A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

Abstract

Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1^(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1^(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1^(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the "bystander effect". This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1^(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.

Figure 1. The phenotypic characterisation of Ank-1^(MRI61689/+) mice.

The morphology of Ank-1^(MRI61689/+) and Ank-1^{(MRI61689/MRI61689)} erythrocytes under light microscopy with Giemsa stain (a) and scanning electron microscopy (b). The osmotic fragility curve of wild-type and Ank-1^(MRI61689/+) erythrocytes when subjected to osmotic stress (c) (n = 5–7 per group). The in-vitro spleen retention rate of wild-type and Ank-1^(MRI61689/+) erythrocytes when passing through filter beds (d) (n = 3 per group). **Indicates P < 0.01, ***indicates P < 0.001, and all error bars are standard error of mean (SEM).

Figure 2. The identification of Ank-1^(MRI61689)mutation and its effects on transcription.

The sequencing of Ank-1^(MRI61689) mutation, showing a T to A transversion (a). Gel electrophoresis of amplified cDNA product from wild-type, Ank-1^(MRI61689/+) and Ank-1^{(MRI61689/MRI61689)}embryonic livers with primers that spanned the exon 17 to 21 of ankyrin-1 cDNA (primer set 1) (b). The sequencing results of both bands, showing exon skipping in the abnormal transcript of Ank-1^{(MRI61689/MRI61689)}embryonic liver (c). Gel electrophoresis of amplified cDNA product from wild-type, Ank-1^(MRI61689/+) and Ank-1^{(MRI61689/MRI61689)}embryonic livers with primers that contained the predicted 11 bp insertion (primer set 2) (d). Sequencing result showing an 11 bp insertion between exon 17 and 18 cDNA of the Ank-1^(MRI61689) transcript (e). The predicted effect of the insertion on the translation of ankyrin-1, showing a frameshift and a premature chain termination (f).

Figure 3. The effect of Ank-1^(MRI61689)mutation on the Ank-1 expression.

Quantitative PCR showing the ankyrin-1 mRNA levels in both Ank-1^(MRI61689/+) and Ank-1^{(MRI61689/MRI61689)}embryonic liver (a). The protein levels of various cytoskeletal proteins examined with Coomassie (b). Representative Western blot images for ankyrin-1 (ANK1), Band 3, alpha-spectrin (SPTA), beta-spectrin (SPTB), protein 4.2 (P 4.2) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene in RBC membrane (c). The quantitative analysis of ankyrin-1 (d) (n = 3 per group). Error bars indicate SEM.

Figure 4. The response of Ank-1^(MRI61689/+) mice to malaria infection.

The parasite load of wild-type and Ank-1^(MRI61689/+) mice when infected with 1 × 10⁴ P. chabaudi (a) and the associated survival curve (b). Parasite intra-erythrocytic growth was assessed through TUNEL assay at 1–10% parasitaemia during late trophozoite stage, as visualised from immunofluorescent images showing presence of parasites and TUNEL-positive parasites (c). The number of TUNEL-positive parasites in both wild-type and Ank-1^(MRI61689/+) mice were counted and expressed as a percentage (d) (n = 3). For parasite invasion and RBC clearance, The IVET assay was done showing the ratio of infected Ank-1^(MRI61689/+) to wild-type erythrocytes over 36 hours (e) and the relative number Ank-1^(MRI61689/+) and wild-type erythrocyte in both infected and uninfected mice (f) during P. chabaudi infection (n = 6–7). **Indicates P < 0.01, ***indicates P < 0.001, ^## and ^###Indicates P < 0.01 and P < 0.001 respectively when compared to wild-type RBC number in infected mice, whereas ^^^indicates P < 0.001 when compared to Ank-1^(MRI61689/+) RBC number in uninfected mice. Error bars indicate SEM.