Disruption of the Toxoplasma gondii Parasitophorous Vacuole by IFNγ-Inducible Immunity-Related GTPases (IRG Proteins) Triggers Necrotic Cell Death

Abstract

Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNγ-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

“…Growth restriction of T. gondii by IFNγ in infected cells (Zhao et al ., 2009b) is directly associated with inhibition of efficient IRG protein loading onto the PVM. We measured intracellular proliferation of T. gondii RHΔ gra7 in bone marrow‐derived macrophages (BMMs) and MEFs in vitro by incorporation of 3 H‐uracil.…”

“…Based upon a unique substitution within the first nucleotide binding motif (GX 4 G K / M S), IRG proteins can be classified into two subfamilies. Activated GTP‐bound effector IRG proteins (the G K S group) accumulate in high densities at the parasitophorous vacuolar membrane (PVM) of avirulent T. gondii type II strains early after invasion, leading to PVM rupture and parasite clearance (Martens et al ., 2005; Ling et al ., 2006; Zhao et al ., 2009b). The loading is cooperative and hierarchical, with two family members serving as pioneers for members loading later (Khaminets et al ., 2010).…”

“…Phosphorylation of multiple family members could be demonstrated, but the target sites identified in Irga6 most closely match the ROP18 substrate phosphorylation motif (Lim et al ., 2013). Certain T. gondii avirulent type II strains also express a virulent allelic form of ROP18 (Saeij et al ., 2006; Niedelman et al ., 2012) but fail to restrict IRG protein accumulation at the PVM to an extent that allows survival in infected mouse cells (Zhao et al ., 2009b). We could demonstrate that for efficient phosphorylation, IRG proteins have to be kept in the inactive GDP‐bound conformation (Fleckenstein et al ., 2012).…”

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

“…Critical mediators of host-directed attacks on PVs are two families of IFNγ-inducible GTPases: immunityrelated GTPases (IRGs) and guanylate binding proteins (GBPs) (2). Members of both GTPase families play roles in host-mediated lysis of PVs, a process resulting in the release of microbes into the host cell cytoplasm, subsequent killing of PV-expelled microbes, and host cell death (3)(4)(5)(6)(7)(8). Additionally, GBPs help deliver cytosolic subunits of the antimicrobial NADPH oxidase NOX2 for assembly on phagosomal membranes, orchestrate the capture of PV-resident microbes inside degradative autophagolysosomes, and promote the activation of canonical and noncanonical inflammasome pathways (5,(8)(9)(10)(11)(12).…”

Maintenance of vacuole integrity by bacterial pathogens

“…Indeed interferon-inducible GTPases target pathogencontaining vacuoles to inhibit pathogen replication via several mechanisms including fusion with lysosomes or autophagosomes [53]. An outcome of recruitment of interferon-inducible GTPases by the Toxoplasma gondii parasitophorous vacuole is membrane disruption [54]. It remains to be seen whether this is also occurs during infection with bacterial pathogens.…”