Graves' disease (GD) is an autoimmune disorder characterized by hyperthyroidism which is mediated by autoantibodies to the thyrotropin receptor (TSHR). Although much is known about the pathophysiology of GD, little is known about the mechanism(s) involved in the events that trigger the production of the autoantibodies. Yersinia enterocolitica is a bacteria that has been postulated to play a role in the induction of GD via molecular mimicry. We have shown that mice challenged with Yersinia enterocolitica or low molecular weight envelope proteins from bacteria develop antibodies that recognize epitopes present on the extracellular domain of human TSHR (ETSHR). Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of autoantibodies capable of reacting with TSHR. Recently, we have extended these findings by purifying and characterizing, for the first time, the Yersinia envelope proteins that are crossreactive with the ETSHR. Interestingly, these crossreactive proteins are chromosomally encoded and mitogenic for mouse B-lymphocytes. The overall goal of this proposal is to investigate the role Y. enterocolitica plays in the induction of GD and to further characterize the crossreactive proteins which are also B-cell mitogens.
The specific aims of the proposed studies are: 1) To clone, express, purify and characterize the Yersinia-derived protein(s) which induce antibodies that recognize the ETSHR. 2) To identify ETSHR epitopes recognized by antibody generated to envelope proteins of Yersinia. 3) To determine if mouse T lymphocytes which recognize envelope proteins of Yersinia can also recognize the ETSHR. 4) To determine if mice immunized with Yersinia or envelope proteins of Yersinia develop altered thyroid/pituitary functions. We will purify the envelope protein(s) of Yersinia that crossreact with ETSHR using HPLC. Sequence data obtained from purified protein(s) will be used for cloning the ETSHR crossreactive envelope protein(s), and we will overexpress the cloned gene(s) using an E. coli expression system. Using a panel of overlapping synthetic peptides that span the entire ETSHR and peptide fragments of ETSHR, we will attempt to map crossreactive epitopes present on ETSHR and Yersinia envelope protein(s). In vitro proliferative responses of T cells generated to ETSHR versus Yersinia envelope proteins will be used for determining T cell recognition of crossreactive epitopes. Finally, we will challenge mice with ETSHR, crossreactive envelope protein(s) of Yersinia, or Yersinia bacteria and assess for altered thyroid functions by: 1) assessing serum levels of thyroxine, 2) measuring in vivo 131I uptake by thyroids, 3) determining the presence of serum antibody to ETSHR, and 4) carrying out pathological evaluations of thyroid glands. Studies proposed in this application will directly test the long held belief that Yersinia infection plays a role in the induction of GD by molecular mimicry and will characterize envelope protein(s) of Yersinia which are mitogenic and could play a role in Yersinia virulence and/or pathogenesis.
