The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii

Abstract

Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine-rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells upregulate immunity-related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N-terminal arginine-rich domain, is critical to its function as a virulence determinant.

Fig. 1

A. Construct design of ROP18 mutants. Schematics are not drawn to scale. Helical wheel projections were drawn using previously defined algorithms (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). For the point mutant, six arginines were mutated to glutamate residues as noted by arrows on the helical wheels. B. Western blot analysis of ROP18 mutant expression in transgenic parasites. Parasite lysates were resolved by SDS-PAGE and blotted for rabbit anti-ROP18 (Rb anti-ROP18) and rabbit anti-GRA7 (Rb anti-GRA7) (top) or mAb BB2 against the Ty tag (Mo anti-Ty) expressed at the C-terminus of ROP18 (bottom). Proteins denoted by brackets. In all transgenic lines, ROP18 exists as two species corresponding to full-length protein (higher molecular weight band) and a mature form that is proteolytically cleaved and is devoid of the signal peptide and prodomain (lower molecular weight band).

Fig. 2

ROP18 mutants traffic properly to rhoptry secretory organelles in the parasite. A. Direct immunofluorescence of parasite infected HFFs stained for a parasite surface marker SAG1 with mAb DG52 and for ROP18-Ty with mAb BB2 directly conjugated to Alexa Fluor 488 and 594, respectively. B. Indirect immunofluorescence of parasite infected HFFs. Cells were stained for the rhoptry marker ROP5 with rabbit anti-ROP5 and for ROP18-Ty with mAb BB2 followed by secondary antibodies conjugated to Alexa Fluor 488 and 594, respectively. Scale = 5μm. Images were acquired using wide field epifluorescent microscopy. ROP18 is detected within the rhoptries, but not at the PVM due to extraction with Triton X-100 used to permeabilize the cells.

Fig. 3

ROP18 mutants are secreted into host cells preceding parasite invasion, but fail to traffic to the PV membrane. A. Localization of ROP18 following secretion into evacuoles. Direct immunofluorescence of parasites pretreated with 1μM Cytochalasin D (CytD) to inhibit parasite invasion into HFFs. Samples were fixed 15 min after infection, permeabilized with saponin and stained for a parasite surface marker SAG1 with mAb DG52 and for ROP18-Ty with mAb BB2 directly conjugated to Alexa Fluor 488 and 594, respectively. White arrows denote secretion of ROP18 into the host cell in “evacuoles”. B. Localization of ROP18 on the surface of the PV membrane shortly after invasion. Samples were fixed 15 min after infection, permeabilized with saponin, and stained for a parasite surface marker SAG1 with mAb DG52 and for ROP18-Ty with mAb BB2 directly conjugated to Alexa Fluor 488 and 594, respectively. Images were acquired using wide field epifluorescent microscopy. Scale = 5μm.

Fig. 4

Localization of ROP18 in the PV membrane of mature vacuoles and following transient transfection of host cells. A. Vacuole localization of ROP18 mutants was determined with laser scanning confocal microscopy of indirect immunofluorescence. Infected HFFs were fixed 24 hpi and permeabilized with digitonin. Samples were stained for a parasite vacuole marker GRA7 with rabbit anti-GRA7 and for ROP18-Ty with mAb BB2 followed by secondary antibodies conjugated to Alexa Fluor 594 and 488, respectively. B. Trafficking of ROP18 following transient transfection of COS-7 cells. Cells transiently expression variants of ROP18 were infected with Type III parasites that naturally lack ROP18 expression, followed by culture for an additional 24 hr. Samples were fixed, permeabilized with saponin, and stained for a parasite vacuole marker GRA7 with rabbit anti-GRA7 and for ROP18-Ty with mAb BB2 followed by secondary antibodies conjugated to Alexa Fluor 594 and 488, respectively. Images were deconvolved in Axiovision v4.5 (Carl Zeiss) using the nearest neighbor algorithm and a single Z-slice is shown. Scale = 5μm.

Fig. 5

Kinase activity remains at wild-type levels in select ROP18 mutants. A. Western blot analysis of ROP18-Ty immunoprecipitation with mAb BB2 (Mo anti-Ty). Unbound (denoted as UB) and bound (denoted as B) fractions were resolved by SDS-PAGE and blotted with rabbit anti-ROP18 (Rb anti-ROP18). B. In vitro kinase assay of immunoprecipitated ROP18-Ty from (A) in the presence or absence of the heterologous substrate dMBP (5μg per reaction) indicated by tracking ³²P radiolabeled ATP. C. In vitro kinase assay of recombinant ROP18 in the presence or absence of the heterologous substrate dephosphorylated myelin basic protein (dMBP) (5μg per reaction) indicated by tracking ³²P radiolabeled ATP. Brackets denote autophosphorylated ROP18 and phosphorylated dMBP in (B) and (C).

Fig. 6

Function of ROP18 vacuole-targeting mutants in subversion of the host IRG pathway. A. Indirect immunofluorescence of IFN-γ-activated RAW 264.7 30 min postinfection. Samples were permeabilized with saponin and stained for a member of the host IRG pathway, either Irgb6 with rabbit anti-Irgb6 or Irga6 with mAb 10D7, and a parasite vacuole marker, either GRA5 with mAb Tg17-113 or GRA7 with rabbit anti-GRA7, followed by secondary antibodies conjugated to Alexa Fluor 488 and 59. Scale = 5μm. IRG positive vacuoles tend to stain weakly for GRA5 or GRA77, hence they appear negative in the composite image. B. Quantification of Irgb6 and Irga6 localization to the vacuolar membrane surrounding ROP18 mutants. Means ± S.D. n = 6 samples from 2 combined experiments. Student’s t test, *P < 0.005. C. Western blot analysis of the phosphorylation state of Irga6 during infection with parasites expressing ROP18 mutants. Infected IFN-γ-activated L929 cells were harvested 2 hr postinfection, resolved by SDS-PAGE, and blotted for phosphorylated T102 with rabbit anti-phospho-Irga6 T102 (555), phosphorylated T108 with rabbit anti-phospho-Irga6 T108 (558), ROP18-Ty1 expression with mAb BB2, or total Irgb6 expression with rabbit anti-Irgb6.