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. 2020 Oct 16;11(1):5258.
doi: 10.1038/s41467-020-18991-8.

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Yifan Wang  1 , Lamba Omar Sangaré  1 , Tatiana C Paredes-Santos  1 , Musa A Hassan  2   3   4 , Shruthi Krishnamurthy  1 , Anna M Furuta  1 , Benedikt M Markus  5   6 , Sebastian Lourido  5   7 , Jeroen P J Saeij  8
Affiliations

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Yifan Wang et al. Nat Commun. .

Abstract

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Toxoplasma gondii genome-wide loss-of-function screen in naïve or IFNγ-activated murine bone-marrow-derived macrophages.
a Screening workflow. At least 5 × 108 Cas9-expressing RH parasites were transfected with linearized plasmids containing 10 sgRNAs against every Toxoplasma gene. Transfected parasites were passaged in HFFs under pyrimethamine selection to remove non-transfected parasites and parasites that integrated plasmids with sgRNAs targeting parasite genes important for fitness in HFFs. Subsequently, the pool of mutant parasites was either continuously passaged in HFFs or passaged for three rounds in murine BMDMs that were left unstimulated or prestimulated with IFNγ. The sgRNA abundance at different passages, determined by illumina sequencing, was used for calculating fitness scores and identifying genes that confer fitness in naïve or IFNγ-activated BMDMs. b Timeline for the generation of mutant populations and subsequent selection in the presence or absence of murine IFNγ. Times at which parasites were passaged (P) are indicated. c Correlation between mean parasite gene fitness scores in Naïve BMDMs and HFF control. Orange dots indicate nine high-confidence candidate genes that confer fitness in naïve BMDMs compared to HFFs (Table 1). d Correlation between mean parasite gene fitness scores in IFNγ-activated and Naïve BMDMs. Red dots indicate 16 high-confidence IFNγ fitness-conferring candidate genes in murine BMDMs and orange dots the nine high-confidence genes conferring fitness in naïve BMDMs compared to HFFs (Table 1).
Fig. 2
Fig. 2. Validation of candidate genes that determine parasite fitness in IFNγ-stimulated murine BMDMs.
a–f Murine BMDMs prestimulated with IFNγ (1 or 5 ng/mL) or left unstimulated for 24 h were infected with luciferase-expressing wild-type (WT) parasites or with parasites in which TGGT1_269620 (a, n = 6), GRA45 (b, n = 5), TGGT1_232670 (c, n = 5), TGGT1_263560 (d, n = 5), GRA22 (e, n = 4), or TGGT1_269950 (f, n = 5) was knocked out (MOI of 0.25). Parasite growth for each strain was measured by luciferase assay at 24 h p.i. Parasite growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. g, h Growth competition assay between GFP-positive WT parasites and GFP-negative g Δgra22 or h Δrom1 parasites was performed in murine BMDMs prestimulated with 1 ng/mL IFNγ or left unstimulated for three passages. The percentage of Δgra22 or Δrom1 was determined at the start of the competition and after three passages by plaque assay measuring the GFP-negative plaques vs. total plaques. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between the ratio of WT vs. knockout before and after competition was analyzed with two-tailed paired t-test.
Fig. 3
Fig. 3. Δgra45 parasites have enhanced susceptibility to IFNγ-mediated growth inhibition in rat BMDMs and human THP-1 macrophages.
a, b Murine BMDMs prestimulated with 5 ng/mL of IFNγ for 24 h were infected with luciferase-expressing WT, Δgra45, Δgra22, ΔTGGT1_263560, or ΔTGGT1_269950 parasites (MOI of 0.5) for 1 h and subsequently fixed, permeabilized, and stained for IRGA6 (a) or IRGB6 (b). A representative image from three independent experiments is shown on the left (Scale bar = 5 μm). Quantification of IRGA6/IRGB6 loading on at least 200 vacuoles is presented on the right panel. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. c, d Brown Norway rat BMDMs prestimulated with or without IFNγ (1 ng/mL or 5 ng/mL) were infected with luciferase-expressing WT, Δgra45 (c), Δgra22 (c), ΔTGGT1_263560 (d), or ΔTGGT1_269950 (d) parasites (MOI of 0.25) for 24 h. Parasite growth for each strain was measured by luciferase assay and the growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. e–h PMA-differentiated THP-1 macrophages prestimulated with or without IFNγ (2.5 or 5 ng/mL) were infected with luciferase-expressing WT, Δgra45 (e, n = 4), Δgra22 (f, n = 3), ΔTGGT1_263560 (g, n = 3), or ΔTGGT1_269950 (h, n = 3) parasites (MOI of 0.25) for 24 h. Parasite growth for each strain was measured by luciferase assay and the growth in IFNγ-activated THP-1 macrophages is expressed relative to growth in naïve THP-1 macrophages. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout analyzed with two-tailed paired t-test.
Fig. 4
Fig. 4. In Δgra45 parasites other GRAs mislocalized after secretion into the vacuole.
