Type III secretion system-dependent translocation of ectopically expressed Yop effectors into macrophages by intracellular Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.