Abstract
The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells.IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.
Highlights
The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV)
To explore the role of ATG in IFN-mediated control of intracellular parasite growth, we examined proteins that interact with ATG5 in an IFN-␥-dependent fashion using proximity-based biotin labeling
We identified 87 interactors of ATG5 in naive HeLa cells and 40 interactors that were enhanced in IFN-␥-stimulated HeLa cells using permissive biotin labeling
Summary
The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole These findings suggest a similar link between interferon-␥ signaling and autophagy that may underlie defense against other intracellular pathogens. Type I strains, which are highly virulent in laboratory mice, largely avoid this fate, while types II and III are highly susceptible [15] These pathways are counteracted by secretion of a parasite family of polymorphic ROP kinases that target IRGs and GBPs to prevent their accumulation on the PV, protecting this intracellular niche [15, 16]. Human cells rely on overlapping and distinct pathways for IFN-␥-dependent control of parasite replication, including depletion of tryptophan via indoleamine 2,3-dioxygenase, select GBPs, generation of reactive oxygen species, ubiquitination, and lysosomal clearance [18]
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