Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Summary Secretion of Yop effector proteins by the Yersinia pes- tis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environ- mental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 ∞ C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-bind- ing domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chap- erone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothet- ical model for how the YopN/SycN/YscB/TyeA com- plex regulates Yop secretion is presented.