RecQ helicases in the malaria parasite Plasmodium falciparum affect genome stability, gene expression patterns and DNA replication dynamics

Abstract

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known 'fragile sites'. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes-including a large proportion of vars-showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

Fig 1. Loss of RecQ helicases affects parasite growth.

(A) PfBLM expression in a panel of ΔPfBLM clones compared to wildtype 3D7 parasites. PfBLM transcript levels were determined by real-time PCR analysis using primer pair PfBLM_F/R. In each clone, relative expression of PfBLM was calculated by comparison to the control gene seryl-tRNA-synthetase. In the wildtype 3D7 line this relative expression level was set to 1 and PfBLM expression in each clone was then expressed as a fraction of this level. PCR was carried out in technical duplicate and the complete lack of PfBLM expression in ΔPfBLM clones was confirmed by agarose gel electrophoresis of PCR reaction products. (B) PfWRN expression in a panel of PfWRN-k/d clones compared to wildtype 3D7 parasites. Primer pair PfWRNjp2F/R, which spans a region of the PfWRN locus that should be absent if gene replacement via double homologous recombination had occurred, was used to detect PfWRN expression. Seryl-tRNA-synthetase and fructose bisphosphate aldolase were used as control genes for normalisation, with PfWRN expression being calculated by comparison to the mean expression level of these two genes. As in (A), relative PfWRN expression was then set to 1 in the wildtype 3D7 control and expression in each clone was expressed as a fraction of this level. PCR was carried out in technical duplicate and the low level of PfWRN expression in PfWRN-k/d clones was confirmed by agarose gel electrophoresis. (C) Parasite growth in the ΔPfBLM, PfWRN-k/d and parent lines, assessed by a standard 48h growth assay. Mean of three biological replicates, each conducted in technical triplicate, is shown; error bars are standard error of the mean and statistical significance was determined using one-tailed t-tests (*, p<0.05; **, p<0.01). (D) Parasite growth counted at 48h intervals over two growth cycles in the ΔPfBLM, PfWRN-k/d and parent lines, after seeding parasites at 0.1% parasitaemia. Mean growth in three biological replicates, each conducted in technical triplicate, is shown; error bars are standard error of the mean.

Fig 2. Clone trees reveal a large increase in micro-indels and SV mutations in PfWRN-k/d line.

(A) A clone tree involves regularly cloning out individual parasites, then growing up and sequencing the bulked-up clones, in order to identify potential de novo mutations. Here each blue disk represents a single parasite genome, a coloured square is a de novo mutation. Cloning by limiting dilution randomly selects one individual parasite, possibly with one or more mutations. Bulking up the culture and sequencing will reveal the new mutation. Note that mutations observed in a progeny sample had occurred within the parental generation, at some point before the clonal dilution. (B) Chromosomal locations of all de novo mutations: BPS (or SNP), micro-indels and Structural Variants (SV). The 14 P. falciparum chromosomes are represented with hypervariable regions (subtelomeric and internal) in darker shades. SNPs and micro-indels are scattered throughout the genome, while SVs are found in hypervariable regions. (C) Mutation rate in 3D7, ΔPfBLM and PfWRN-k/d lines. The micro-indel and SV rates increased by 2.3 and 4.2 fold in PfWRN-k/d lines compared to their wild-type counterpart, respectively.

Fig 3. Disruption of RecQ helicases affects genome-wide transcriptional patterns.

(A) Bar graphs showing the number of up-or down-regulated genes (filled and hatched bars respectively) in ΔPfBLM and PfWRN-k/d parasites at ring, trophozoite and schizont stages. (B) Plots showing the genomic locations of all differentially-expressed (DE) genes in ΔPfBLM and PfWRN-k/d parasites compared to locations of PQSs across the 14 chromosomes. Circles representing PQSs are scaled in diameter according to the number found within each ~64kb of the genome, represented by 1° of the 360° in this circular schematic. Overlapping circles occur in places where many PQSs lie within a single segment, making the circle large enough to overlap with adjacent segments. (C) Venn diagrams showing the number of genes differentially expressed in ΔPfBLM (upper panel) or PfWRN-k/d (lower panel) parasites at one or more time points (R, rings; T, trophozoites; S, schizonts). (D) Box-plot showing the percentage tandem-repeat (TR) content of differentially expressed genes in RecQ helicase mutants at rings (R), trophozoites (T) and schizonts (S). Genes are divided into Up and Down regulated subsets. Lines indicate medians, box and whiskers indicate interquartile and full ranges. Shaded boxes represent a statistically significant difference in the mean from that of the null dataset (all genes in the genome) by 2-tailed t-test: *, p<0.05; **, p<0.01; ***, p<0.001. (E) Box-plot as in (A), showing the percentage low-complexity-region (LCR) content of differentially expressed genes in RecQ helicase mutants. (F) Box-plot as in (A), showing distances between PQSs and genes differentially expressed in RecQ helicase mutants.

Fig 4. DNA replication parameters in RecQ helicase mutants.

(A) Representative DNA fibres from synchronous blood-stage parasites. Upper panel: DNA fibres are in blue, IdU in red and CldU in green; lower panel: the IdU and CldU tracks extracted from the upper panel. 50 kb scale bars are indicated. (B, C and D) Comparative analysis of replication fork speed (B) inter-origin distances (C) and asymmetric forks (long fork to short fork ratios) (D) from synchronous blood-stage parasites. Black bars on dot plots indicate median values. The two-tailed Mann-Whitney test was used to calculate the corresponding P values (ns, not statistically significant P value; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). (E) Percentage of symmetric, asymmetric and unidirectional replication forks counted in the wildtype and RecQ helicase mutants. The groups were significantly different by Chi-square test (P < 0.00001).

Fig 5. DNA replication parameters in RecQ helicase mutants challenged with G4 stabilising drugs.

(A, B and C) Comparative analysis of replication fork speed (A) inter-origin distances (B) and asymmetric forks (long fork to short fork ratios) (C) from synchronous blood-stage parasites, treated with 0.75μM TMPyP4 or TMPyP2. Data from wildtype parasites untreated with drugs are also shown for comparison. Black bars on dot plots indicate median values. The two-tailed Mann-Whitney test was used to calculate the corresponding P values (ns, not statistically significant P value; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). (D) Percentage of symmetric, asymmetric and unidirectional replication forks counted in the wild type and RecQ helicase mutants challenged with G4 stabilising drugs. The groups were significantly different by Chi-square test (P < 0.00001).

Fig 6. Schematic summarising the roles of RecQ helicases in P. falciparum.

The schematic shows proposed helicase roles at replication forks (A), transcribed genes (B) and telomeres (C).