The Gamma Interferon (IFN-γ)-Inducible GTP-Binding Protein IGTP Is Necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN-γ-Stimulated Astrocytes

Toxoplasma gondii is a protozoan parasite commonly causing a self-limiting, usually asymptomatic infection in immunocompetent hosts but can cause serious disease in the central nervous system in individuals with immunocompromised immune systems, such as AIDS patients, or immature immune systems, such as newborns (19). The parasite is usually controlled by an effective host immune response mediated by the cytokine gamma interferon (IFN-γ). IFN-γ activates antitoxoplasmacidal activity in macrophages and microglia in the brain and in a variety of nonprofessional immune effector cells, including fibroblasts, epithelial cells, endothelial cells, and astrocytes. IFN-γ-stimulated activities of both professional and nonprofessional immune effector cells are necessary for resistance to T. gondii (36). Several IFN-γ-induced antitoxoplasmocidal mechanisms have been described, including induction of indole-2,3-dioxygenase, nitric oxide production, production of reactive oxygen intermediates, and iron deprivation (1, 7, 10, 11, 27, 35). Our previous studies found that IFN-γ significantly inhibits growth of T. gondii in astrocytes via an IFN-γ-inducible GTP-binding protein (IGTP)-dependent mechanism (13-15). IGTP has also been found to regulate survival of T. gondii in activated macrophages (5). IGTP is a member of the p47 GTPase family of IFN-γ response proteins, a diverse family of 46- to 47-kDa GTPases that are strongly induced by IFN-γ; these GTPases are also known as the immunity-related guanosine triphosphatase, or IRG, family (20, 32). The p47 GTPases are located on organelles of the endocytic pathway and have recently been recognized as an important mechanism by which the host can target intravacuolar pathogens (21, 22, 26, 30, 32). The p47 GTPases are found in all vertebrates from fish, dogs, mice, and humans (20). In mice, over 20 p47 GTPases have been identified, with six p47 GTPases (IGTP, IRG-47, LRG-47, IIGPI, GTPI, and TGTP) cloned and sequenced. In humans, they are less diverse, with only three identifiable genes, but one human p47 GTPase, IRGM, has also been found to be involved in resistance to intravacuolar pathogens (3, 28). The p47 GTPases are related to the dynamin family of GTPases, which mediate membrane tubulation and vesicle fission, and it has been postulated that the p47 GTPases may function in a similar manner in the targeting of intravacuolar pathogens (20). The precise mechanism(s) mediated by the p47 GTPases, however, is not well understood. T. gondii is an intravacuolar pathogen which resides in a unique compartment called the parasitophorous vacuole (PV), which is resistant to host cell endocytic and exocytic trafficking (25). The nonfusogenic PV has been viewed as an adaptive mechanism for intracellular survival, as fusion with the lysosomes results in degradation of the parasite. The PV is also actively modified by the parasite and forms a replicative niche through which the parasite acquires nutrients from the host cell for growth and replication. Four p47 GTPases, IGTP, IRG-47, LRG-47, and IIGPI, have been found to be involved in resistance to T. gondii in vivo (8, 23, 31). Recent in vitro studies indicate that the p47 GTPase-mediated inhibition of T. gondii is via disruption of the vacuole, which results in destruction of the parasite within the host cell (18, 23, 30). IFN-γ-stimulated, p47 GTPase-mediated disruption of the PV has been described to occur in both macrophages and astrocytes (18, 23). In macrophages, vacuolar disruption was found to involve vesiculation of the PV membrane (PVM), subsequent release of the parasite into the host cell cytoplasm, and stripping of the parasite plasma membrane, resulting in degradation of parasites (18). In macrophages, disrupted vacuoles were observed in autophagosomal compartments and autophagous delivery to the lysosomes was found to be involved in parasite elimination (18). Vesicles containing IGTP were found in association with the PV, although the role of IGTP in autophagy and PV stripping was not understood. In murine astrocytes, multiple p47 GTPases, including IGTP, were found to associate with the PV in a maturation-like process leading to vacuolar disruption (23). IIGPI was found to contribute to vacuolar maturation and parasite killing but was not totally responsible, implying involvement of the other p47 GTPases in this process. In astrocytes, no evidence for autophagous engulfment of the disrupted PVs or delivery to the lysosomes was found (23). Thus, there appear to be some differences between the IFN-γ-induced PV disruption in astrocytes and that in macrophages. For neither cell type is the mechanism(s) by which IGTP or the other p47 GTPases mediate vacuolar disruption well understood. In the current study, we have further investigated the mechanism of IFN-γ inhibition and the role of IGTP in the disruption of the PV in murine astrocytes. A synchronous-invasion method for infecting cells was used to establish a homogenous population of vacuoles to analyze IGTP association with the PV and live-cell imaging of green fluorescent protein (GFP)-IGTP used to analyze the early events in PV disruption. In this study, we report that intravacuolar movement of the parasite occurred just prior to PV disruption, and in some instances, IFN-γ induced parasite egression. These studies provide new insights into the crucial events leading to PV disruption, and on the basis of these results, we postulate a mechanism of IGTP-mediated PV disruption that involves a role for the host cell endoplasmic reticulum (ER) both in mediation of PV disruption and in parasite egression.