ANALYSIS OF PROTEIN MODIFICATION USING EASTERN BLOTTING TECHNIQUE IN EHRLICHIA MAY EXPLAIN THE IMMUNE RESPONSES IN AN ANIMAL MODEL OF EHRLICHIOSIS

Sunil Thomas, E.Crossley, N.Thirumalapura, Ph.D, and D. H. Walker, M.D.

Dept. of Pathology, Keiller Building, UTMB, Galveston, TX-77550

Introduction: Ehrlichia muris and Ixodes ovatus ehrlichia (IOE) are genetically related Ehrlichia species used in mouse models of ehrlichial disease. Though E. muris is mildly virulent, the IOE pathogen causes severe disease in mice. To develop effective vaccination strategies against Ehrlichia, we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed with E. muris are resistant to lethal infection with IOE. Objective: To determine antigenic variations in two strains of closely related Ehrlichia species – E. muris and IOE. Methods: We employed a novel technique, Eastern blotting to detect posttranslational protein modifications. Results: Cross reactions of antigenic proteins were observed when we probed both E. muris and IOE cell lysates with IOE- and E. muris- specific polyclonal antibody. Analysis of the total proteins of E. muris and IOE by two dimensional gel electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Eastern blotting showed that E. muris proteins are more lipoylated and glycosylated than those of IOE. Analysis of T cell costimulators showed greater expression of CD28, OX40 and 4-1BB in both CD4 and CD8 T cells in the E. muris model compared to the IOE model or uninfected controls. Conclusions: E. muris antigenic proteins are more posttranslationally modified than those of IOE. This research was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI031431).