Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

Aditi Alaganan, Sarah J. Fentress, Keliang Tang, Qiuling Wang, and L. David Sibley1

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Ralph R. Isberg, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA, and approved December 9, 2013 (received for review July 20, 2013)

The intracellular parasite enjoys a wide host for destruction by relocating to the parasite vacuole and vesic- range and is adept at surviving in both naive and activated macro- ulating the PVM (13). Under normal conditions, immunity- phages. Previous studies have emphasized the importance of the related GTPase family M (IRGM) proteins that are found on host active serine-threonine protein kinase rhoptry protein 18 (ROP18), endomembranes bind IRGs and maintain them in an inactive which targets immunity-related GTPases (IRGs), in mediating mac- state (14). Upon infection, IRGs are preferentially recruited to rophage survival and acute virulence of T. gondii in mice. Here, we -containing vacuoles due to an absence of IRGM demonstrate that ROP18 exists in a complex with the pseudoki- proteins (14). However, virulent strains of T. gondii that express a type I allele of ROP18, an active serine/threonine kinase, are nases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein Δ Δ able to phosphorylate IRGs, and thus to decrease their re- 7 (GRA7). Individual deletion mutant gra7 or rop18 was par- cruitment to the PVM (15, 16). The activity of ROP18 is reg- tially attenuated for virulence in mice, whereas the combined ulated by a related pseudokinase ROP5 (17), which also binds Δ Δ gra7 rop18 mutant was avirulent, suggesting these proteins to monomeric Irga6 and blocks its assembly in vitro (18, 19). act together in the same pathway. The virulence defect of the Recent evidence implicates ROP5 and ROP18 in avoidance of double mutant was mirrored by increased recruitment of IRGs guanylate binding proteins, which also contribute to restriction and clearance of the parasite in IFN-γ–activated in of T. gondii in IFN-γ–activated macrophages (20). vitro. GRA7 was shown to recognize a conserved feature of IRGs, Kinases often interact with pseudokinases and scaffolding MICROBIOLOGY binding directly to the active dimer of immunity-related GTPase a6 proteins for regulation or substrate recruitment (21, 22). Con- in a GTP-dependent manner. Binding of GRA7 to immunity-related sequently, we were interested in whether ROP18 forms a com- GTPase a6 led to enhanced polymerization, rapid turnover, and plex with other parasite proteins that might influence its activity eventual disassembly. Collectively, these studies suggest that or function. Our studies establish that ROP18 is part of a com- ROP18 and GRA7 act in a complex to target IRGs by distinct mech- plex with the related pseudokinases ROP8/2 and the dense anisms that are synergistic. granule protein GRA7, the latter of which acts synergistically with ROP18 in controlling acute virulence in the mouse. pathogenesis | innate immunity | cooperative polymerization Results Characterization of ROP18-Containing Complexes. To identify ROP18 he apicomplexan parasite Toxoplasma gondii has a re- interacting partners, we immunoprecipitated ROP18 from cells Tmarkable host range and is capable of infecting most infected with a transgenic type III strain parasite expressing active warm-blooded animals (1). The cellular life cycle of this op- ROP18 (i.e., CTG + ROP18-Ty) vs. CTG, which lacks ROP18 portunistic pathogen involves active invasion of nucleated expression (9). Tandem MS (MS/MS) analysis revealed that ROP18 host cells and establishment of an intracellular niche within the parasitophorous vacuole. This compartment is demarcated Significance by the parasitophorous vacuole membrane (PVM), which avoids fusion with the host endomembrane system, although being Toxoplasma gondii is a widespread parasite of animals that studded with many parasite proteins (2). PVM-localized proteins frequently infects humans. Infection in mice, a natural host for derive from two major organelles: the rhoptries (ROP proteins) transmission, is strongly dependent on IFN-γ, which up-regu- and the dense granules (GRA proteins), which are sequentially lates innate defenses. The parasite secretory kinase rhoptry secreted upon invasion (3). The strategic location of GRAs and protein 18 (ROP18) phosphorylates immunity-related GTPases ROPs on the PVM positions them to play important roles in (IRGs) and prevents recruitment to the vacuole surrounding interacting with the host. virulent parasites. Here, we demonstrate that ROP18 is found ROP proteins are secreted directly into the host cell at in a complex with other pseudokinases (i.e. ROP8/2) and the the time of invasion, after which they target to the PVM or other dense granule protein GRA7. Our studies reveal that GRA7 locations within the cell (4). Although many ROP proteins contain a kinase fold, nearly half of these are predicted to be binds to oligomers of Irga6, resulting in increased polymeri- pseudokinases because they lack the critical catalytic residues zation initially but then leading to rapid disassembly. The that are normally required for phosphate transfer (5). Often, activity of GRA7 may prevent proper IRG loading onto the these pseudokinases (i.e., ROP5, ROP8/2, ROP4/7) exist as parasite-containing vacuole and may make substrates available tandem gene duplications and show evidence of positive selec- for ROP18, thus explaining its contribution to virulence. tion (5). Following secretion, ROP2 family members are targeted to the cytoplasmic face of the PVM via a series of amphipathic Author contributions: A.A., S.J.F., and L.D.S. designed research; A.A., S.J.F., and Q.W. α-helical regions in their N termini (6, 7). performed research; K.T. contributed new reagents/analytic tools; A.A. and L.D.S. ana- ROP proteins generated renewed interest when it became lyzed data; and A.A. and L.D.S. wrote the paper. evident that some ROPs confer critical strain-specific virulence The authors declare no conflict of interest. in mice (8–12). In particular, the active kinase ROP18 and the This article is a PNAS Direct Submission. pseudokinase ROP5 defend the parasite vacuole by blocking the 1To whom correspondence should be addressed. E-mail: [email protected]. function of mouse immunity-related GTPases (IRGs) (4). Fol- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lowing up-regulation by IFN-γ, IRGs target susceptible parasites 1073/pnas.1313501111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1313501111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 (detected by 26 unique peptides) was specifically associated with the X-100 was used to permeabilize all membranes fully, positive rhoptry pseudokinase ROP2 (detected by 15 unique peptides) and staining for SAG1 was observed, as well as staining for GRA7 in GRA7 (detected by 11 unique peptides) (Table S1). A majority of dense granules and ROP18-Ty in rhoptries (Fig. 1B, Lower; both peptides matched ROP2, whereas four peptides also matched proteins are extracted from the PVM under these conditions). ROP8, a closely related protein that is located immediately up- Combined with the MS/MS data above, these findings support stream in the same locus, referred to here as ROP8/2 (Fig. S1). Two the existence of a complex consisting of ROP18, GRA7, and peptides of ROP4/7 were also identified in the ROP18-Ty immu- ROP8/2 on the surface of the PVM in infected cells. noprecipitation (IP), which may reflect the previously described interaction between GRA7 and ROP4/7 (23). To validate the Analysis of ROP18 Complex Deletion Mutants. To explore the ROP18 binding partners identified by MS/MS, GRA7 was immu- functions of GRA7 and ROP8/2, we made single deletions of Δ noprecipitated from WT RH strain vs. gra7 mutant parasites un- each gene by homologous recombination (Fig. S1 and Table der similar conditions and analyzed by MS/MS. This reciprocal IP S3). Furthermore, ROP8/2 and ROP18 were each separately revealed an almost identical set of proteins comprising the bait deleted in the Δgra7 parasite line to create two double-deletion protein GRA7 (14 peptides), ROP2 (14 peptides), ROP4 (8 pep- mutants (Fig. S2). Drug-resistant parasite pools were cloned tides),ROP7(5peptides),ROP18(4peptides),andROP5(19 by limiting dilution and screened by PCR (Figs. S1 and S2 and peptides) (Table S2). These proteins were enriched in the IP Δ Table S3)andWesternblotting(Fig.1C) for their respective samples from WT parasites but not in those from gra7 mutant gene deletions. The Δgra7Δrop18 parasites were then com- parasites (Table S2). Although ROP5 was not seen in the ROP18- plemented with either GRA7 or ROP18-Ty, and single-cell Ty IP, its presence in the GRA7 complex, together with ROP18, is clones were confirmed for proper protein expression and lo- consistent with the observation that ROP5 regulates the kinase activity of ROP18 (17). To confirm the interaction shown by MS, we calization (Fig. S3). + GRA7 was previously implicated in nutrient acquisition (24), immunoprecipitated ROP18 from CTG ROP18-Ty parasites vs. Δ CTG parasites and performed Western blotting to detect interact- so we were interested in whether the gra7 parasites would ex- ing partners. ROP2 and GRA7 were specifically coprecipitated with hibit any growth defect in vitro. Plaque assays were performed on ROP18 (Fig. 1A), whereas GRA5, another secretory protein that the WT parasites and various mutant parasites to test whether also localizes to the PVM, did not associate with ROP18 (Fig. 1A), deletion caused a growth defect. At 7 d postinfection, the number demonstrating the specificity of this complex. of plaques was comparable for WT and all mutant lines (Fig. 1D and Fig. S4A). Relative plaque size measurements and replication ROP18 and GRA7 Localize to the Cytosolic Face of the PVM. The assays further confirmed the absence of an in vitro growth defect identification of GRA7 as part of a complex with ROP18 sug- of the Δgra7 mutant (Fig. S4 B and C). We also analyzed the ul- gested that it might localize to the external surface of the PVM trastructure of WT and Δgra7Δrop18 parasite vacuoles by EM and after secretion, similar to ROP18 (6). To determine whether observed no differences in either the presence of the tubulove- GRA7 also localizes to this interface, digitonin was used to sicular network (TVN) or the general architecture of the network permeabilize infected host cells selectively. Under these semi- (Fig. S5). In addition, we observed normal localization of both permeabilized conditions, antibodies to both ROP18 and GRA7 ROP18 and ROP5 on Δgra7 vacuoles (Fig. S6 A and B). Hence, stained the cytoplasmic surface of the PVM at both 30 min and the defects described below for the Δgra7Δrop18 parasites are 12 h, whereas staining of the parasite surface antigen (SAG1) notrelatedtoadverseeffectsongrowthordifferencesin remained negative (Fig. 1B, Upper). In contrast, when Triton vacuole morphology or composition.

