Determining Cellular and Molecular Targets of The Yersinia Tyrosine Phosphatase YopH During Animal Infection.

摘要

The gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion system that is required to translocate proteins essential for virulence, called Yops (Yersinia outer proteins), into host cells. Yops are efficiently translocated to a wide variety of cells grown in culture and most interact with many different mammalian proteins in different cell types; however their cellular and molecular targets in animal infection were unknown. Using a fluorescence-based assay that employs a membrane permeable dye, CCF2-AM, that is a substrate for a YopH-beta-lactamase fusion protein, I identified the types of cell targeted by Yersinia by FACS analysis. Professional phagocytes, i.e. neutrophils, macrophages and dendritic cells, were selectively targeted by Yersinia for translocation of Yops in the Peyer's patches, mesenteric lymph nodes and spleen after oral-gastric inoculation. In the absence of these cells, only low levels of Yop translocation were detected indicating that during infection Yersinia specifically chooses these cells. Interestingly, when the architecture of the spleen was destroyed, Yersinia retained its preference for translocation into professional phagocytes compared to B and T cells from splenocytes at low multiplicity of infection (MOI) but not at high MOI. In addition, Yersinia bound preferentially to professional phagocytes versus B and T cells from splenocytes and used two adhesins, YadA and Invasin, to enhance its association with all splenocytes. Together these results indicate that Yersinia discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.;After identifying neutrophils as a significant cell target of Yersinia, I determined a molecular target of YopH in neutrophils during murine infection. YopH is a tyrosine phosphatase that inhibits diverse cellular functions such as phagocytosis and ROS production in tissue culture models. To identify the molecular target(s) of YopH during mouse infection, neutrophils containing translocated with Yops from mice infected with wild-type Yersinia or yopH Yersinia were collected and analyzed using an anti-phospho tyrosine antibody by Western blot. SLP-76 was dephosphorylated in the presence of YopH indicating that YopH interferes with signal-transduction in Slp-76 controlled pathway(s) in neutrophils. Changes in Syk phosphorylation, an upstream activator of Slp-76 were not detected suggesting that Slp-76 was a direct target of YopH. We also show that neutrophils translocated with YopH have reduced ability to flux calcium, which is required for ROS production, and that YopH translocation leads to a reduction of IL-10 production during infection. In general, phagocytes producing ROS have increased levels of secreted IL-10. Finally, a yopH mutant was better able to colonize spleens in mice lacking neutrophils indicating that YopH inactivates neutrophils in infection. Combined these data strongly suggest that Slp-76 is a molecular target of YopH and its function in neutrophils is required to advert the immune system during animal infection. To our knowledge, we have identified for first time cellular targets of Type III secretion during murine infection.