Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
First Committee Member
Gregory V. Plano, Committee Chair
Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental 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, I 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 with TyeA. The YopN mutants did not seem to affect the structural integrity of the T3S apparatus and required an intact TyeA-binding domain and TyeA to block secretion. However, the ability of these mutants to block Yop secretion constitutively did not depend on their N-terminal secretion signal, their intact chaperone-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 hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.
Biology, Microbiology; Chemistry, Biochemistry
Ferracci, Franco, "Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion" (2005). Dissertations from ProQuest. 2264.