Fig. 1. Interaction of ROP18 and GRA7 and generation of KO and complemented lines. (A) Co-IP of proteins with ROP18. Lysates from CTG or CTG + ROP18- Ty parasites were immunoprecipitated with mAb BB2 to Ty (Mo αTy). Western blotting (WB) of the pellet (Pell, 100% loaded) and supernatant (Sup, 25% loaded) fractions was probed for rabbit anti-ROP18 (Rb αROP18), rabbit anti-ROP2 (Rb αROP2), rabbit anti-GRA7 (Rb αGRA7), or mAb to GRA5 (Mo αGRA5). (B) Immunofluorescence localization of ROP18 and GRA7 in selectively permeabilized (digitonin) vs. fully permeabilized (Triton X-100) cells. Permeabilized cells were incubated with rabbit anti-GRA7 (red), mAb BB2 to Ty tag to detect ROP18-Ty (blue), and mAb DG52 to SAG1 (green). (Scale bars: 5 μm.) (C) Lysates from WT or mutant parasites were resolved by SDS/PAGE and probed with Rb αROP18 and rabbit anti-T. gondii actin (Rb αTgACT) as a loading control (Upper)or with Rb αROP2 and Rb αGRA7 (Lower). (D) Plaque assay of in vitro growth of the WT and mutant parasites.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1313501111 Alaganan et al. Downloaded by guest on September 26, 2021 Combined Deletion of ROP18 and GRA7 Leads to Severe Attenuation more effectively in IFN-γ–activated macrophages. Although of Acute Virulence. Because RO18 plays an important role in the Δrop18 parasites were more efficiently cleared than WT par- virulence of T. gondii in mice (15–17), we were interested in asites at 20 h postinfection, the survival of Δgra7 mutants was whether the ROP8/2 and GRA7 proteins might also contribute unchanged (Fig. 3E). Consistent with the higher level of IRG to this phenotype. When challenged with Δrop8/2 parasites, recruitment to vacuoles surrounding the double mutant, sig- outbred CD-1 mice died with almost identical kinetics to WT nificantly fewer Δgra7Δrop18 parasites survived compared with parasites (Fig. 2A). In comparison, CD-1 mice infected with the single Δrop18 mutant (Fig. 3E). Complementation with either Δgra7 and Δgra7Δrop8/2 parasites experienced a slight delay in GRA7 or ROP18 restored the survival defect seen in the double time to death that was not as severe as that seen with Δrop18 mutant (Fig. 3E). parasites (Fig. 2A). In contrast, all CD-1 mice challenged with The enhanced recruitment and clearance of the double mutant the Δgra7Δrop18 parasites survived (Fig. 2A). To establish the ex- suggested that ROP18 and GRA7 act together to block the IRG tent of this attenuation, we examined the dose-dependent mortality pathway, albeit with ROP18 having a more dominant role. To of mice following challenge with Δgra7Δrop18 parasites. Although test this prediction, we examined the survival of Δgra7 and − − adoseof103 parasites led to survival of CD-1 mice, higher doses Δgra7Δrop18 parasites in Irgm3 / mice, which are defective in resulted in a dose-dependent mortality (Fig. 2B). Complementation loading of IRGs onto the PVM (25). Although we observed of the Δgra7Δrop18 parasites with either GRA7 (Δrop18/GRA7) or a slight delay in time to death of outbred CD-1 mice challenged ROP18-Ty (Δgra7/ROP18-Ty) restored virulence to the approxi- with Δgra7 mutants (Fig. 2A), this effect was eliminated in the − − mate levels of the single mutants (Fig. 2C). more susceptible C57BL/6 strain and in Irgm3 / mice (Fig. 3F). To determine the importance of immunity in the attenuation Consistent with the