Toxoplasma’s Arms Race with the Host Interferon Response: A Ménage à Trois of ROPs

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The Toxoplasma gondii virulence factors ROP 5 and ROP18 both target immunity-related GTPases (IRGs) to evade immunity. In this issue of Cell Host and Microbe, Etheridge et al. identify a third virulence factor ROP17, which forms a complex and synergizes with ROP5/ROP18 to fully disable the IRG-system of anti-parasite defense.

Toxoplasma gondii is a protozoan parasite transmitted a sexually through the food chain through carnivorous feeding in wild and domesticated animals, but the parasite also undergoes a well-defined sexual cycle exclusively in felines. Rodents, being the natural prey of cats are considered to be important intermediate hosts for the parasite, while infections occurring in humans, although clinically important in the context of acquired immunodeficiency or congenital transmission, represent a dead end for the parasite’s transmission cycle.

Beginning with genetic studies pioneered by David Sibley in the early 90’s, progress in unraveling the genetic and molecular basis for the virulence of T. gondii in the laboratory mouse has, in the recent decade, gained great momentum. Using crosses generated between highly virulent type I or intermediate virulence type II and a virulent type III parental parasites, genetic mapping analyses identified an active rhoptry kinase ROP18 and an enzymatically inactive pseudokinase ROP5 as principal determinants of T. gondii virulence (Reese et al., 2011; Saeij et al., 2006). A key set of ROP18 kinase substrates belong to the family of immunity-related GTPases or IRGs (Fentress et al., 2010). During the Th1-dominated immune response to T. gondii, IFNγ greatly induces expression of IRGs, which associate with the nascent parasitophorous vacuole membrane (PVM) formed by the parasite following invasion. In host cells infected with non-virulent strains, which express low levels of ROP18, loading of IRGs, through a yet to be determined receptor(s) on the PVM, somehow results in the vesiculation and physical disruption of the PVM and subsequent death of the parasite(Zhao et al., 2009). However, in the case of virulent strains, ROP18 phosphorylates specific residues in switch region I of the GTPase domain of IRGs, inactivating enzymatic activity, IRG oligomerization and assembly on the PVM(Steinfeldt et al., 2010). Although the type II strain expresses a ROP18 allele capable of increasing virulence in the type III strain, the PVM formed by type II parasites are permissive for IRG loading and are susceptible to this IFNγ-induced cellular defense mechanism. This observation indicated that interactions with another gene present in the virulent type I or the a virulent type III strain is needed for immune evasion.

Subsequent studies identified and characterized another virulence locus that encodes the polymorphic pseudokinase ROP5, which is similar between type I and type III strains, but is distinct in type II strains and helped explain how the Toxoplasma virulence associated evasion mechanism works optimally in vivo. The virulence-associated allele of ROP5 interacts with IRGs, favoring the inactive GDP-dependent conformation of the IRGs(Fleckenstein et al., 2012). Association of IRGs with ROP5 also exposes the target threonines located in the switch I loop for ROP18 phosphorylation. In vitro studies also provided evidence that interaction with the ROP5 pseudokinase enhances the kinase activity of ROP18(Behnke et al., 2012). Altogether these data are consistent with the genetic evidence that ROP5 and ROP18 operated along the same pathway to target IRGs and act to forestall destruction of the PVM. However, the fact that deletion of ROP18 in the virulent type I background only resulted in slight attenuation, while ROP5 deficiency results in complete loss of ability to cause lethal infection in mice raised the question of whether ROP5 regulated the activity of additional virulence factors. Deletion of either ROP5 or ROP18 alone resulted in approximately the same levels of IRG loading and type I parasite destruction in IFNγ activated macrophages, suggesting that the putative ROP5-dependent actors may promote virulence independently of the ROP18-mediated evasion of IRG clearance of the parasite.

To identify additional ROP5 regulated virulence effectors, Etheridge et al.(2014) utilized tandem affinity purification of native complexes containing ROP5. Mass spectrometry analysis identified ROP18 as well as a new ROP kinase ROP17. Examination of the sequences of ROP17 indicated a shared allele between a virulent type II and type III strains and a unique type I sequence. Similar to ROP18, ROP 17 contains a conversed catalytic triad that predicted active serine/threonine kinase activity. The kinase activity of native ROP-5 or ROP17-containing complexes was greater compared to recombinant ROP17 alone, suggesting the presence of additional enhancing factors in vivo. Nevertheless, the enzyme activity of native ROP17 complexes was not strictly dependent on the presence of ROP5.

Genetic deletion of ROP17, similar to the case of ROP18, resulted in only slight attenuation of virulence. Deletion of both ROP17 and ROP18, however, generated parasites that are completely a virulent. Nevertheless, ROP17/18 double mutants regained their lethality in mice lacking the IRG lrgm3, suggesting the surprising conclusion that ROP17 may also be operating in the same IRG pathway targeted by ROP18. Using a positional-scanning peptide array to construct a probability matrix motif for ROP17, it was determined that ROP17 preferred to phosphorylate Thr102 in the switch region I of IRGs, while the ROP18 motif centered on Thr108. This differential preference was confirmed experimentally using MS analysis of phosphorylation sites in the Irgb6 protein incubated with ROP17. Using native gel analysis of lrgb6 protein incubated with GTP to promote oligomerization, Etheridge at al. (2014) demonstrated that ROP17 preferred phosphorylation of polymerized IRGs and suggested that phosphorylation leads to disassemblage of the IRG polymers accumulating on the PVM. Given the likelihood that assembled complexes of ROPs exposed on the surface of the PVM are highly heterogenous, it is uncertain how the observed preference for Thr 102 vs. Thr 108 or for different members of the IRG family translates in vivo. Nevertheless, given previous findings that both residues are critical for enzyme activity and for oligomerization, the authors propose a model where ROP17 and ROP18 together phosphorylate and prevent assembly of IRGs on the PVM. They further suggest a scenario where phosphorylation of polymerized IRGs by ROP17 would lead to generation of monomeric substrates for ROP5-ROP18 complexes. Even though the in vitro activitation of ROP17 appears to be independent of ROP5, complexation of ROP17 with ROP5 will likely facilitate IRG-substrate localization and indirectly lead to enhanced ROP17 activity in vivo.

The current discovery of the tripartite ROP5:ROP18:ROP17 virulence complex that appears to principally target the mouse-specific IRG system underscores the importance of rodents in the life cycle of Toxoplasma. Paradoxically, virulence leads to lethality while a virulence will result in persistence and transmission in nature. Recently, the Howard laboratory in Cologne reported a very interesting analysis of the IRG system in wild mice, which revealed a great degree of complexity and polymorphism rivaling that observed for the MHC(Lilue et al., 2013). They also discovered a wild-derived CIM mouse strain, which is resistant to T. gondii strains that are typically lethal for laboratory mice. In this wild-derived mouse, a divergent IRG, designated as lrgb2-b1_CIM bound to ROP5 and itself becomes a target for the ROP5-ROP18 complex, but blocks the phosphorylation of effector IRGs, leading to destruction of the parasite vacuole. In this case, the mouse immune system has been selected to resist virulent T. gondii strains to allow encystment and latency. However, the same virulent strains have a disadvantage when infecting less resistant mouse strains (exemplified by laboratory mice). Given the loss of the IRG system in primates(Bekpen et al., 2005), it is unlikely that same counter-regulatory mechanisms are operative when Toxoplasma infects humans.