FIG. 1.

(A) Scheme of translational protein fusions of YopE with the L. monocytogenes-derived CD4 and CD8 target antigen LLO. C-terminal ends of these hybrid proteins were M45 epitope tagged, and they are encoded by the designated plasmids. Both plasmids bear the genetic information for the chaperone SycE. Transcription of vector-borne genes and gene fusions was achieved under the control of the wild-type yopE/sycE promoters. (B) Translocation of hybrid YopE/LLO proteins into macrophage-like cells. P388D₁ cells were infected with wild-type Y. pseudotuberculosis pIB102 or the isogenic yopK-null mutant strain pIB155 carrying the indicated plasmid. The presence of chimeric YopE in different fractions was examined as described in Materials and Methods. Lanes 1, whole-cell lysate of non-cell-associated bacteria; lanes 2, bacterium-free infection medium; lanes 3, Triton X-100-soluble P388D₁ cell lysate containing translocated proteins. The total protein amounts obtained from all three fractions were loaded. Hybrid YopE proteins were detected by protein immunoblotting with a MAb to M45.