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. 2010 Dec 16;8(6):484-95.
doi: 10.1016/j.chom.2010.11.005.

Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence

Sarah J Fentress  1 , Michael S Behnke, Ildiko R Dunay, Mona Mashayekhi, Leah M Rommereim, Barbara A Fox, David J Bzik, Gregory A Taylor, Benjamin E Turk, Cheryl F Lichti, R Reid Townsend, Wei Qiu, Raymond Hui, Wandy L Beatty, L David Sibley
Affiliations

Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence

Sarah J Fentress et al. Cell Host Microbe. .

Abstract

Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.

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Figures

Figure 1
Figure 1. ROP18 promotes growth in Gr1+ inflammatory monocytes
(A) In vivo parasite expansion following i.p. injection with 200 parasites. Comparison of Type I (ROP18 expressing), type III (ROP18-deficient), transgenic type III parasites expressing active type I ROP18 (Type III + ROP18) or kinase-dead ROP18 (Type III + ROP18 D/A) (Taylor et al., 2006). Mean ± SD, n=3 animals (representative of two experiments), (†=succumbed). (B) Flow cytometry analysis of cell recruitment to the peritoneum at 6 days postinfection. Plots from a single representative animal. Numbers indicate % Gr1+ F480+ inflammatory monocytes. (C ) Number of Gr1+ F4/80+ monocytes in the peritoneum at 6 days post infection. Mean ± SD, n = 3 animals (*P < 0.005, Student's t test), representative from 3 experiments. (D) Gr1+/F4/80+ monocytes from C were gated and analyzed for infection with T. gondii based on staining for surface antigen 1 (SAG1). Plots represent a single representative animal. Numbers indicate % Gr1+ inflammatory monocytes that were infected. (E) Number of infected Gr1+ F4/80+ monocytes in the peritoneum at 6 days postinfection. Mean ± SD, n = 3 animals (*P < 0.005, Student's t test), representative from 3 experiments. (F) In vitro parasite clearance in elecited Gr1+ monocytes, which were identified by surface staining. Mean ± SEM, n=3 experiments (**P < 0.0005, Student's t test).
Figure 2
Figure 2. ROP18 mediates protection of the parasite-containing vacuole
Electron microscopy of parasite-containing vacuoles within Gr1+ monocytes at 2 h postinfection. Type I and Type III + ROP18 parasites resided in normal vacuoles (first column), Type III and Type III + ROP18 D/A vacuoles showed vesiculation of the membrane (arrows, center column), followed by degeneration (final column). Scale=1 μm.
Figure 3
Figure 3. ROP18 blocks Irgb6 loading
(A) Immunofluorescence localization of Irgb6 on the parasite-containing vacuole in infected Gr1+ monocytes examined at 30 min post infection. Irgb6 localized with rabbit polyclonal Ab (Alexa Fluor 488, green); GRA5 localized with mAb Tg 17-113(Alexa Fluor 594, red); and nuclei stained with DAPI (blue). Scale = 5 μm. (B) Quantification of Irgb6 localization to the PV in Gr1+ monocytes. Mean ± SEM, n=3 experiments (*P < 0.005, Student's t test). (C) CryoimmunoEM localizing Irgb6 to the parasite-containing vacuole membrane (arrowheads) in macrophages. Scale = 0.5 μm.
Figure 4
Figure 4. ROP18 coprecipitates and phosphorylates IRGs
(A) Immunoprecipitation (IP) of Irgb6 from macrophages infected with type III, type III expressing active ROP18 (Type III + ROP18), or type III expressing the kinase-dead form of ROP18 (Type III +ROP18 D/A). IP with specific rabbit antiserum (Rb) against Irgb6; proteins were resolved by SDS-PAGE and Western blotted with goat (Gt) αIrgb6 and mouse (Mo) αTy to detect ROP18. Doublet of Irgb6 corresponds to an unknown modification. (B) Phosphorylation of ROP18 and Irgb6 in a kinase reaction in vitro. Immunoprecipitated ROP18 (Mo αTy) and Irgb6 (Rb αIrgb6) were incubated with 32P-ATP in a kinase reaction, resolved by SDS-PAGE, and signals detected by phosphorimaging. * corresponds to Irgb6. Multiple autophorphorylation bands in ROP18 represent different processing forms as described previously (Qiu et al., 2008); the intensity of labeling characteristically changes in the presence of substrate. ROP18 migrates slightly faster in the presence of Irgb6; however, no other proteins were