idea that GRA7 and ROP18 act synergisti- of the double mutant, we injected the Δgra7Δrop18 parasites into cally to avoid IRG-mediated killing, the normally attenuated − − − − IFN-γ receptor (IFN-γR) / mice, which are highly susceptible to Δgra7Δrop18 mutant killed Irgm3 / mice within 10 d (Fig. 3F). Δrop18 and Δrop5 mutants of T. gondii (17). Mutant Δgra7Δrop18 −/− parasites killed IFN-γR mice within 10 d postinfection but GRA7 Binds to and Stimulates Rapid Turnover of the GTP-Activated remained avirulent in control C57BL/6 mice (Fig. 2D). This Irga6 Oligomer in Vitro. Increased recruitment of IRGs to the finding indicates that the attenuation of Δgra7Δrop18 parasites is vacuole surrounding Δgra7Δrop18 parasites, as well as the − − a consequence of immunological control and not due to intrinsic complete reversal of the Δgra7Δrop18 avirulence in Irgm3 / differences in growth. mice, led us to test whether GRA7 directly affects IRG function. Irga6 oligomerization was monitored by 90° light scattering, as Δgra7Δrop18 Parasites Show Increased IRG Recruitment and Are described previously (26). Addition of GTP to Irga6 alone caused Cleared by Activated Macrophages. The observation that the at- an increase in light scattering consistent with polymerization (Fig. MICROBIOLOGY tenuation of Δgra7Δrop18 parasites relies on IFN-γ signaling 4A). Addition of substochiometric amounts of GRA7 resulted in suggested that the double mutant may be more susceptible to the an increase in light scattering (Fig. 4A), suggesting it promotes IRG pathway, which is a known target of ROP18 (15, 16). oligomerization and/or cross-links and bundles Irga6. Sedimenta- Consistent with this, we observed a significant increase in the tion assays revealed more Irga6 in the pellet fraction at the peak of extent of Irga6 (Fig. 3 A and B) and Irgb6 (Fig. 3 C and D) lo- light scattering when GRA7 was present compared with Irga6 calization to Δgra7Δrop18 parasite vacuoles in IFN-γ–activated alone (Fig. S7A). The subsequent decrease in light scattering, macrophages. When complemented with GRA7 or ROP18, which corresponds to depolymerization (26), occurred much faster levels of Irga6 and Irgb6 recruitment to the vacuole were re- and dropped to a much lower level in the presence of GRA7 (Fig. stored to WT levels (Fig. 3 A–D). 4A and Fig. S7A). Altered kinetics of polymerization and turnover Based on the increased levels of IRG recruitment, we hy- of Irga6 showed a dose-dependent response with addition of pothesized that the double-mutant parasites might also be cleared GRA7 (Fig. 4B). To determine the mechanism of this disruption, we examined the interaction of His6-tagged GRA7 with recombinant Irga6. GRA7 coprecipitated recombinant Irga6 in the presence of GTP but not GDP (Fig. 4C), suggesting that it recognizes GTP-bound dimers and/or oligomers but not GDP-bound monomers of Irga6 (26, 27). Precipitation was not due to formation of large olig- omers or aggregates of Irga6 and GRA7, as shown by the failure of these proteins to pellet in the absence of Ni beads (Fig. S7B). The interaction between GRA7 and Irga6 was also not the result of nonspecific binding to Ni beads, because an untagged version of GRA7 did not bring down Irga6 in the presence of beads; however, it was still able to alter the polymerization of Irga6 (Fig. S7B). Moreover, His6-tagged aldolase, an unrelated gly- colytic