Proximal and Distal Cell Autonomous Immune Responses to Intracellular Pathogens

Abstract

The cell-autonomous response describes any cell’s singular ability to protect itself against pathogens. This response is strongly regulated by the cytokine interferon gamma (IFN-γ) produced upon infection with various microbial pathogens. Engagement of IFN-γ with its receptor in any cell induces expression of hundreds of interferon-stimulated-genes (ISG) that orchestrate antimicrobial responses through various modes of action. ISG mode of action can be characterized as having proximal functions and distal functions. Proximal functions describe the requirement for an ISG to come in close contact with a given pathogen to fulfill its mode of action, while distal functions describe the ability to promote antimicrobial activities without the need for close association. In this dissertation, I explore novel and established proximal and distal functions for various ISGs. Specifically, I described the novel antiparasitic function of RNF213 against T. gondii by directly targeting the parasitophorous vacuole for destruction, and GBPs distal role in modulating immune signaling pathways during infection with cytosolic pathogens. IFN-γ-inducible antiparasitic defense programs comprise nutritional immunity, the production of noxious gases, and the ubiquitylation of the Toxoplasma-containing parasitophorous vacuole (PV). PV ubiquitylation prompts the recruitment of host defense proteins to the PV and the consequential execution of antimicrobial effector programs, which reduce parasitic burden. However, the ubiquitin E3 ligase orchestrating these events has remained unknown. Here I demonstrate that the IFN-γ-inducible E3 ligase RNF213 translocates to Toxoplasma PVs and facilitates PV ubiquitylation in human cells. Toxoplasma PVs become decorated with linear and K63-linked ubiquitin and recruit ubiquitin adaptor proteins in a process that is RNF213 dependent but independent of the linear ubiquitin chain assembly complex (LUBAC). IFN-γ priming fails to restrict Toxoplasma growth in cells lacking RNF213 expression, thus identifying RNF213 as a potent executioner of ubiquitylation-driven antiparasitic host defense. IFN-γ can also induce expression of a subset of dynamin-like GTPases named p65 guanylate-binding proteins (GBPs). The antimicrobial function of GBPs has been extensively studied as having proximal functions given their ability to associate with the lipid membranes of vacuolar pathogens and cytosolic bacteria. GBPs, however, can modulate LPS-induced pyroptosis, an inflammatory form of cell death, and this is independent of direct association with gram-negative bacteria. Here I also demonstrate that GBPs can also modulate the type I interferon response during infection with cytosolic gram-positive bacteria which are not directly targeted by GBPs. The work described in this thesis demonstrates the ability of IFN-γ-induced ISGs to promote a multitude of antimicrobial functions to intracellular pathogens. It highlights the importance and requirement for the cell-autonomous response during containment of pathogens and proper execution of the innate-immune response during infection.