detected, nor modifications other than phosphorylation, were detected by MS analysis. (C) In vivo phosphorylation of Irgb6 by ROP18. IFN-γ-activated macrophages were labeled with 32P orthophosphate, infected for 30 min, and IP with Rb αIrgb6. * corresponds to Irgb6 as verified by MS/MS identification. Additional bands reflect contaminating host kinase activity that is unrelated to ROP18. (D, E) IP of Irga6 (D) or Irgb10 (E) from IFN-γ-activated macrophages with either mouse antiIrga6 (Mo αIrgb6) or rabbit anti-Irgb10 (Rb αIrgb10) at 1 h post infection. Proteins were resolved by SDS-PAGE and blotted with rabbit anti-ROP18 (Rb αROP18) or mAb to the Ty epitope tag (Mo α ROP18-Ty). Multiple bands in ROP18 represent processing as described previously (Qiu et al., 2008). (F, G) Phosphorylation of Irga6 (F) or Irgb10 (G) by ROP18 in an in vitro kinase reaction. ROP18 was immunoprecipitated (IP: Mo αTy) from extracellular parasites expressing either active kinase (ROP18) or the inactive mutant (ROP18 D/A) and combined with immunoprecipitated Irga6 (IP: Mo αIrga6) or Irgb10 (IP: Rb αIrgb10) isolated from IFN-γ activated macrophages. Proteins were incubated with 32P-ATP in a kinase reaction, resolved by SDS-PAGE, and signals detected by phosphorimaging. Multiple bands in ROP18 represent processing as described previously (Qiu et al., 2008).
Figure 5
Figure 5. ROP18 substrate preference identifies a motif in SWI of IRGs
(A) Peptide array probing ROP18 substrate specificity by radiolabeled kinase assay. (B) Sequence WebLogo of ROP18 substrate preference, phosphoacceptor site at position 0. Top five amino acids at each position are shown, height is reflective of probability. (C) ClustalX alignment of select IRG proteins. Numbering based on Irgm1. SWI, switch region I; SWII, switch region II. (D) MEME sequence logo of region surrounding SWI from the IRGs shown in C. Full MEME motif alignments shown in Figure S4 A,B. (E) Mass spectrometry of in vitro phosphorylation of a peptide of Irgb6 by ROP18. MS1 spectra of unphosphorylated peptide (m/z 1247.6123), monophosphorylated, and mixed diphosphorylated peptide. Supporting data in Figure S4 C,D. (F) Nano-LC-Orbitrap-MS of a tryptic phosphopeptide derived from recombinant Irgb6 incubated with ROP18. The MS2 spectrum was acquired from the [M+2H]2+ ion (left inset) in the Orbitrap. The high resolution CID spectrum obtained for this phosphopeptide showed a series of b and y ions that were consistent with the amino acid sequence shown (* denotes the phosphorylated residue). Complete methods and supporting data are given in the Supplemental Information (Figure S4, Table S2).
Figure 6
Figure 6. ROP18 is necessary to block Irgb6 recruitment
(A) Deletion of ROP18 (Δrop18) in a Δku80 type I strain as shown by Western blotting using rabbit anti-ROP18 (Rb αROP18) and rabbit anti-actin (Rb αACT1). Faint upper band represents processing form of ROP18 (Qiu et al., 2008). (B) Survival of mice infected by i.p. inoculation with type I Δku80 strain and Δrop18 KO. Representative of two similar experiments (P < 0.0001), n = 8 animals each. (C) Deletion of ROP18 resulted in increased recruitment of Irgb6 in IFN-γ-activated peritoneal macrophages at 30 min post infection. Irgb6 localized with rabbit polyclonal Ab (Alexa Fluor 488, green); GRA5 localized with mAb Tg 17-113 (Alexa Fluor 594, red); and nuclei stained with DAPI (blue). Mean ± SEM, n=3 experiments (*P < 0.005, Student's t test). (D) Immunofluorescence staining of IFN-γ-activated RAW 246.7 cells 48 h after addition of siRNAs targeting Irgb6 compared to control siRNA. Thin white lines indicate path for intensity profile analysis. Scale = 20 μm. (E) Mean intensity analysis of cells treated for RNAi. Whole cell intensities were measured using Volocity software, and mean intensities for each cell were plotted. Population mean, *P < 0.005, Mann-Whitney test. (F) Enhanced clearance of Δrop18 parasites in IFN-γ-activated RAW 264.7 macrophages was overcome by specific depletion of Irgb6 by RNAi. Mean ± SEM, n=3 experiments, *P < 0.005, Student's t test.
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