 and 19 kDa peptidoglycan-associated lipoprotein. In conclusion, for the first time we have identified some low molecular weight proteins in the Omps of Salmonella which are T cells immunoreactive in patients with salmonella induced ReA/uSpA.

Fig. 1

Protein profile (sodium dodecyl sulphate-polyacrylamide gel electrophoresis, 10% gel, Coomassie stain) of the eight repooled Prep-Cell fractions (I–VIII) after SDS removal. MWM lane: molecular weight marker (in kDa); Omp lane: whole outer membrane proteins fraction. Molecular weight range and finally recovered amount of proteins of each fraction in the bottom of figure.

Fig. 2

Human synovial fluid mononuclear cells response to fractions of Omp. Synovial fluid mononuclear cells of Salmonella induced reactive arthritis patients (n = 7) were stimulated in vitro with I–VIII antigenic fractions of Omp of S typhimurium. Each graph represents one individual. Stimulation index (SI) of ≥ 2·5 was taken as a positive response and is indicated by a horizontal dotted line.

Fig. 3

Protein profile (sodium dodecyl sulphate-polyacrylamide gel electrophoresis, 15% gel, Coomassie stain) of the four immunodominant Omp fractions (I–IV). Each protein band (marked by numbered arrow) from every fraction was picked and processed for protein identification. MWM lane: molecular weight marker (in kDa), Omp lane: whole outer membrane proteins fraction.