Manipulation of the mTOR signaling pathway by the Shigella effector protein OspB

Massachusetts General Hospital, Boston MA

Investigators

Abstract

Project Summary/Abstract Shigella spp. and other pathogens target host cell signaling pathways to create an environment conducive to survival and replication. Identifying these signaling pathways and how they are manipulated by pathogens is essential for understanding pathogenesis. Gram-negative bacterial pathogens such as Shigella deliver effector proteins into host cells that modify the cellular environment. OspB is an effector introduced into host cells by the Shigella type 3 secretion system, a protein apparatus that delivers effectors directly from Shigella into the host cell cytoplasm. Our lab recently found that OspB activates the mTORC1 signaling pathway and sensitizes cells to rapamycin inhibition of mTORC1, the master regulator of cell growth. How OspB activates mTORC1 is unknown. I found that OspB is predicted to be structurally homologous to the cysteine protease domain of a family of toxins that includes RtxA from Vibrio cholerae and TcdB from Clostridium difficile. The putative catalytic sites and substrate binding pocket residues of RtxA are conserved in OspB. We found that expression of OspB in the yeast Saccharomyces cerevisiae induces sensitivity of yeast cells to caffeine and rapamycin and that the putative catalytic cysteine and histidine are required to induce sensitivity, suggesting that OspB function is conserved across divergent branches of life, that the putative catalytic site cysteine and histidine are required for OspB enzymatic activity, and that the target(s) of OspB may be the same in human and yeast cells. Based on these results, I hypothesize that OspB is a cysteine protease and that OspB enzymatic activity modifies mTORC1 and/or an associated protein(s) in a manner that leads to its activation and to sensitization to rapamycin inhibition of mTORC1. The proposed project is designed to determine how OspB alters the mTORC1 signaling pathway. I will (1) test its function as a cysteine protease using activity-based probes and in vivo assays in mammalian cells and (2) define host factors required for OspB function using biochemical and genetic approaches. Identification of a mechanism for OspB function would guide our understanding of other bacterial proteases and provide insights into fundamental mechanisms of mTORC1 regulation.