Abstract
Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.
Highlights
Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival
IRGM and GKS proteins differ in their subcellular localization: IRGM proteins associate with endomembranes, whereas monomeric GDP-bound GKS proteins predominantly reside within the host cell cytoplasm [4, 17, 21, 22]
PVs containing the type II T. gondii strain Prugniaud A7 (Pru) became ubiquitin-labeled upon IFNγ priming in both mouse embryonic fibroblasts (MEFs) (> 20% Ub-positive PVs) and primary bone marrow-derived macrophages (BMMs) (Fig. 1B and Fig. S1)
Summary
In IRGMdeficient cells, GKS proteins enter the active state prematurely, form protein aggregates, mislocalize, and fail to bind to PVs [17, 21]. These previous observations help explain how IRGM proteins promote the delivery of GKS proteins to PVs, IRGM proteins control the subcellular localization of GBPs through an uncharacterized mechanism [6, 17, 24,25,26]. Experimental removal of the IFNγ-inducible ubiquitination pathway dramatically diminishes the p62-dependent delivery of GBPs to PVs and thereby renders host cells more susceptible to infections. Our observations imply that ubiquitin serves as a host-induced pattern that marks intracellular structures as immune targets for members of the GBP family of host defense proteins
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