A systematic map of genetic variation in Plasmodium falciparum

Abstract

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.

Figure 1. Mapping the Recombination Breakpoints across the 14 P. falciparum Chromosomes in the Chloroquine-Sensitive F1 Progeny Strain C188

A total of 14,289 SFPs were identified over three hybridizations by comparing HB3, Dd2, or the recombinant line C188 to 3D7. SFPs were recorded as HB3 (green bars) in the recombinant line if the probe was scored as polymorphic in both C188 and HB3 but not Dd2, or were recorded as Dd2 (red bars) if scored as polymorphic in Dd2 and C188 [19] but not HB3. Regions with a large number of markers scored as both HB3 and Dd2 are shown in yellow and are associated with the known locations of var, rifin, and stevor genes (shown as blue dots). Markers determined by microsatellite length mapping (MLP) are coloured purple and blue [7].

Figure 2. Linkage Disequilibrium surrounding pfcrt (MAL7P1.27) in FCB and Dd2 on Chromosome 7

Scores were generated by calculating the probability of observing the same genotype by chance over a moving 40-kbp window (with the probability of observing the same genotype for any one SFP by chance placed at 0.33). The plot shows the ratio between the probability and the maximum possible probability for regions with at least four SFPs, with 1 indicating the best possible score. The position of antigenic variation clusters (vars, stevors, or rifins), are shown in blue and are marked. SFPs mapping to these genes were excluded from the calculations because our data indicate that mitotic recombination may be occurring in these genes. pfcrt, which is located between bases 307,926 and 311,020 on Chromosome 7, is shown as a black triangle. The trough at pfcrt is likely to be due to the strong selective pressure on pfcrt, and is consistent with the observation that FCB and Dd2 have different alleles of pfcrt even though published SNP data also shows disequilibrium in surrounding regions [5]. Data for other chromosomes are shown in Figure S1.

Figure 3. The Heat Map Shows the Base 2 Logarithm of the Ratio of the Normalized, Background Subtracted Probe Signal for the Listed Isolate (18.02, Dd2, 7G8 or HB3), Relative to the Sequenced Isolate, 3D7

All probes on the array were selected to be unique within 3D7. Each horizontal bar represents a single gene on the right arm of Chromosome 12. Dark probes, with low signal intensities, do not match any region in the genome of the listed isolate, while light probes, displaying high signal intensities, such as those near the GTP-cyclohydrolase gene (PFL1155w) in Dd2, HB3, and 7G8 contain matches to multiple regions of the genome. The signal intensity and the consecutive nature of the pattern suggest that strains 7G8, HB3, and Dd2 contain more than one copy of the GTP-cyclohydrolase gene PFL1155w. An arrow represents the location of the GTP-cyclohydrolasePFL1155w gene on Chromosome 12.

Figure 4. Example of a Deleted Region

Hybridization levels were calculated with the MOID algorithm as previously described [59]. The figure shows the signal for 26 genes beginning with PFB0010w and ending with PFB0120w for three strains (41.02, 3D7, and C188). The background level (zero) was determined by analyzing the signal of non-P. falciparum genes on the array. Data for all genes and all strains are available from Table S12.