Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice

Abstract

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

Figure 1

S1823F mutation in ApiAP2 TF of PbNK65 does not alter the progression of infection. (A–D) C57BL/6 mice were inoculated intraperitoneally with WT PbANKA^F-, WT PbNK65^S- and mutant PbNK65 ^F iRBCs (10⁶/mouse). Peripheral parasitemia given as percent of RBC that are infected (A), hemoglobin levels (g/dl) (B), clinical symptoms measured by evaluating motor abilities based on a 10 point scale (higher scores correspond more advanced disease) outlined in methods (C), and survival (D) are shown for each group with time (days) post infection. Each circle in (A–C) represents an individual mouse and lines represent mean values. Data is representative of three independent experiments each carried out with at least 10 mice per group.

Figure 2

S1823F mutation in ApiAP2 TF of PbNK65 does not induce experimental cerebral malaria. (A, B) Mice were inoculated with WT PbNK65^S, mutant PbNK65^F or WT PbANKA^F iRBCs as in Fig. 1 and brains were collected from infected mice and uninfected controls at various time points. Sites of brain hemorrhage are shown as patchy dark blue colorations on brain photos of Evans blue injected mice. Photos are representative of more than 10 mice per group (A). (B) Tissue sections taken from different parts of infected mouse brains were stained with Hematoxylin and Eosin and then evaluated under light microscopy for histological signs of brain pathology. Sites of hemorrhage in 10 × magnified slides are shown with black arrow heads. RBC congested areas and signs of edema are shown in 40 × magnified slides with green and blue arrow heads respectively. 3 mice/group/day were used for collecting tissue sections.

Figure 3

PbNK65^F- and PbNK65^S-infected mice have similar quantities of CD8⁺ T cells and iRBCs in their brains. (A, B) Mice were infected as indicated in Fig. 1. At days 10 or 21 post infection mice were injected intravenously with AF-488 conjugated CD45 specific Abs (in vivo) to label intravascular leukocytes. Brains were harvested shortly after and processed as detailed in methods section. Lymphocytes were then incubated with a second Ab including BV421 labelled CD45 specific Abs (in vitro) which stains both vascular and extravascular leukocytes. CD11b was used to gate out microglia and CD8 was used to gate CD8⁺ T cells. Within the CD8⁺ T cell gate cells that are double positive for both CD45 stains are gated as intraluminal cells (purple gate) and cells that are single positive for only in vitro CD45 stain were gated as adluminal cells (green gate) (A). Absolute counts of parenchymal CD8⁺ T cells are graphed (B). Each circle represents an individual mouse n.s. = P > 0.05 (One way ANOVA with Dunnet’s multiple comparisons test). (C) qPCR comparisons of parasite levels in brain specimens taken at day 21 post infection from mice infected as indicated in Fig. 1. Each circle represents an individual mouse. n.s. = P > 0.05 (Welch’s T-test).

Figure 4

Mutating the ApiAP2 TF of PbANKA to ApiAP2 of PbNK65 does not alter its ECM inducing ability. (A, B) Mice were infected with 10⁶ iRBC/mouse dose of mutant PbANKA^S and WT PbANKA^F parasites microanatomic changes showing dark colored hemorrhagic areas in Evans blue stained brains (A) and histologic changes showing hemorrhagic areas (black arrow heads) and RBC congested capillaries (green arrow heads) in hematoxylin and Eosin stained brain sections are shown (B). (C, D) Two different inoculation doses were used to infect mice and changes in parasitemia (C) and survival (D) are graphed. Each circle represents a single mouse and line represents mean for (C) Data is representative of two independent experiments.