This study aimed to elucidate the mechanism by which the p47 GTPases confer resistance to intracellular pathogens, using T. gondii
as a model. We made the surprising observation that many p47 GTPases, including IIGP1, assemble together at the T. gondii
vacuole followed by the disintegration of the vacuolar compartment and killing of T. gondii.
This distinguishes the mechanism of T. gondii
killing clearly from phagosomal maturation. Indeed we did not detect IIGP1 on phagosomes containing dead T. gondii
(G). The accumulation of IIGP1 is therefore dependent on the active invasion of host cells by the parasite and is unlikely to be initiated by receptors such as Toll-like receptors recognizing pathogen-associated molecular patterns on the T. gondii
]. The PV formed by T. gondii
represents a distinctive compartment [4
]. Most host cell proteins are excluded from the PVM while a number of parasite-encoded proteins are inserted into the PVM [6
]. It will be of great interest to see how cells in general and the p47 GTPases in particular recognize these alien compartments in the cytoplasm of infected cells.
A recently published study failed to show an association of IGTP and LRG-47 with vacuoles containing live or dead T. gondii
]. We also failed to detect LRG-47 at the vacuole, but we report the clear localization of IGTP at vacuoles formed by live parasites. This discrepancy might be due to the different cell types used in the two studies (mouse astrocytes versus mouse bone marrow-derived macrophages). However in our system the association of IGTP with PVs was indeed less marked than that of the other p47 GTPases and it is thus possible that this association was missed in the experimental system of Butcher et al [23
In IFN-γ-induced uninfected cells the p47 GTPases localize to overlapping but distinct compartments [35
]. In T. gondii
infected cells however, IIGP1, TGTP, IRG-47, GTPI, and IGTP are closely co-localized at the PVM. In the specific cases of IGTP and IIGP (and probably also TGTP), which are primarily located at the ER in interferon-treated cells in the absence of infection [35
], it might be argued that the vacuolar association observed in our studies reflects, not repositioning of the GTPases, but rather the well-known accumulation of ER cisternae at the T. gondii
]. In the case of IIGP1, however, the accumulation of the GTPase is far more intense than that of any of three other ER proteins examined as markers for ER accumulation at the vacuole (). The case that IGTP is repositioned from the ER to the PV is less clear than that for IIGP1 since the intensity of this p47 at the PV is less striking than that of the other GTPases, though still relatively more intense than the ER markers. Evidently accumulation of ER cisternae at the PV also fails to account for the PV accumulation of the Golgi-localized GTPI (Figure S2
) or of the cytosolic IRG-47 [35
]. Although the trigger for the striking relocation of the p47 GTPases is currently unknown, it requires GTP binding at least for TGTP1 and IIGP1 since IIGP1K82A and TGTPK69A fail to relocate (B, unpublished data). We previously reported the relocation of LRG-47 from Golgi membranes to plasma membrane ruffles triggered by phagocytosis [35
], and another study reported a brefeldin A-sensitive association of LRG-47 with Mycobacterium tuberculosis-
containing phagosomes [26
]. It therefore appears that the initial location of the p47 GTPases in cells is a resting or storage location from which the p47 GTPases are recruited to plasma membrane-derived compartments upon pathogen uptake or invasion.
It is remarkable that a proportion of T. gondii-
containing PVs with apparently normal released GRA7 do not appear to accumulate p47 GTPases, while other vacuoles in the same cell are intensely labeled. In astrocytes we report approximately 70% of vacuoles carrying IIGP1 and a slightly higher percentage carrying TGTP. This stochastic behavior is unlikely to have a purely kinetic basis since no difference was observed when T. gondii
infections were synchronized by centrifugation of the parasites onto the cell layers (unpublished data). We have been able to show in reconstruction experiments in fibroblasts that the p47 GTPases are the only interferon-inducible components necessary for the initiation of p47 accumulation at the PV (unpublished data). Nevertheless other constitutive cellular components required for PV accumulation may be limiting. It is also not excluded that T. gondii
itself may resist the accumulation of p47 GTPases at the PV through one or more of the many components known to be secreted into the host cell during the infection process [49
IGTP and LRG-47 have already been shown to be required for resistance against T. gondii
in astrocytes and/or bone marrow-derived macrophages [22
] and we now add IIGP1 to the list of p47 GTPases mediating cell-autonomous resistance against this organism. Furthermore IGTP, LRG-47, and IRG-47 are required for resistance against T. gondii
in infected mice [24
]. The participation of so many highly diversified members of the p47 GTPase family in resistance to T. gondii
and their co-localization on the PV suggests that they may act in a cooperative manner. Furthermore the individual p47 GTPases are non-equivalent against different pathogens [24
] suggesting a complex relationship between individual members of the p47 family. We have been able to show that interactions with other p47 GTPases are required for the accumulation of IIGP1 at the PV during T. gondii
infection (unpublished data). Thus the normal function of the p47 GTPases is indeed interactive, with some members of the family possibly fulfilling a regulatory function and others an effector function. Such a situation could explain the apparent anomaly that the two p47 GTPases, whose elimination leads to the strongest phenotypes in T. gondii
infection, are IGTP and LRG-47, one of which, IGTP, is only weakly associated with the PV, possibly via ER accumulation, and the other (LRG-47) is probably not associated with the PV at all.
