Research ArticleIMMUNOLOGY

A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity

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Science Advances  05 Feb 2020:
Vol. 6, no. 6, eaaw6957
DOI: 10.1126/sciadv.aaw6957
  • Fig. 1 ApiAP2F mutation alters the DNA sequence specificity of the PBANKA_011210 ApiAP2 protein.

    (A) DNA sequences that received the highest hit in DNA binding microarray analysis of the first domain of ApiAP2S- and ApiAP2F-mutated PBANKA_011210 ApiAP2 proteins. (B to D) PbNK65S or PbNK65F parasites were used to infect C57BL/6 mice. RNA-seq analysis was carried out from blood samples of infected mice. Principal components analysis (B), genes whose expressions were significantly altered between the two groups (C), and the pie chart representation of multigene families that these genes belong to (D) are shown. Numbers under bar graphs in (C) indicate the gene annotation in (PBANKA_######) format. Gene families for each gene are indicated in parenthesis. Pseudogenes are shown with asterisks. (E and F) Density graphs showing the relative distribution of the middle hexamer regions of DNA sequences in (A) in the 1500-bp promoter region of randomly sampled (E) or differentially regulated (F) genes.

  • Fig. 2 PbNK65F infection leads to stronger IFN-γ and TNF-α responses in infected mice.

    (A to C) C57BL/6 mice were infected with either PbNK65F or PbNK65S using 106 iRBCs per mouse delivered intraperitoneally. Changes in survival (A), parasitemia (B), and hemoglobin levels (C) were monitored during the course of infection. Each circle represents an individual mouse. Data represent three independent experiments each carried out with at least 10 animals per group. (D) Sera collected at different time points of mice infected, as described above. Time-dependent changes in serum levels of various cytokines and chemokines are quantified using a multiplex enzyme-linked immunosorbent assay (ELISA) strategy. Data represent two independent experiments. Each circle refers to a single mouse and lines denote mean values. Statistically significant differences are shown with asterisk (* = 0.01 < P ≤ 0.05; ** = 0.001 < P ≤ 0.01; *** = 0.0001 < P ≤ 0.001). Statistical significance was calculated using Welch’s t test (F)

  • Fig. 3 PbNK65F-induced IFN-γ is linked to formation of stronger adaptive immune responses.

    (A to C) C57BL/6 mice were infected with either PbNK65F or PbNK65S using 106 iRBCs per mouse delivered intraperitoneally. Spleens were harvested at different time points, and GC B cells within the B cell gate (A), plasma cell (PC) lineage cells within the splenocyte gate (B), and TFH within the TH gate (C) are analyzed in flow cytometry. Representative flow cytometry plots (left) and bar graphs of actual cell numbers (right) are shown for each cell type. Data are pooled from four independent experiments. p.i., post-infection. (D to F) Age- and sex-matched IFN-γ KO and WT (C57BL/6) mice were infected side by side with either PbNK65F or PbNK65S as outlined above. Mice were sacrificed at day 19 after infection, and their spleens were analyzed. Each circle represents an individual mouse. Bars indicate the mean values. Statistical significance was calculated using Welch’s t test (A to C) or one-way analysis of variance (ANOVA) with Sidak’s multiple comparison test (D to F). Significant values are shown with asterisks (* = 0.01 < P ≤ 0.05; ** = 0.001 < P ≤ 0.01; *** = 0.0001 < P ≤ 0.001).

  • Fig. 4 Infection with PbNK65F causes more persistent GC response and less disruption in splenic architecture.

    Mice were infected as outlined in Fig. 2. Spleens harvested at different time points were stained with hematoxylin and eosin (H&E) to show overall splenic structure, B220/CD4 to localize follicles, PNA/IgM to point out the GC foci, and Ki-67 to determine the proliferation activity in GCs. Representative spleen sections belonging to either PbNK65S parasite– or PbNK65F parasite–infected mice are shown for day 10 (A) and day 19 (B). Arrowheads and dashed circles indicate GC foci and proliferative activity, respectively. (C) Histopathologic evaluation of the spleen samples taken on days 6, 10, 15, and 19 after infection of mice with either PbNK65S or PbNK65F and processed as described above. At least two mice were evaluated per group per day. p.i., postinfection.