a, b PBS or NP-40-treated pellet (P) and supernatant (S) fraction of extracellular parasite were detected with antibodies against GRA2 (a) or GRA7 (b). SAG1 was used as the parasite loading control. The image is representative of 4 independent experiments. c–f HFFs infected with indicated parasites for 4 h (e) or 24 h (c, d, f) were stained with antibodies against GRA5 (c), GRA7 (d), or MAF1 (e, f). The images are representative of results from three independent experiments (scale bar = 5 μm). The percentage of vacuoles with only PV-lumen staining was quantified as shown on the right. g HFFs infected with indicated parasites transiently expressing GRA23-HA were stained with antibodies against SAG1 (green) and the HA epitope (red). The images are representative of results from three independent experiments for WT and Δgra45 parasites with two experiments for Δmyr1 parasites (scale bar = 5 μm). The percentage of vacuoles with only PV-lumen GRA23 staining was quantified as shown on the right. h HFFs infected with indicated parasites transiently expressing GRA44-Myc were stained with antibodies against the Myc epitope. The images are representative of results from three independent experiments (scale bar = 2 μm). The percentage of vacuoles with only cytosolic GRA44 staining was quantified as shown on the right. i, j HFFs infected with indicated parasites transiently expressing GRA16-HA-FLAG (i) or GRA24-HA-FLAG (j) were subjected to the immunofluorescent assay. The images are representative of results from three independent experiments (scale bar = 10 μm). The nuclear intensity of GRA16 (i) or GRA24 (j) was quantified as shown on the right. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. Statistical analysis was done by one-way ANOVA with Tukey’s multiple comparisons test (c–f, h–j) and two-tailed paired t-test (g) due to different n between Δmyr1 and Δgra45.
Fig. 5
Fig. 5. The α-Crystallin domain (ACD) and I/VxI/V motifs are critical for the chaperone-like function of GRA45.
a Schematic illustration of the constructs used for generation of the parasite expressing full-length GRA45 or GRA45 with indicated mutations. ACD and TL indicate predicted α-Crystallin domain and transthyretin-like fold, respectively. The conserved VxV or IxV motifs are indicated accordingly. b Extracellular Δgra45 parasites or Δgra45 parasites complemented with C-terminal HA-tagged wild-type or indicated mutant version of GRA45 were fixed with methanol and staining with indicated antibodies. The images are representative of results from two independent experiments (scale bar = 2 μm). c HFFs infected with indicated parasite strains for 24 h were fixed and stained with antibodies against HA epitope. The images are representative of results from two independent experiments (scale bar = 5 μm). d HFFs infected indicated parasite strains for 4 h were stained with antibodies against MAF1. The images are representative of results from two independent experiments (scale bar = 2 μm). e Δgra45 or Δgra45 complemented with wild-type or indicated mutant version of GRA45 were transiently transfected with GRA16-Ty (upper) or GRA24-Ty (lower) expressing plasmids and immediately used to infect HFFs and fixed at 24 h p.i. and subjected to the immunofluorescent assay with antibodies against Ty epitope. The images are representative of at least 40 host cells containing a single parasitophorous vacuole with four or more parasites (scale bar = 10 μm). f Murine BMDMs prestimulated with 5 ng/mL of IFNγ or left unstimulated for 24 h were infected with indicated parasite strains. Twenty-four hours p.i. parasite growth for each strain was measured by luciferase assay. Parasite growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between WT and knockout (or mutant strains) was analyzed with one-way ANOVA with Tukey’s multiple comparisons test and the p values are indicated above the columns.
Fig. 6
Fig. 6. Compared to Δmyr1 parasites, Δgra45 parasites are more susceptible to IFNγ and less virulent in mice.
a Toxoplasma genes that have at least four sgRNAs present after the 3rd passage in naïve BMDM in the three independent screens are rank-ordered according to their IFNγ vs. Naïve BMDM fitness scores. Data are displayed as average fitness scores (black plots) ± SEM (gray lines). b Mean IFNγ vs. Naïve BMDM fitness of GRA45, MYR1, MYR2, MYR3, and MYR4. Data are displayed as mean ± SEM with independent screens (n = 3) indicated by the same color dots (S1: white; S2: blue; S3: red). The significant difference was analyzed with one-way ANOVA with Tukey’s multiple comparisons test. c Murine BMDMs prestimulated with or without IFNγ (1, 5, 10, or 50 ng/mL) were infected with luciferase-expressing WT, Δmyr1, Δgra45, or Δgra45 + GRA45HA parasites (MOI of 0.25) for 24 h. Parasite growth for each strain was measured by luciferase assay and the growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as connecting lines with mean of four independent experiments ± SEM. The significant difference was analyzed with two-way ANOVA with Tukey’s multiple comparisons test. d CD-1 mice were i.p. infected with 100 tachyzoites of WT, Δmyr1, Δgra45, or Δgra45 + GRA45HA parasites and survival of mice was monitored for 30 days. Data are displayed as Kaplan–Meier survival curves (n = 9 mice infected with WT or Δgra45 parasites, n = 5 mice infected with Δmyr1 parasites, and n = 4 mice infected with Δgra45 + GRA45HA parasites). The significant difference was analyzed with Log-rank (Mantel–Cox) test.
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