IIGP1 directly associates with the PVM and localizes to apparently PVM-derived vesicles with an electron-dense coat (A and ). In view of the intense concentration of p47 GTPases on the PVM it is plausible that the electron-dense coat indeed consists of p47 GTPases. Biochemically, IIGP1 shows micromolar affinities for nucleotides, nucleotide-dependent oligomerization, and cooperative GTP hydrolysis [38
]. These properties relate IIGP1 to the dynamin family of GTPases with a well established role in membrane fission and deformation processes [40
]. It is therefore conceivable, though not yet formally shown, that IIGP1 and also probably other p47 GTPases act directly on the PVM causing its deformation and vesiculation and thereby disruption. Vesiculation on the scale observed may lead to the net abstraction of enough material from the PVM to cause loss of membrane integrity. The sequestration by T. gondii
of ER cisternae to the PVM [10
] may allow the supply of new lipids to the PVM at a high rate as a defense mechanism. Transport of membrane material carrying IIGP1 and T. gondii
proteins from the PV ( and ) could entail active interaction with microtubules, and association has been shown between IIGP1 and the microtubule motor binding protein, Hook-3 [50
]. Active transport of PVM-derived material could give rise to the observed long filamentous structures emanating from the vacuole (). However, filamentous projections containing T. gondii-
derived proteins, which were still connected to the PVM, have also been detected in cells not induced with IFN-
γ permissive for T. gondii
The parasite itself deteriorates after the disruption of its vacuole. It remains to be established whether the p47 GTPases also contribute to perforation of the T. gondii
plasma membrane or whether the p47 GTPase-dependent removal of the protective PVM renders the parasite accessible to cytosolic factors mediating its disintegration. We have been unable to find convincing evidence that autophagy participates at this stage in pathogen elimination. LC3 accumulates in the vicinity of disrupted vacuoles () but does not appear to surround the pathogen and no profiles resembling autophagic vacuoles were seen engulfing disrupted vacuoles by cryo-electron microscopy. While it is not clear why the cytosol may be an inimical environment for T. gondii,
there are precedents showing that bacteria that normally replicate in modified phagosomes are inviable when introduced into the cytosol [52
]. Interestingly, the anti-microbial activity of the host cell cytosol was cell-type dependent and, in RAW 264.7 macrophages, induced by the pre-exposure of the host cell to the pathogen [53
]. There is no independent experience of the viability of T. gondii
released into the cytosol. However, the intensive modification of the PV by the parasite surely indicates a high level of co-adaptation between the organism and its constructed microenvironment.
The disruption of T. gondii
PVs accompanied by cytoplasmic dissemination of parasite-encoded proteins such as GRA7 () may also accelerate presentation of antigens to the adaptive immune system via the class I pathway. Release of GRA7 into the cytosol and association with host cytoplasmic structures has been reported before in the absence of interferon treatment. In this case, however, GRA7 release occurred much later, possibly coincident with the tachyzoite-bradyzoite transition. The possible implication of this late GRA7 release for class I antigen presentation was also noted [42
]. A recent study showed the MHC class I restricted, TAP-dependent presentation of a T. gondii
-encoded protein that was secreted into the vacuolar space by splenocytes of infected mice . It is conceivable that the p47 GTPase-dependent disruption of the PVs contributes to the accessibility of this protein to the antigen presentation machinery.
We have identified a previously unknown mechanism of cell-autonomous resistance in mice against the intracellular protozoan pathogen T. gondii.
This involves a direct attack on the PVM leading to vesiculation and ultimately disruption of the membrane, and requires the participation of multiple interferon-inducible p47 GTPases. In view of the wide sequence divergence and distinctive resistance properties associated with the different p47 GTPases [28
] it will be of interest to find out whether a role in membrane vesiculation is their exclusive mode of action on pathogen-containing vacuolar compartments, including phagosomal derivatives, or whether their contribution to pathogen resistance is more diverse than this. Thus MacMicking et al. [26
] have concluded from their own studies on resistance in mice to Mycobacterium tuberculosis
that LRG-47 acts in the interferon-stimulated cell to accelerate phagosomal acidification, an activity which is difficult to reconcile with the phenomena observed here, where the T. gondii
PVs never enter the lysosomal system as defined by the acquisition of LAMP1.
As we have shown [28
], there are 23 p47 GTPase genes in mice, of which at least 14 are inducible by interferon. Only four of these (IGTP, LRG-47, IRG-47, and IIGP1) have yet been analyzed experimentally to any significant extent and all these have been shown to be active, each in its distinctive way, in resistance to vacuolar pathogens. They have so far been implicated in resistance to a remarkable range of major pathogens (Gram negative bacteria: Salmonella typhimurium;
Gram positive bacteria: Listeria monocytogenes;
Mycobacteria: M. tuberculosis
and Mycobacterium avium;
Protozoa: T. gondii, Leishmania donovani, Trypanosoma cruzi
]. There is no reason to doubt that the complexity and functional diversity of the p47 resistance system will be further extended as new family members are analyzed in detail. It is evident that the entire structure of immunity in mice against vacuolar pathogens is critically dependent on the effector mechanisms delivered by the p47 GTPases. Against this background, therefore, it is quite an extraordinary fact that the p47 resistance system is completely absent in man [28
]. The implications of this remarkable difference in the deployment of immune mechanisms between man and his principal experimental model organism, the mouse, will require extensive experimental analysis.