Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens
- Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, United Kingdom
- Host-Pathogen Interactions Laboratory, Gulbenkian Institute for Molecular Medicine, Portugal
- Whitehead Institute, Massachusetts Institute of Technology, United States
- Institute of Immunology and Infection Research, University of Edinburgh, United Kingdom
Figures
Figure 1 with 1 supplement
MYR1 and MYR1-dependent factors are not contributing to in vitro Toxoplasma survival in IFN-γ-activated macrophages.
(a) BMDMs were stimulated with 100 U/ml IFN-γ for 24 hr or left untreated, before infection with mCherry-expressing Toxoplasma strains for 24 hr. Cells were fixed and imaged by high-content imaging, and the percentage of infected cells in IFN-γ-treated BMDMs compared to untreated controls is shown. WT refers to PruΔUPRT. The box-plot shows the median value ± SD and the whiskers show minimum and maximum values. Significance was tested with the One-way Anova test with the Benjamini, Krieger and Yekutieli FDR correction, n=5. (b) Schematic of the flow cytometer-based growth competition assay of an IFN-γ-dependent effector (e.g. GRA12). HFFs and BMDMs were co-infected with equal amounts of colourless WT and mCherry-expressing ΔGRA12 or ΔMYR1 strains. BMDMs were stimulated with 100 U/ml IFN-γ for 24 hr or left untreated before infection. The mCherry signal was quantified by flow cytometry analysis and the ratio between the strains after two passages (output) was compared to the input. (c) Growth of competing mutant and WT parasites was assessed by flow cytometry. The normalised ratios of output versus input are shown. The average of two independent experiments with technical triplicates is shown. Bars display mean ± SD. (d) Violin plot of the plaque size of parental Pru∆KU80 (n=202) and derived Pru∆MYR1 (n=95) strains assessed in two independent experiments. The bar represents the median value and representative images are reported on the right. Significance was tested using a two-tailed unpaired Welch’s t-test, ** p<0.01, **** p<0.0001.
-
Figure 1—source data 1
Raw data of the high-content imaging quantification of Toxoplasma-infected BMDMs pre-treated or not with IFN-γ displayed in Figure 1a.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-data1-v1.xlsx
-
Figure 1—source data 2
Raw data of the flow cytometry-based competition assay experiments in BMDMs pre-treated or not with IFN-γ, and human fibroblasts displayed in Figure 1c.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-data2-v1.xlsx
-
Figure 1—source data 3
Combined plaque sizes data of Pru∆MYR1 parasites compared to the parental strain displayed in Figure 1d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-data3-v1.xlsx
-
Figure 1—source data 4
PDF file containing the original plaque images analysed for Figure 1d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-data4-v1.zip
-
Figure 1—source data 5
Original cropped plaque images displayed in Figure 1d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-data5-v1.zip
Figure 1—figure supplement 1
Validation of the created PruΔIST strain.
PCR validation of the PruΔIST strain compared to parental PruΔKu80 (WT).
-
Figure 1—figure supplement 1—source data 1
PDF file containing the original agarose gel image displayed in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-figsupp1-data1-v1.zip
-
Figure 1—figure supplement 1—source data 2
Original agarose gel image displayed in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
ΔMYR1 parasites expand in the murine host and form cysts in vivo.
(a) Mice were infected i.p. with luciferase-expressing Toxoplasma strains, WT-Luc or ΔMYR1-Luc, and whole-body intravital imaging was performed at days 3, 5, and 7 p.i. (b) Representative whole-body intravital imaging. (c) Graph shows the total bioluminescence signal converted to a logarithmic scale from two independent experiments. Statistical differences between the two strains at each timepoint were tested with the Mixed-effects model (REML) with Sidak post-hoc tests (continued lines). Significance for the ΔMYR1-Luc growth over time was tested with a One-Way Anova test (dotted line). Bars display mean ± SEM. Number of mice per group: day 3 p.i.: n=14; day 5 p.i.: n=11; day 7 p.i.: n=8. * p<0.05, *** p<0.001. (d) Representative fluorescent image of a brain cyst from a mouse infected with ΔMYR1-Luc. mCherry-expressing ΔMYR1-Luc parasites were detected by microscopy. Cyst wall was stained with the FITC-conjugated lectin DBA. The scale bar represents 25 µm.
-
Figure 2—source data 1
PDF containing the intravital images displayed in Figure 2b.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-data1-v1.zip
-
Figure 2—source data 2
Original images displayed in Figure 2b.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-data2-v1.zip
-
Figure 2—source data 3
Raw data of the total bioluminescent signal from intravital imaging displayed in Figure 2c.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-data3-v1.xlsx
-
Figure 2—source data 4
PDF containing the original image of the Toxoplasma cyst displayed in Figure 2d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-data4-v1.zip
-
Figure 2—source data 5
Original image of the Toxoplasma cyst displayed in Figure 2d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-data5-v1.zip
Figure 2—figure supplement 1
Validation of the created Luciferase-expressing strains.
PCR validation of the luciferase-expressing WT-Luc and ΔMYR-Luc strains compared to their respective parental strains PruΔKu80 (WT) and PruΔMYR1 (ΔMYR1).
