Invasion bySalmonella typhimuriumInduces Increased Expression of the LMP, MECL, and PA28 Proteasome Genes and Changes in the Peptide Repertoire of HLA-B27

Abstract

We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion withSalmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimuriuminduced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28α and -β genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Infection with Salmonella typhimurium has no effect on the composition and cleavage specificity of the 20S proteasome in human lymphoid cells

“…These results are in contrast with a previous report 15 in which infection of HeLa cells with S. typhimurium increased the reverse transcription‐polymerase chain reaction values of the β 1 i, β 2 i and β 5 i subunits, and the amount of β 1 i protein as detected by Western blot analysis, although in that study the expected changes in the mature proteasomes purified from infected cells were not analysed. These differences might be attributed to the different cell lines used in both studies, and to the different behaviour of the bacteria in both cell types, because S. typhimurium proliferates inside HeLa cells, but not, or very little, inside C1R cells.…”

“…It has been reported that infection of HeLa cells by S. typhimurium results in an increase of the inducible subunit b 1 i, and concomitant changes in the HLA-B27bound peptide repertoire. 15 Changes in the B27-bound peptide repertoire were also reported in L cells infected with Shigella flexneri. 16 In contrast, in the lymphoid cell line C1R, the HLA-B27-bound peptide repertoires from S. typhimurium-infected and non-infected cells are very similar.…”

“…It has been reported that infection of HeLa cells by S. typhimurium results in an increase of the inducible subunit β 1 i, and concomitant changes in the HLA‐B27‐bound peptide repertoire 15 . Changes in the B27‐bound peptide repertoire were also reported in L cells infected with Shigella flexneri 16 .…”

Downregulation of the proteasome subunits, transporter, and antigen presentation in hepatocellular carcinoma, and their restoration by interferon‐γ

“…All of the primer pairs were previously used for the successful detection of each of the genes. 16,17,[30][31][32][33] The sizes of the PCR products were confirmed to be identical to the predicted sizes ( Table 1) by 1.2% agarose gel electrophoresis. The amplification program used was: preheating (95 ° C, 9 min); 35 cycles (95 ° C, 1 min; 55 ° C, 1 min; 72 ° C, 1.5 min); last extension (72 ° C, 10 min).…”

“…The sequences of PCR primers used here are shown in Table 1. All of the primer pairs were previously used for the successful detection of each of the genes 16,17 , 30–33 . The sizes of the PCR products were confirmed to be identical to the predicted sizes (Table 1) by 1.2% agarose gel electrophoresis.…”