enzyme, did not precipitate Irga6 (Fig. 4C). In addition, a GRA7 mutant (GRA7 MUT) that was modified to eliminate the hydrophobic transmembrane domain (Fig. S7C) was also able to precipitate Irga6 efficiently (Fig. 4C), suggesting that the interaction does not depend on the putative transmem- brane domain of GRA7. To test the specificity of the GRA7– Fig. 2. Virulence of WT and mutant parasites in laboratory mice. (A)WT Irga6 interaction further, we took advantage of previous studies and deletion mutants were injected into CD-1 mice (100 parasites, s.c. in- that have defined the importance of GTP hydrolysis on the oculation), and survival was followed for 30 d (n = 10 mice for all strains ′ ′ except Δrop8/2 and Δgra7Δrop8/2, where n = 5 mice). ****P < 0.0001, formation of Irga6 oligomers. Irga6 binds to 2 dGTP and 3 – Δ Δ dGTP but only oligomerizes with 2′dGTP (27). GRA7 bound to Gehan Breslow Wilcoxon test. (B) Dose-dependent mortality of gra7 rop18 ′ ′ parasites. CD-1 mice (n = 5) were injected i.p with varying doses of parasites, Irga6 in the presence of 2 dGTP but not 3 dGTP (Fig. 4D), and survival was followed for 30 d. (C) Δgra7Δrop18 parasites and their indicating that GRA7 binds to oligomers of Irga6. In addition, singly complemented strains (i.e., Δrop18/GRA7, Δgra7/ROP18-Ty) were two Irga6 point mutants, T102A and T108A, which are in- injected into C57BL/6 mice (100 parasites, s.c. inoculation), and survival was capable of hydrolyzing GTP or oligomerizing (16), were also monitored for 30 d (n = 10 mice for all strains). ****P < 0.0001, Gehan– unable to bind to GRA7 (Fig. 4D). In accordance with the in- − − Breslow Wilcoxon test. (D) Survival of WT C57BL/6 mice vs. IFN-γR / mice crease in light scattering of Irga6 in the presence of GRA7, challenged with Δgra7Δrop18 parasites. Mice (n = 10) were injected i.p. with addition of GRA7 stimulated higher phosphate release from the 100 Δgra7Δrop18 parasites, and survival was followed for 30 d. GTPase activity by Irga6 compared with Irga6 alone (Fig. 4E).

Alaganan et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 Fig. 3. IRG recruitment and clearance in activated macrophages. (A) Immunofluorescence staining of Irga6 on parasite-containing vacuoles in activated RAW macrophages at 30 min postinfection stained with mAb 10D7 for Irga6 (green), rabbit polyclonal antibody to ROP5 (red), and DAPI (blue). (Scale bar: 5 μm.) (B) Quantification of Irga6 localized to the parasite vacuole in IFN-γ–activated RAW cells. Mean ± SD of the population (SDP) (n = 3 experiments). *P < 0.05 and **P < 0.001, Student t test. (C) Immunofluorescence staining of Irgb6 on parasite-containing vacuoles in IFN-γ–activated RAW macrophages at 30 min postinfection stained with rabbit polyclonal α-Irgb6 (green), mAb Tg17-113 to GRA5 (red), and DAPI (blue). (Scale bar: 5 μm.) (D) Quantification of Irgb6 localized to the parasite vacuole in activated RAW cells. Mean ± SDP (n = 3 experiments). *P < 0.05, Student t test. (E) Quantification of parasite survival in IFN-γ–activated RAW macrophages. Mean ± SD values for two experiments with three replicates each are given (n = 6). *P < 0.05 and **P < 0.001, Mann– − − Whitney test. (F) Survival of Irgm3 / vs. WT C57BL/6 mice injected s.c. with 100 parasites (n = 7 mice for Δrop18 and Δgra7Δrop18, n = 5 mice for Δgra7). ****P < 0.0001, Gehan–Breslow Wilcoxon test.

