Analysis of genome instability and implications for the consequent phenotype in Plasmodium falciparum containing mutated MSH2-1 (P513T)

Abstract

Malarial parasites exhibit extensive genomic plasticity, which induces the antigen diversification and the development of antimalarial drug resistance. Only a few studies have examined the genome maintenance mechanisms of parasites. The study aimed at elucidating the impact of a mutation in a DNA mismatch repair gene on genome stability by maintaining the mutant and wild-type parasites through serial in vitro cultures for approximately 400 days and analysing the subsequent spontaneous mutations. A P513T mutant of the DNA mismatch repair protein PfMSH2-1 from Plasmodium falciparum 3D7 was created. The mutation did not influence the base substitution rate but significantly increased the insertion/deletion (indel) mutation rate in short tandem repeats (STRs) and minisatellite loci. STR mutability was affected by allele size, genomic category and certain repeat motifs. In the mutants, significant telomere healing and homologous recombination at chromosomal ends caused extensive gene loss and generation of chimeric genes, resulting in large-scale chromosomal alteration. Additionally, the mutant showed increased tolerance to N-methyl-N'-nitro-N-nitrosoguanidine, suggesting that PfMSH2-1 was involved in recognizing DNA methylation damage. This work provides valuable insights into the role of PfMSH2-1 in genome stability and demonstrates that the genomic destabilization caused by its dysfunction may lead to antigen diversification.

Keywords: MutS homologue 2; PfMSH2-1; Plasmodium falciparum; malaria; mutation accumulation test; mutator.

Fig. 1.

Mutation rates estimated with the indel data obtained from MA clones of Mut and WT. (a) Genomic category-dependent mutation rates. (b) Allele size-dependent mutation rates. (c) Repeat motif-dependent mutation rates. There was a statistically significant difference between means with different alphabets (P<0.05; Tukey–Kramer test) when compared within the same parasite-type clones. Error bars represent standard error of the mean. The asterisk indicates a statistically significant difference when comparing means between Mut and WT (^* P<0.05, ^** P<0.01; Welch’s t-test).

Fig. 2.

Distribution of deletion and insertion mutation rates at ‘AT’ repeats by repeat numbers. The mutation rates were calculated for each class of five ‘AT’ repeats in Mut (a) and WT (b). Error bars represent standard error of the mean. The asterisk indicates a statistically significant difference between insertion and deletion mutation rates (^* P<0.05, ^** P<0.01; Welch’s t-test).

Fig. 3.

Circos plots depicting genome variations in Mut (a) and WT (b). The outermost track shows the 14 chromosome end regions of Plasmodium falciparum in which the letters ‘L’ and ‘R’ denote left and right ends, respectively. One scale label indicates 4 kbp. The locations of genes are shown with black lines. The inner six rings represent chromosome ends of MA clones represented as follows: intact sub-telomere regions (light grey), intact core genome regions (grey), large deletions (dark red), and large deletions shared with progenitor (light blue) or some MA clones (light blue with asterisk). Solid orange circles indicate breakpoints repaired by telomere healing. Ribbons show homologous recombination, and the colour indicates the chromosome from which the donor region to repair was derived. Open orange circles indicate breakpoints where the repair mechanism was not identified.

Fig. 4.

Susceptibility of Mut and WT to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). The ring-stage synchronized parasitized RBCs (pRBCs) were grown in malaria complete medium with 0 µg ml⁻¹ (control), 1.5 µg ml⁻¹ and 3 µg ml⁻¹ MNNG for 24 h, and microscopic observation was performed 2‒5 days post-exposure. (a) The representative cell morphology of MNNG-treated and untreated parasites. The parasites in the control group developed normally, while those in the 1.5 µg ml⁻¹ and 3 µg ml⁻¹ MNNG-treated groups showed abnormal morphology with reddish colour and indistinct cytoplasm and nuclei. This abnormal morphology was not observed in the control group. Conversely, abnormal morphology of parasites with cell shrinkage due to overgrowth was observed on day 5 in the control group. (b) The pRBCs with normal morphology were counted under a microscope 2–5 days post-exposure. The percentage survival of the MNNG-exposed group relative to that of the control group was assessed and plotted. Error bars represent standard error of the mean. The asterisk indicates a statistically significant difference when comparing means between Mut and WT (^* P<0.05, ^** P<0.01; Welch’s t-test).