Antigenic protein modifications in Ehrlichia

Abstract

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 were resistant to lethal infection with Ixodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming with E. muris and later injected with IOE-detected antigenic proteins in E. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both the 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 electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed that E. muris proteins are more lipoylated and glycosylated than those of IOE.

Figure 2

Western blot of one dimensional gel electrophoresis probed with polyclonal antibodies against (a) E. muris/IOE (b) E. muris and (c) IOE (1 : 100). Five micrograms of cell lysate from supernatant of DH82 cell line, supernatant of DH82 cell line infected with E. muris, DH82 cell lysate and E. muris infected DH82 cell lysate were used in the study. Representative images based on three independent experiments.

Figure 1

Western blot of one dimensional gel electrophoresis probed with polyclonal antibodies against (a) E. muris/IOE (b) E. muris and (c) IOE (1 : 100). Five micrograms of cell lysate from mouse spleen, spleen infected with E. muris or IOE was used in the study. Representative images based on three independent experiments.

Figure 4

Western blot of two dimensional gels (E. muris infected spleen and IOE infected spleen) probed with polyclonal antibodies against (a) E. muris/IOE and (b) E. muris. Fifty micrograms of protein was used to run the first dimensional gel. The proteins of interest are marked in the image (rectangle).

Figure 3

Two dimensional gel of (a) uninfected spleen (b) spleen infected with E. muris, and (c) spleen infected with IOE stained with Coomassie blue. Fifty micrograms of protein was used to run the first dimensional gel. Representative images based on three independent experiments.

Figure 5

Two dimensional gels of (a) E. muris infected spleen and (b) IOE infected spleen probed with Cholera Toxin B subunit for the detection of lipids. Fifty micrograms of protein was used to run the first dimensional gel. Representative images based on three independent experiments. The proteins of interest are shown in boxes.

Figure 6

Two dimensional gels of (I) uninfected spleen (II) E. muris-infected spleen and (III) IOE infected spleen probed with (a) wheat germ agglutinin and (b) Con-A for the detection of glucose moieties. For the first dimension 50 µg protein was used to run gel. The protein of interest is marked in the image.

Figure 7

Two dimensional gels of (I) uninfected spleen (II) E. muris infected spleen and (III) IOE-infected spleen probed for phosphoproteins. Fifty micrograms of protein was used to run the first dimensional gel. The images are the representative of five independent experiments. The proteins of interest are marked in the images (rectangle).