Discussion time to death in CD-1 outbred mice, although not in more sus- Our findings reveal that ROP18 exists in a protein complex on ceptible C57BL/6 mice, suggesting there is no intrinsic growth the PVM with the parasite pseudokinases ROP8/2 and GRA7. defect of the Δgra7 mutant. Although loss of GRA7 alone had Previous co-IPs also identified an interaction between the a minimal effect on survival, when combined with deletion of pseudokinases ROP4/7, ROP8/2, and GRA7 and demonstrated ROP18, it led to enhanced IRG recruitment and clearance in that these proteins become phosphorylated within the host cell IFN-γ–activated macrophages in vitro and a profound loss of (23). Deletion of either GRA7 or ROP8/2 alone did not decrease virulence in both CD-1 and C57BL/6 mice. The dramatic change virulence, unlike the loss of ROP18, suggesting that GRA7 and in virulence is unlikely to be due to defects in growth in vivo, ROP8/2 are not involved in regulating ROP18 function. How- because the double-Δgra7Δrop18 mutant showed normal growth in vitro. Importantly, the decreased virulence of the Δgra7Δrop18 ever, the combined loss of ROP18 and GRA7 led to a much − − greater attenuation than the loss of either gene alone. Double- mutant was reversed in IFN-γR / mice, leading to a similar time Δgra7Δrop18 mutants showed increased recruitment of IRGs and to death as that observed previously with WT parasites (17). The clearance in IFN-γ–activated macrophages. The avirulence decreased virulence of the Δgra7Δrop18 mutant was also reversed − − − − phenotype of this double mutant was reverted in Irgm3 / mice, in Irgm3 / mice, implicating IRGs in this control mechanism. implicating the complex in avoidance of the IRG pathway. The IRG proteins form the major IFN-γ–dependent resistance GRA7 bound to Irga6-GTP in vitro, enhancing the initial rate of mechanism to T. gondii in the mouse (13). Collectively, these polymerization but eventually leading to a rapid disruption of the findings indicate that GRA7 and ROP18 act synergistically to oligomeric complex. These findings suggest a unique role for block the IRGs and that in the absence of this host defense GRA7 in combating cell-autonomous defenses by altering IRG pathway, they are no longer required. polymerization kinetics. GRA7 dramatically increased polymerization of Irga6 in vitro, Despite a previously suggested role of GRA7 in nutrient ac- as revealed by 90° light scattering and sedimentation. The accel- quisition (24), we did not observe any defect in growth of the erated rate of Irga6 assembly suggests that GRA7 may act as Δgra7 mutant under conditions examined here. Moreover, the a nucleation or stabilizing factor for polymer formation. Alterna- Δgra7 mutant showed a normal PVM and TVN structure and tively, GRA7 may bind to small oligomers of Irga6 and cross-link normal distribution of the virulence factors ROP18 and ROP5 or bundle them, leading to greater light scattering and sedi- on the PVM. Loss of GRA7 did not directly affect IRG loading mentation. Intriguingly, GRA7 only bound to forms of Irga6 that or clearance in IFN-γ–activated macrophages, unlike deletion of were competent for oligomerization. Structural and modeling GRA2, which leads to alterations in the TVN and increased IRG studies have defined a dimer interface in Irga6 that is necessary recruitment (18). Deletion of GRA7 did lead to a slight delay in for homo-oligomerization and mediates binding to heterodimer

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1313501111 Alaganan et al. Downloaded by guest on September 26, 2021 These results suggest that GRA7 may recognize the active dimer that leads to IRG assembly, thereby taking advantage of a natural regulatory feature to alter polymerization kinetics. The function of GRA7 contrasts with that of known mediators of IRG function or GTPase effectors. IRGs exist as monomers when bound to GDP, and they oligomerize in the presence of GTP to undergo hydrolysis by a cooperative mechanism (26). IRGs have intrinsic GTPase activating protein (GAP) activity and exchange nucleotides readily; as such, they do not rely on GAPs or guanine nucleotide exchange factors (GEFs) to regu- late their functions (26). Instead, IRGM proteins are thought to regulate IRGs by preventing their assembly through guanylate dissociation inhibitor activity that maintains them in a GDP- bound state (28, 29). Because GRA7 binds only to oligomers of Irga6-GTP and not to Irga6-GDP monomers, it is unlikely to act as a GEF. Binding of GRA7 to Irga6 oligomers slightly enhanced GTP hydrolysis and phosphate release, consistent with the in- creased amount of polymer formed. In addition, GRA7 greatly increased the rate of depolymerization of Irga6, resulting in faster turnover. The precise interactions between GRA7 and Irga6 that drive increased assembly and faster turnover remain to be defined by future studies. The in vivo significance of enhanced Irga6 polymerization in the presence of GRA7 is presently unclear, although several models can be considered based on our in vitro findings. If GRA7 on the PVM were recognized by the host as a target for IRGs, we would expect to see higher recruitment to vacuoles surrounding WT parasites. This model can be ruled out because there was little change in IRG recruitment in the Δgra7 mutant,

