Article Figures & Data
Figures
- FIG 1
In vivo and in vitro (invasion) parasite growth rates, host survival rates, and host body weights after infection with parasites carrying PyEBL C741 or Y741 allele. (A) Parasitemias of parental and allele-exchanged P. yoelii clones. (B) Mouse survival rates after infection with different parasite clones. (C) Body weights of mice infected with parasites as indicated. (A to C) Mean values + standard deviations (SD) from 5 mice in each group. One-way analysis of variance (ANOVA): *, P < 0.05, and ***, P < 0.001, for all comparisons. (D) Smears of in vitro cultures with RBCs reinvaded by P. yoelii N67 or N67C ring-stage parasites. (E) Parasitemias of N67 and N67C parasites 18 h after addition of purified merozoites to red blood cells. (F) Percentages of ring, trophozoite, and schizont stages 18 h after invasion. (G) Parasitemias over time after in vitro invasion of N67 and N67C merozoites. (H) Percentages of ring, trophozoite, and schizont stages at different time points after invasion. (E and G) Mean values + SD were plotted.
- FIG 2
PyEBL protein expression and localization in merozoites and infected red blood cells. (A to L) IFA was performed as described in Text S1. Red, anti-PyEBL antibody; blue, DAPI (4′,6-diamidino-2-phenylindole); green, anti-TER119 antibody for red blood cell (RBC) membrane. (A to F) PyEBL expression in merozoites of N67, N67C, and allele-exchanged parasites as marked. (G to L) PyEBL expression in RBCs infected with ring or trophozoite stages. Note weak staining of iRBC membranes compared with those of uninfected RBCs. (M to R) Images from N67 and N67C parasites with HA::Flag-tagged EBL. Green, signals from anti-HA antibody; red, anti-TER119 antibody; blue, DAPI. (M and N) Membrane-bound vesicles (green dots) in the cytoplasm of red blood cells infected with a single parasite (M) or three N67 parasites (N). (O and P) Similar images from N67C-infected RBCs. (Q and R) Schizonts of N67 (Q) or N67C (R) parasites.
- FIG 3
Protein expression and phosphorylation of iRBCs carrying the Y741 or C741 allele. (A) Ponceau S staining of iRBC lysates and Western blotting detection of PyEBL protein expression using anti-PyEBL antibodies in iRBC lysates of mixed parasite stages. Ponceau S staining of total proteins, and anti-histone H3 antibody (α-H3) to show similar protein loading. (B) The same staining as described for panel A using lysates of schizont cultures incubated overnight. (C) Ponceau S staining of iRBC lysates and Western blotting to detect PyEBL protein expression using anti-HA antibody. (D) Detection of PyEBL in pellets and supernatants (Sup) from iRBCs of N67 and N67C parasites. (E) Western blot detection of band 3 protein immunoprecipitated in anti-HA antibody protein complex from saponin-lysed N67 or N67C iRBC supernatants (Sup) or pellets. (F) Western blot detection of phosphotyrosine (pTyr) proteins in samples as described for panel E. (G) Western blot detection of TER119 in samples as described for panel E.
- FIG 4
Dynamics of osmotic lysis of RBCs and iRBCs carrying Y741 or C741 allele. (A) Dynamics of hemolysis of uninfected RBCs (uRBCs) and RBCs infected with N67 and N67C schizonts in different NaCl concentrations (0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%). (B) The same experiments described for panel A using NaCl concentrations from 0.5% to 0.7% (0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, and 0.7%). (C and D) The same experiments as described for panel B comparing N67 and N67Y-C (C) and N67C and N67CC-Y (D) parasites. Experiments were repeated independently (different mice at different times) at least twice. Mean values and SD are from 3 to 5 measurements. Two-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 5
Spleen pathology and serum cytokine/chemokine levels after infection with parasites carrying C741 or Y741 allele. (A to J) Images of H&E-stained spleen tissues from mice infected with different allele-exchanged parasite clones, magnified at 4× (A, C, E, G, and I) and 20× (B, D, F, H, and J). (K to P) Cytokine/chemokine levels in mice infected with parasites with either the C741 or Y741 allele on day 4 postinfection. Cytokines/chemokines in plasma collected from mice infected with N67, N67C, N67CC-Y, and N67CC-C parasites on day 4 postinfection were measured using a bead array (Bio-Rad). Mean values and standard errors of the means (SEM) from 4 or 5 mice. One-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 6