  • Fig. 5 PbNK65F infection leads to a stronger TH1 type immune response.

    (A to G) C57BL/6 mice were infected with either PbNK65F or PbNK65S as described in Fig. 2. Sera were collected at days 7 and 19 after infection and were assayed for total IgM and IgG (A) using ELISA kits. Parasite-specific IgM (B), IgG (C), IgG1 (D), IgG2b (E), IgG2c (F), and IgG3 `(G) were quantified on ELISA plates coated with iRBC lysates of WT parasite– or mutant parasite–infected mice. Arbitrary units (AU) were calculated using a titration curve generated by serially diluting a mixture of serum samples as explained in Materials and Methods. n.s. = P > 0.05; * = 0.01 < P ≤ 0.05; ** = 0.001 < P ≤ 0.01; *** = 0.0001 < P ≤ 0.001 (Welch’s t test).

  • Fig. 6 The SNP affects the disease progression and formation of immunologic memory in infected mice.

    (A to D) C57BL/6 mice were infected with 106 iRBCs (intraperitoneally) coming from either PbNK65F- or PbNK65S-infected donors. A group of mice were treated with chloroquine (10 mg/kg) daily between days 8 and 16 after infection, after which the treatment was ceased and disease progression was observed. The experimental design is illustrated in (A). Progression of parasitemia (B) and Kaplan-Meier survival curve (C) for the initial infection are shown. (D) In a parallel experiment, some of the surviving mice (after being confirmed as parasite free by blood smear) were re-infected on day 37 and observed until day 80. Kaplan-Meier survival chart (left) of the groups of mice that were infected 106 iRBCs (intraperitoneally) as outlined in the table (right) is shown. (E to G) C57BL/6 (E and F) or RAG-2 KO (G) mice were infected with 102 iRBCs (intraperitoneally) taken from either PbNK65S- or PbNK65F-infected donors. The parasitemia (E) and survival (F) curves of infected C57BL/6 mice and parasitemia curves of infected RAG-2 KO mice are shown. Data represent two independent experiments each carried out with 10 to 15 mice per group. Circles refer to individual mice.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaaw6957/DC1

    Fig. S1. CRISPR-Cas9 gene modification strategy is outlined.

    Fig. S2. The SNP in PBANKA_011210 ApiAP2 TF does not interfere with the progression of sexual and pre-erythrocytic stages of the P. berghei life cycle.

    Fig. S3. Flow cytometry gating strategy.

    Fig. S4. Graphs comparing mice infected with PbNK65F and PbNK65S.

    Table S1. Summary of changes observed between the WT and CRISPR-Cas9–mutated parasites based on whole-genome sequencing analysis.

    Supplementary information 2. The 900-nucleotide synthetic sequence.

    Data file S1. Supplementary information 1: DNA binding analysis of ApiAP2 domain 1.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. CRISPR-Cas9 gene modification strategy is outlined.
    • Fig. S2. The SNP in PBANKA_011210 ApiAP2 TF does not interfere with the progression of sexual and pre-erythrocytic stages of the P. berghei life cycle.
    • Fig. S3. Flow cytometry gating strategy.
    • Fig. S4. Graphs comparing mice infected with PbNK65F and PbNK65S.
    • Table S1. Summary of changes observed between the WT and CRISPR-Cas9–mutated parasites based on whole-genome sequencing analysis.
    • Supplementary information 2. The 900-nucleotide synthetic sequence.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.csv format). Supplementary information 1: DNA binding analysis of ApiAP2 domain 1.

    Files in this Data Supplement:

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