whereas the Δgra7Δrop18 mutant showed much greater re- MICROBIOLOGY cruitment of Irga6 and Irgb6. Instead, it seems likely that GRA7 disrupts IRG function by altering the kinetics of polymerization, which might occur in several different ways. First, binding of GRA7 to small oligomers or dimers may prevent them from assembling correctly on the PVM. Second, GRA7 binding to IRGs may lead to enhanced assembly but then drive rapid dis- assembly into inactive GDP-bound monomers. Such activity by GRA7 would be expected to make substrates available to ROP18, which phosphorylates residues in switch region I (SWI) (15, 16), or to ROP5, which is thought to bind to monomeric IRGs to prevent their assembly (19). Further studies are needed to define how these various virulence factors work cooperatively in vitro and in vivo to thwart the IRG system and protect the parasite from destruction. IRGs target a variety of intracellular , including T. gondii, where they oligomerize onto the PVM and result in its vesiculation and destruction (13). The virulence factor ROP18 Fig. 4. Interaction of GRA7 and Irga6 in vitro. (A) Oligomerization of 50 μM phosphorylates key residues in SWI of IRGs, and this is associ- Irg6 ± 20 μM GRA7 in a 90° light scattering assay, in which 1 mM GTP was ated with preventing their accumulation on the PVM (15, 16). added at the location indicated by the arrow at 37 °C. AU, arbitrary units; PK, The function of ROP18 is augmented by the pseudokinase proteinase K-treated GRA7. (B) Oligomerization of 50 μM Irg6 ± GRA7 in ROP5, which can directly bind to IRGs and prevent assembly in a 90° light scattering assay in which 1 mM GTP was added at the location vitro (18, 19), while also enhancing the activity of ROP18 (17). indicated by the arrow at 37 °C. (C) GTP-dependent binding of WT (GRA7) vs. Our findings suggest that GRA7 acts in vitro to disrupt IRGs by double-point mutant (GRA7 MUT) to Irga6. Supernatants (S; 10%) and pel- a unique biochemical mechanism of increasing the turnover of lets (P; 50%) were resolved by SDS/PAGE and stained with SYPRO Ruby. His- Irga6 oligomers, thus potentially complementing the functions of tagged ALD (His-Ald) was included as a negative control. (D) Nucleotide- other known virulence determinants ROP18 and ROP5. This dependent binding of GRA7 to Irga6. Recombinant His-tagged GRA7 (His- example reveals multiple layers of defense used by the parasite to GRA7) was incubated with WT or mutant Irga6 and various nucleotides for 10 counteract innate immunity in the host. min at 37 °C and then immunoprecipitated with Ni beads. The supernatants were resolved by SDS/PAGE and stained with SYPRO Ruby. (E) Phosphate re- Materials and Methods lease during oligomerization of Irga6 (50 μM); incubation ± 20 μMGRA7, Parasite Propagation. Parasite strains (referred to in detail in Table S3)were 20 μM ALD (Ald), or PK-treated GRA7 (+PK); and addition of final 1 mM GTP passaged as tachyzoites in human foreskin fibroblast (HFF) monolayers in DMEM (+GTP). [Pi], cumulative phosphate released (micromolar) (n = 3). **** P < containing 10 mM Hepes, 10 μg/mL gentamicin, 6 mM L-glutamine, 1 mM py- 0.0001, two-way ANOVA test. ruvate, and 10% (vol/vol) FBS, cultured at 37 °C in 5% (vol/vol) CO2. Plaque assays were carried out on confluent monolayers of HFF cells as described in SI Materials and Methods. formation with IRGM proteins that negatively regulate polymer assembly (27). GRA7 was unable to bind to mutants (T102 and Antibodies and Microscopy. Rabbit antisera that were specific to recombinant T108) that fail to hydrolyze GTP, and hence do not undergo GRA7 or ROP2 were generated as described in SI Materials and Methods. oligomerization (16). Similarly, GRA7 was only able to bind to Immunoprecipitation and Western blotting assays were performed as de- Irga6 in the presence of 2′dGTP, which induces polymerization scribed previously (15) (SI Materials and Methods). Immunofluorescence more slowly than GTP, whereas it did not bind to Irga6 in the microscopy to localize parasite proteins in infected cells and EM to evaluate presence of 3′dGTP, which fails to drive oligomerization (27). the morphology of infected cells were performed as described previously

