Functional analysis of sirtuin genes in multiple Plasmodium falciparum strains

Abstract

Plasmodium falciparum, the causative agent of severe human malaria, employs antigenic variation to avoid host immunity. Antigenic variation is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. The histone-modifying 'sirtuin' enzymes PfSir2a and PfSir2b have been implicated in this process. Disparate patterns of var expression have been reported in patient isolates as well as in cultured strains. We examined var expression in three commonly used laboratory strains (3D7, NF54 and FCR-3) in parallel. NF54 parasites express significantly lower levels of var genes compared to 3D7, despite the fact that 3D7 was originally a clone of the NF54 strain. To investigate whether this was linked to the expression of sirtuins, genetic disruption of both sirtuins was attempted in all three strains. No dramatic changes in var gene expression occurred in NF54 or FCR-3 following PfSir2b disruption, contrasting with previous observations in 3D7. In 3D7, complementation of the PfSir2a genetic disruption resulted in a significant decrease in previously-elevated var gene expression levels, but with the continued expression of multiple var genes. Finally, rearranged chromosomes were observed in the 3D7 PfSir2a knockout line. Our results focus on the potential for parasite genetic background to contribute to sirtuin function in regulating virulence gene expression and suggest a potential role for sirtuins in maintaining genome integrity.

Fig 1. Polarized expression of var genes subsets in different strains.

A: Analysis of var subset expression within clones of 3D7, NF54 and FCR-3 from disparate sources, demonstrating skews in var sub-class expression. 3D7 clones were derived from three different sources (MR4 3D7, 3D7 HR1.2 and 3D7 KW). NF54 clones were derived from the MR4 strain and var gene expression was compared to that previously obtained from clones of a distinct source of NF54 [31]. Var expression data for FCR-3 was obtained from clones previously examined from two parental parasite sources [11]. Var expression patterns are group representations that were determined by qRT-PCR using gene-specific primers for the entire var family in 3D7/NF54, normalized to seryl-tRNA-synthetase and sorted by ups-group. Var expression patterns were measured by qRT-PCR using gene-specific primers for the majority of the family in FCR-3, sorted by ups-group and normalized to the average abundance of the transcript of adenylosuccinate lyase. Independent clones are shown for each strain (different sources are denoted by underline). B: Relative copy numbers (RCN) for all of the genes within each 3D7 and NF54 strain were summed, and the proportions of expression of each subset within each genetic background are indicated. RCN was calculated by comparison to the abundance of transcripts encoding seryl-tRNA-synthetase (results were also similar if compared to the ring-stage-specific genes encoding SBP1 (PF3D7_0501300) and MAHRP (PF3D7_1370300)). Pie charts show proportions of each var transcript and the size of the chart is proportional to the total RCN. * denotes significant difference of p < 0.01.

Fig 2. Sirtuin expression and genetic targeting in NF54 and FCR-3.

A: Quantitative RT-PCR analysis of PfSir2a and PfSir2b expression in schizont-stage 3D7, NF54 and FCR-3. RCN is calculated by comparison to the average abundance of transcripts encoding two control genes: seryl-tRNA-synthetase and myosin. Plot shows average of 2 biological replicates, measured in technical duplicate. B: Table showing sirtuin gene disruptions attempted and achieved in 3D7, NF54 and FCR-3. C: Southern blots showing disruption of PfSir2b in NF54, PfSir2a and PfSir2b in FCR-3, and attempted but unsuccessful disruption of PfSir2a in NF54. The expected sizes for the FCR-3Δsir2a and NF54Δsir2a endogenous genomic locus, integrated flank 1, integrated flank 2 and concatamerized plasmid following AccI/NdeI digestion are 1.1 kb, 3.9 kb, 2.4 kb and 5.2 kb, respectively. The expected sizes for the FCR-3Δsir2b and NF54Δsir2b endogenous genomic locus, integrated flank 1, integrated flank 2 and concatamerized plasmid following ScaI/HpaII digestion are 1.7 kb, 4.2 kb, 2.1 kb and 4.5 kb. D: Var expression patterns in two clones each of NF54 wt, NF54Δsir2b, FCR-3 wt, FCR-3Δsir2a and FCR-3Δsir2b measured by qRT-PCR using gene-specific primers for the majority of the var gene family, sorted by ups-group. RCN was calculated by comparison to the abundance of transcripts encoding the average of the three control genes seryl-tRNA synthetase, arginyl-tRNA synthetase and glutaminyl-tRNA synthetase. Individual clones denoted in black and brown bars. Pie charts define percentage of each ups class expressed, and are sized proportional to the total RCN. ns = no significant difference between total var gene expression levels in the individual strains.

Fig 3. Telomere length is slightly altered in NF54 and FCR-3 sirtuin mutants.

A: Schematic showing structure of a P. falciparum telomere. The genome is digested with frequent-cutting restriction enzymes which do not cut within the telomere repeat to make a Telomere Restriction Fragment (TRF) Southern blot. B: TRF Southern blots of sirtuin-mutant lines compared to parental lines. Representative blots are shown and represent more than 3 experiments for each strain. The average median telomere lengths +/- S.D. from replicate blots are listed below each smear.

Fig 4. Complementation of 3D7ΔSir2a results in reduced var gene expression.

A: Schematic of PfSir2a complementation plasmid. B: PfSir2a expression levels measured by qRT-PCR in schizonts of 3D7, 3D7ΔSir2a and 3D7ΔSir2a_comp. RCN is calculated by comparison to the average abundance of transcripts encoding two control genes: seryl-tRNA-synthetase and myosin. Plot shows average of 2 biological replicates measured in technical duplicate. C: TRF Southern blot of 3D7, 3D7ΔSir2a and 3D7ΔSir2a_comp. D: Var expression patterns in 2 clones each of 3D7ΔSir2a (red, pink) and 3D7ΔSir2a_comp (black, grey), measured as in Fig. 2D. 3D7ΔSir2a_comp shows significant reduction in var expression levels as compared to 3D7ΔSir2a. Pie charts represent var expression by ups class. * denotes significant difference of p < 0.01.

Fig 5. Dramatic chromosomal rearrangements in 3D7ΔSir2a gene disruption mutants.

A: Southern blots of genomic DNA from 3D7 and 3D7ΔSir2a, digested with Taq1 and probed for the first sub-telomeric repeat, TARE1, or the conserved ‘ATS’ region of var genes. Arrows indicate bands that differ between the two lines. B: Pulsed-field gel electrophoresis showing altered karyotype in 3D7ΔSir2a compared to 3D7. C: Comparative genomic hybridization plots of 3D7 and 3D7ΔSir2a. Only those chromosomes containing significant CNVs are shown. The log₂ratio of Cy3/Cy5 value is plotted against chromosomal position. D: Chromosomal rearrangements in 3D7ΔSir2a detected by array CGH. The location of each var gene is shown in orange in the schematic chromosomes. The two locations that show CNV in NF54 are indicated with arrows. Deletions and amplifications are highlighted with boxes.