B-224. Macrophages, Dendritic Cells and Neutrophils Are the Primarily Targets of YopH During Oral Infection with Yersinia pseudotuberculosis

E. A. Durand, C. Castillo, J. Mecsas;
Tufts Univ., Boston, MA.

The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system to translocate effectors, called Yops, into mammalian cells. Translocation of Yops is essential for virulence. However, the cells targeted by Y. pseudotuberculosis are not known. We identified the host cells in the Peyer’s patches (PP), mesenteric lymph nodes (MLN), and spleen targeted for injection of YopH during oral-gastric route of infection. Using a fluorescence-based assay that employs a membrane permeable dye, CCF2, that is a substrate for a YopH-TEM fusion protein, we identified the cell types targeted by YopH-TEM by labeling infected host cells with antibodies specific for various cell markers. Five days post-infection we observed that levels of neutrophils and macrophages increased by 5 and 10 in PP, MLN and spleen. In all tissues the levels of B and T cells remained constant. YopH preferentially targeted macrophages, neutrophils and dendritic cells in the spleen, PP and MLN. In contrast, when spleen cells were harvested and then infected with Yersinia, all cell types were equally susceptible to injection of YopH at high MOI. Interestingly, at low MOI, macrophages and neutrophils were also preferentially target ex vivo. Furthermore, this preferential targeting was not dependent on the phagocytic activity of host cells as treatment of host cell with cytochalasin D did not affect preferential binding of these cell types. Histological analysis indicates that microcolonies localized in the spleen primarily at the site of infiltration of neutrophils, macrophages. We also tested whether other Yops were translocated into the same cells that have been injected with YopH-TEM (blue cells) or into non-targeted cells (green cells). We found that YopE is in the same cells injected with YopH-TEM. We conclude that neutrophils, macrophages and dendritic cells are preferentially targeted due to ligand-receptor interactions that confer binding specificity to these cells and this targeting may be enhanced because bacteria co-localize with these cells. Furthermore, several Yops are translocated into the same cells in vivo suggesting that Yops are expressed and translocated coordinately.