Date of Award
Spring 4-7-2025
Document Type
Thesis (Ph.D.)
Department or Program
Microbiology and Immunology
First Advisor
James Bliska
Abstract
Bacteria utilize sophisticated secretion systems to inject effectors into host cells in order to facilitate their survival and replication in the host. Some pathogenic species of Gram-negative bacteria use a conserved contact-dependent T3SS to inject a wide array of effectors. While the effectors diverge in their structures, functions and cell-localization, the T3SS is well conserved. Several effectors discussed in this thesis target key host inflammasome responses. The focus of this work is how the Yersinia effector YopM inhibits the pyrin inflammasome. During Yersinia infection, two effectors, YopE and YopT inadvertently activate the pyrin inflammasome while targeting RhoA to avoid phagocytosis. Pyrin acts as a guard, sensing bacterial modifications to RhoA and assembles a caspase-1 inflammasome. When activated, pyrin is dephosphorylated and interacts with the inflammasome adaptor ASC through homotypic PYD-PYD interactions. However, YopM counteracts this YopE/T-triggered assembly of the pyrin inflammasome by hijacking host kinases RSK and PRK to phosphorylate pyrin, locking it in an inactive state. Here, I determined that YopM specifically binds to the N-terminal PYD of human pyrin using x-ray crystallography. I identified key residues in both YopM and the hPYD involved in their interactions. I also show that mutation of key residues on YopM abolishes its binding to the hPYD and inhibition of the human pyrin inflammasome in THP-1 cells. Interestingly, YopM does not bind to the mouse PYD and seems to target the mouse pyrin inflammasome using a different pathway. The YopM-hPYD complex presented here is the first, to my knowledge, of a virulence factor targeting a member of the DDF of proteins. I expand on current knowledge of protein-protein interactions of the PYD subfamily by identifying a role for electrostatic patches and key residues discussed herein. The complex structure identifies the central LRRs 5-6 on YopM as the key binding site for hPYD. I also provide insights on YopM by solving the structure of the 13-LRR YopMPestA isoform. Additional biochemical analyses of YopMPestA determined that YopM is monomeric under neutral pH conditions, and that low pH acts as a conformational switch, triggering tetramer formation.
Recommended Citation
Mwaura, Bethany Wairimu, "Biochemical and Structural Insights into the Yersinia Effector YopM and its Negative Regulation of the Pyrin Inflammasome" (2025). Dartmouth College Ph.D Dissertations. 391.
https://digitalcommons.dartmouth.edu/dissertations/391