PyEBL Y741 allele is associated with elevated type I interferon response, differentiation of T-helper cells, and Ig isotype switching. (A) Enriched biological process ontologies from differentially expressed genes in spleens of mice infected with parasites containing the Y741 (N67CC-Y) or C741 (N67C) allele. Lists of differentially expressed genes with a cutoff at Log2(norm.reads x/norm.reads y) ≥ 1 of normalized reads per kilobase per million (RPKM) were uploaded onto Gene Ontology (GO) tools (http://geneontology.org/page/go-enrichment-analysis), and search results in GO tree view images from the website are presented. Enriched terms are for genes expressed at higher levels in N67CC-Y-infected mice than in N67C-infected mice. differentiat., differentiation; resp., response. (B) Mice infected with parasites carrying the Y741 allele express consistently higher levels of Stat1 (t test, P = 0.045) and Stat2 (P = 0.002) transcripts from RNA-seq. Diff.LSmean, differences in least-squares means. (C) Protein phosphorylation detected by Western blotting using anti-pSTAT1 and anti-pSTAT2 antibodies. Bone marrow-derived macrophages (BMDMs) were incubated with red blood cells infected with parasites having the Y741 (N67 and N67CC-Y) or C741 (N67C and N67Y-C) allele for the indicated times. β-Actin is included as a protein loading control. (D) Western blotting of spleen lysates on days 1 and 4 postinfection with parasites as indicated. Proteins were detected using antibodies as indicated on the left side of the gels. β-Actin is included as a protein loading control. (E) Levels of IFN-α in the blood of mice infected with N67, N67C, N67Y-C, and N67CC-Y parasites on day 1 postinfection. (F) Levels of IFN-β in the blood of mice infected with N67, N67C, N67Y-C, and N67CC-Y parasites on day 1 postinfection. For panels E and F, mean values and SEM are from 3 to 5 replicates. Mann-Whitney U test: **, P < 0.01; ***, P < 0.001.
- FIG 7
PyEBL Y741 allele is associated with increased annexin V binding and PS-CD36-mediated phagocytosis. (A) Percentages of RBCs infected with individual parasite lines carrying the Y741 or C741 allele phagocytized by bone marrow-derived dendritic cells (BMDCs). (B) Mean percentages of phagocytized iRBCs from C741 or Y741 parasite groups. (C and D) The same experiments as described for panels A and B but performed using bone marrow-derived macrophages (BMDMs). (E) Percentages of iRBCs bound by annexin V. RBCs infected with different parasites are as marked. NI, noninfected RBCs. (F) Percentages of phagocytized RBCs or iRBCs with or without blocking antibodies. Anti-CD16/CD32 antibodies were added to block nonspecific binding through CD16/CD32. Iso, isotype antibody control. (G) Percentages of red blood cells infected with N67 or N67C schizonts stained by anti-complement 3 (C3) antibody and counted using flow cytometry. (H) Serum levels of IgG and IgM in uninfected mice (Unin) and mice infected with N67 or N67C on day 6 postinfection, measured using ELISA with 1:100,000 antibody dilution. OD, optical density. (I) The same measurements as described for panel G but using plates coated with P. yoelii parasite lysates (1:64 dilution). Mean values and SD are from 3 to 5 replicates. ANOVA and Mann Whitney (B and D) tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Supplemental Material
FIG S1
Diagram of PyEBL protein domains, linear constructs for allelic exchange, and confirmation of C741Y allelic exchange. Download FIG S1, TIF file, 0.9 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S1
Summary of phenotypic changes in PyEBL C741Y allele-exchanged parasites. Download Table S1, DOCX file, 0.01 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S2
CRISPR/Cas9-mediated HA::Flag tagging of EBL proteins in Plasmodium yoelii nigeriensis N67 and N67C parasites. Download FIG S2, TIF file, 0.3 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S3
Detection of Ly6G+ cells in the spleens and lungs of mice infected with N67, N67C, N67CC-Y, and N67Y-C parasites and additional enriched biological process ontologies for genes expressed at lower levels in mice infected with C741 parasites. Download FIG S3, TIF file, 15.9 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S2
(A) Read counts, relative gene expression levels, and comparisons of gene expression for mice infected with N67, N67C, and allele-exchange parasites day 4 postinfection. (B) Genes used for GO term enrichment analysis comparing N67CC-Y and N67C parasites, leading to the plot in Fig. 6A. Download Table S2, XLSX file, 7.0 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S3
(A) Activated and inhibited upstream activators between N67CC-Y and N67C (N67CC-Y-N67C). (B) Differentially expressed genes between parasites with PyEBL C741 or Y741 allele. Download Table S3, XLSX file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S4
Py235 and PyEBL protein expression and localization in merozoites using rabbit anti-PyEBL serum and anti-Py235 monoclonal antibody. Download FIG S4, TIF file, 1.7 MB.
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TEXT S1
Supplemental results and methods. Download Text S1, DOCX file, 0.05 MB.
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FIG S5
Summary of putative mechanisms of differential immune responses mediated by PyEBL C741 and Y741 alleles. Download FIG S5, TIF file, 1.3 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S4
Oligonucleotides and sequences used in construction of gene knockout cassettes and typing of recombinant parasites. Download Table S4, XLSX file, 0.01 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