Alaganan et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 (15) (SI Materials and Methods). Immunofluorescence microscopy to evalu- followed by the addition of GTP (final concentration of 1 mM), and moni- ate the recruitment of Irga6 and Irgb6 to parasite-containing vacuoles and tored over time with a PTI Quantmaster spectrofluorometer (Photon Tech- the clearance of parasites in IFN-γ–activated RAW cells was performed as nology International). Curves were processed using Prism (GraphPad). described previously (15) (SI Materials and Methods). In Vitro Binding Assay. Recombinant GRA7 and T. gondii aldolase (ALD) were MS. Proteins were eluted from protein G Sepharose beads by incubation in expressed in Escherichia coli and purified as described in SI Materials and 2% RapiGest SF surfactant (Waters) with 20 mM DTT at 95 °C for 10 min, Methods. Ni beads (Sigma–Aldrich) were either charged with His-tagged GRA7 followed by 5 min at room temperature. Eluates were collected by centri- or His-tagged ALD or left uncharged in buffer A. Irga6 (50 μM) was added to fugation at 1,000 × g and digested with 2 μg of trypsin for 16 h at 37 °C. the Ni beads in a final volume of 50 μL of buffer B and various nucleotides Digested samples were dried and resuspended in 10 mL of 5% (vol/vol) (final concentration of 1 mM) and incubated at 37 °C for 10 min. Beads were acetonitrile in 0.1% formic acid and analyzed by liquid chromatography MS/ washed in buffer B, and 10% (vol/vol) of the unbound supernatant fraction MS using the LTQ-Orbitrap Velos (Thermo Scientific) with a 2-h gradient. The and 50% (vol/vol) of the bound-bead fraction were resolved on 10% (vol/vol) m/z spectra data were searched using MASCOT (Matrix Science) against the acyrlamide gels by SDS/PAGE and stained with SYPRO Ruby (Invitrogen). GenBank nr (nonredundant) and ToxoDB 8.1 databases. GTP Hydrolysis Assays. To measure phosphate release, 50 μM purified Irga6 Generation of Plasmids and Transgenic Parasites. Primers used for generation was incubated with either 20 μM GRA7, 20 μM proteinase K-treated GRA7, of plasmids for KO or complemented strains used in this study are available on 20 μM ALD, or proteinase K-treated buffer A in the presence of 1 mM GTP in request. To generate the KO and complemented lines, plasmids were line- buffer B at 37 °C. As negative controls, Irga6 and GRA7 without GTP and arized and transfected into the WT parasites, and single-cell clones were GRA7 alone were incubated similarly. At time intervals, reactions were isolated (Table S3) as described in SI Materials and Methods. sampled and phosphate release was measured using a GTPase colorimetric

− − kit (Innova Biosciences). Pi release from incubation of GTP alone was sub- In Vivo Infection. CD-1, IFN-γR / , and C57BL/6 mice were obtained from tracted from the samples containing GTP. a commercial vendor (Charles River Laboratories) and housed in an American Association for Assessment and Accreditation of Laboratory Animal Care- − − Statistics. Student t tests were performed under the assumption of equal approved facility. The Irgm3 / mice were obtained from Greg Taylor (Duke variance and with a two-tailed test, where P ≤ 0.05 was considered signifi- University, Durham, NC) (30) and bred locally. Female mice between 8 and 10 cant. Graphs were plotted with mean ± SD of the population or SD as noted. wk of age were challenged with freshly lysed tachyzoites and monitored for – 30 d postinfection, as described previously (15). Infections were done in Scatter plots were analyzed using the Mann Whitney test for statistical groups of five mice per strain and repeated two (n = 10) or more times. significance under nonparametric conditions not assuming Gaussian distri- ≤ Animal studies were approved by the Institutional Animal Studies Commit- bution and a two-tailed test, where P 0.05 was considered significant. – – tee (School of Medicine, Washington University in St. Louis). Kaplan Meier plots that were analyzed using the Gehan Breslow Wilcoxon test, where P ≤ 0.05 was considered significant. For the phosphate release ≤ Irga6 Oligomerization Assays. Recombinant Irgs6 was expressed as GST-fusion assay, curves were analyzed by a two-way ANOVA test, where P 0.05. protein, cleaved, and purified as described in SI Materials and Methods. Oligomerization of Irga6 was monitored by 90° light scattering in a total ACKNOWLEDGMENTS. We thank Vern Carruthers and Mae Huynh (Univer- Δ reaction volume of 150 μL containing 40 mM Tris·Cl (pH 8.0), 5 mM MgCl2, sity of Michigan) for the ku80 strain, Greg Taylor (Duke University) for Irgm3−/− mice and antibodies, and members of the Sibley laboratory for and 2 mM DTT (buffer B). Polymerization of 50 μM Irga6 alone was com- helpful comments. Sophie Alvarez (Danforth Plant Sciences Center) per- pared with that of Irga6 combined with different concentrations of GRA7 or formed the MS studies. Wandy L. Beatty (Microbiology Imaging Facility, proteinase K-treated GRA7 (SI Materials and Methods). Proteinase K-treated Washington University in St. Louis) performed the EM studies. This study buffer A [40 mM Tris·Cl (pH 8.0), 15 mM NaCl] was added to the Irga6-only was supported by the National Institutes of Health (Grants AI34036 and reaction as a control. The protein solutions were first equilibrated to 37 °C, AI36629).

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