Major Histocompatibility Complex Class I Peptide Presentation afterSalmonella entericaSerovar Typhimurium Infection Assessed via Stable Isotope Tagging of the B27-Presented Peptide Repertoire

Abstract

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.

Proteasome-independent HLA-B27 Ligands Arise Mainly from Small Basic Proteins

“…This approach, which used techniques of quantitative expression proteomics (43) that were recently applied to identifying MHC ligands (44,45), was based on metabolic labeling of cellular proteins with [ 15 N]Arg (46). This method allows the labeling of virtually all HLA-B27 ligands because Arg at P2 is a nearly universal motif of B27-bound peptides (10,47).…”

Identification of Endogenously Presented Peptides from Chlamydia trachomatis with High Homology to Human Proteins and to a Natural Self-ligand of HLA-B27

“…Although it is unclear how and what chlamydial proteins may reach the processing-loading pathway of MHC class I molecules and to what extent they may be processed in the cytosol or other cell compartments, we specifically addressed the question of whether intracellular processing of the endogenously synthesized DNA primase might lead to generation of the P-(211-222) peptide and/or other HLA-B27 ligands with homology to human protein sequences. This approach was also undertaken because a direct mapping of HLA-B27-restricted chlamydial epitopes from infected cells by MS techniques is exceedingly difficult due to very low expression of bacterial epitopes on infected cells (19,20) and to downregulation of MHC class I expression induced by C. trachomatis on infected cells (6,7).…”

“…However, a critical test for the relevance of the DNA primasederived peptide was to determine whether this peptide, or a closely related one, was actually processed and presented in vivo by HLA-B27. A direct molecular approach aiming at directly mapping chlamydial peptides presented by HLA-B27 in infected cells by biochemical methods is hardly feasible due to the exceedingly low expression of bacterial antigens on these cells as reported, for instance, with Salmonella (19,20). In the case of Chlamydia, this approach is further complicated by the down-regulation of MHC class I expression induced by the bacteria shortly after infection (6,7).…”

Pathogenesis of ankylosing spondylitis and reactive arthritis

“…New analytical techniques have been applied to probing synovial fluids and tissue for evidence of prior or current microbes [52,53]. Invasion by Salmonella, however, did not alter the B27 peptide presentation profile [54]. Quantitative analysis of invasion of gram-negative bacteria into human synoviocytes did not correlate with the B27 status of the target cells, in contrast to prior studies using B27-transfected cells as targets [55].…”