-
Figure 2—figure supplement 1—source data 1
PDF file containing the original agarose gel image displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-figsupp1-data1-v1.zip
-
Figure 2—figure supplement 1—source data 2
Original agarose gel image displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
The in vivo growth defect of ΔMYR1 is rescued by the presence of MYR1-competent parasites, independently of functional host adaptive immunity.
(a) Mice were infected i.p. with a mixed inoculum of Toxoplasma tachyzoites, containing a 20:80 ratio of luciferase-expressing ΔMYR1-Luc to WT or ΔMYR1 strains that do not express luciferase. Growth of ΔMYR1-Luc was monitored at 3, 5, and 7 days p.i. by whole-body intravital imaging. (b) Representative whole-body intravital imaging. (c) Total bioluminescence signal converted to a logarithmic scale from mice co-infected with ΔMYR1-Luc:WT (n=18) or ΔMYR1-Luc:ΔMYR1 (n=17). Graph shows cumulative data from four independent experiments. Bars display mean ± SEM. Significance was tested with a Two-way Repeated Measures ANOVA with Sidak post-hoc tests. (d) IL-12p40, CCL2/MCP-1, TNF and IFN-γ levels were detected in peritoneal lavage and serum of animals infected with mixed Toxoplasma inoculum (20:80 ratio) at day 7 p.i. by ELISA. The violin-plots show the median (continued black line) and quartiles (dotted grey lines). Significance was tested with the Welch’s t-test (TNF, CCL2/MCP-1 and IL-12p40 ELISAs) and Mann-Whitney test (IFN-γ ELISAs). Samples from two to three independent experiments were assayed. Number of samples for CCL2: WT n=13 and ΔMYR1 n=12. Number of samples for other cytokines: peritoneal lavage samples: WT n=8 and ΔMYR1 n=7; serum samples: WT n=4 and ΔMYR1 n=3. Data with higher differences between inocula (CCL2 and IFN-γ) are displayed in a logarithmic scale in Figure 3—figure supplement 1. (e) Representative whole-body intravital imaging at day 7 p.i. of C57BL/6Ntac (WT) or RAG2-deficient (Rag2-/-) mice infected i.p. with a mixed inoculum containing a 20:80 ratio of luciferase-expressing ΔMYR1-Luc tachyzoites to WT or ΔMYR1 strains that do not express luciferase. (f) Total bioluminescence signal converted to a logarithmic scale from mice co-infected with ΔMYR1-Luc:WT (n=10) or ΔMYR1-Luc:ΔMYR1 (n=8). Graph shows cumulative data from two independent experiments. Bars display mean ± SEM. Significance was tested with the Multiple Mann-Whitney test with a False Discovery Rate approach by a Two-stage step-method. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
-
Figure 3—source data 1
PDF containing the intravital images displayed in Figure 3b.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data1-v1.zip
-
Figure 3—source data 2
Original images displayed in Figure 3b.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data2-v1.zip
-
Figure 3—source data 3
Raw data of the total bioluminescent signal from intravital imaging displayed in Figure 3c.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data3-v1.xlsx
-
Figure 3—source data 4
Raw data of the ELISA results displayed in Figure 3d.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data4-v1.xlsx
-
Figure 3—source data 5
PDF containing the intravital images displayed in Figure 3e.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data5-v1.zip
-
Figure 3—source data 6
Original images displayed in Figure 3e.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data6-v1.zip
-
Figure 3—source data 7
Raw data of the total bioluminescent signal from intravital imaging displayed in Figure 3f.
- https://cdn.elifesciences.org/articles/102592/elife-102592-fig3-data7-v1.xlsx
Figure 3—figure supplement 1
Infection with ΔMYR1 parasites elicits less IFN-γ and CCL2 compared to wild-type paraites.
(a–c) IFN-γ at peritoneal lavage (a) and CCL2 at both peritoneal lavage (b) and serum (c), were detected by ELISA at day 7 p.i. with mixed Toxoplasma inocula (20:80 ratio) of luciferase-expressing ΔMYR1-Luc to WT or ΔMYR1 strains that do not express luciferase. The violin-plots display data shown in Figure 3d converted to a logarithmic scale, with the median (continued black line) and quartile values (dotted grey lines) highlighted. Significance was tested with the Mann-Whitney test for IFN-γ, and by Welch’s t-test for CCL2 data. The number of biological replicates (n) in each graph is the same as mentioned in the original graphs on Figure 3d. *** p<0.001, **** p<0.0001.
Figure 4
Schematic of the difference between single-strain Toxoplasma ΔMYR1 infections and CRISPR pool infections in vivo.
ΔMYR1 parasites do not expand within the peritoneum and are rapidly cleared (left panel). ΔMYR1 parasites within a CRISPR pool are rescued by MYR-competent parasites in a paracrine manner (right panel). MYR-dependent factors cross the MYR-translocon and affect growth within the peritoneal environment in trans, through cytokine secretion and recruitment of host cells. However, Toxoplasma virulence factors that act at the cell-autonomous level, such as GRA12, cannot be rescued in trans.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/102592/elife-102592-mdarchecklist1-v1.docx
-
Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/102592/elife-102592-supp1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens
eLife 13:RP102592.
https://doi.org/10.7554/eLife.102592.3
