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Toxoplasma GRA7 Effector Increases Turnover of Immunity-Related Gtpases and Contributes to Acute Virulence in the Mouse

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Toxoplasma GRA7 Effector Increases Turnover of Immunity-Related Gtpases and Contributes to Acute Virulence in the Mouse 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 Toxoplasma gondii 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 pathogen-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 macrophages 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 cytosol 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.
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