New insights into the Plasmodium vivax transcriptome using RNA-Seq.

Historically seen as a benign disease, it is now becoming clear that Plasmodium vivax can cause significant morbidity. Effective control strategies targeting P. vivax malaria is hindered by our limited understanding of vivax biology. Here we established the P. vivax transcriptome of the Intraerythrocytic Developmental Cycle (IDC) of two clinical isolates in high resolution by Illumina HiSeq platform. The detailed map of transcriptome generates new insights into regulatory mechanisms of individual genes and reveals their intimate relationship with specific biological functions. A transcriptional hotspot of vir genes observed on chromosome 2 suggests a potential active site modulating immune evasion of the Plasmodium parasite across patients. Compared to other eukaryotes, P. vivax genes tend to have unusually long 5' untranslated regions and also present multiple transcription start sites. In contrast, alternative splicing is rare in P. vivax but its association with the late schizont stage suggests some of its significance for gene function. The newly identified transcripts, including up to 179 vir like genes and 3018 noncoding RNAs suggest an important role of these gene/transcript classes in strain specific transcriptional regulation.

Inferred relatedness and heritability in malaria parasites.

Malaria parasites vary in phenotypic traits of biomedical or biological interest such as growth rate, virulence, sex ratio and drug resistance, and there is considerable interest in identifying the genes that underlie this variation. An important first step is to determine trait heritability (H(2)). We evaluate two approaches to measuring H(2) in natural parasite populations using relatedness inferred from genetic marker data. We collected single-clone Plasmodium falciparum infections from 185 patients from the Thailand-Burma border, monitored parasite clearance following treatment with artemisinin combination therapy (ACT), measured resistance to six antimalarial drugs and genotyped parasites using 335 microsatellites. We found strong relatedness structure. There were 27 groups of two to eight clonally identical (CI) parasites, and 74 per cent of parasites showed significant relatedness to one or more other parasites. Initially, we used matrices of allele sharing and variance components (VC) methods to estimate H(2). Inhibitory concentrations (IC(50)) for six drugs showed significant H(2) (0.24 to 0.79, p = 0.06 to 2.85 x 10(-9)), demonstrating that this study design has adequate power. However, a phenotype of current interest--parasite clearance following ACT--showed no detectable heritability (H(2) = 0-0.09, ns) in this population. The existence of CI parasites allows the use of a simple ANOVA approach for quantifying H(2), analogous to that used in human twin studies. This gave similar results to the VC method and requires considerably less genotyping information. We conclude (i) that H(2) can be effectively measured in malaria parasite populations using minimal genotype data, allowing rational design of genome-wide association studies; and (ii) while drug response (IC(50)) shows significant H(2), parasite clearance following ACT was not heritable in the population studied.

Adaptive copy number evolution in malaria parasites.

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

The transcriptome of Plasmodium vivax reveals divergence and diversity of transcriptional regulation in malaria parasites.

Plasmodium vivax causes over 100 million clinical infections each year. Primarily because of the lack of a suitable culture system, our understanding of the biology of this parasite lags significantly behind that of the more deadly species P. falciparum. Here, we present the complete transcriptional profile throughout the 48-h intraerythrocytic cycle of three distinct P. vivax isolates. This approach identifies strain specific patterns of expression for subsets of genes predicted to encode proteins associated with virulence and host pathogen interactions. Comparison to P. falciparum revealed significant differences in the expression of genes involved in crucial cellular functions that underpin the biological differences between the two parasite species. These data provide insights into the biology of P. vivax and constitute an important resource for the development of therapeutic approaches.

Plasmodium vivax trophozoites insensitive to chloroquine.

BACKGROUND: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax. METHODS: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment. RESULTS: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM. CONCLUSION: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

In vitro activity of ferroquine (SSR 97193) against Plasmodium falciparum isolates from the Thai-Burmese border.

BACKGROUND: On the borders of Thailand, Plasmodium falciparum has become resistant to nearly all available drugs, and there is an urgent need to find new antimalarial drugs or drug combinations. Ferroquine (SSR97193) is a new 4-aminoquinoline antimalarial active against chloroquine resistant and sensitive P. falciparum strains in vivo and in vitro. This antimalarial organic iron complex (a ferrocenyl group has been associated with chloroquine) is meant to use the affinity of Plasmodium for iron to increase the probability for encountering the anti-malarial molecule.The aim of the present study was to investigate the activity of ferroquine against P. falciparum isolates from an area with a known high multi-drug resistance rate. METHODS: Parasite isolates were obtained from patients with acute falciparum malaria attending the clinics of SMRU. In vitro cultures of these isolates were set-up in the SMRU-laboratory on pre-dosed drug plates, and grown in culture for 42 hours. Parasite growth was assessed by the double-site enzyme-linked pLDH immunodetection (DELI) assay. RESULTS: Sixty-five P. falciparum isolates were successfully grown in culture. The ferroquine mean IC50 (95% CI) was 9.3 nM (95% C.I.: 8.7 - 10.0). The mean IC50 value for the principal metabolite of ferroquin, SR97213A, was 37.0 nM (95% C.I.: 34.3 - 39.9), which is four times less active than ferroquine. The isolates in this study were highly multi-drug resistant but ferroquine was more active than chloroquine, quinine, mefloquine and piperaquine. Only artesunate was more active than ferroquine. Weak but significant correlations were found between ferroquine and its principal metabolite (r2 = 0.4288), chloroquine (r2 = 0.1107) and lumefantrine (r2 = 0.2364). CONCLUSION: The results presented in this study demonstrate that the new ferroquine compound SSR97193 has high anti-malarial activity in vitro against multi-drug resistant P. falciparum.

In vitro antimalarial drug susceptibility and pfcrt mutation among fresh Plasmodium falciparum isolates from the Lao PDR (Laos).

Recent drug trials in Laos have shown high levels of Plasmodium falciparum resistance to chloroquine, but there are no published data on in vitro antimalarial drug susceptibility. We used the double-site enzyme-linked pLDH immunodetection (DELI) assay to estimate the in vitro antimalarial drug susceptibility of 108 fresh P. falciparum isolates from southern Laos. The geometric mean (95% confidence interval) 50% inhibitory concentration values (nmol/L) were 152.4 (123.8-187.6) for chloroquine, 679.8 (533.8-863.0) for quinine, 45.9 (37.9-55.7) for mefloquine, 5.0 (4.4-6.4) for artesunate, 6.3 (4.5-8.9) for dihydroartemisinin, and 59.1 (46.4-75.3) for lumefantrine. The proportion of isolates defined as resistant were 65%, 40%, and 8% for chloroquine, quinine, and mefloquine, respectively. Of 53 isolates genotyped for the pfcrt T76K chloroquine-resistance mutation, 48 (91%) were mutants. P. falciparum in Laos is multi-drug resistant; antimalarial immunity resulting from the use of ineffective chloroquine before 2005 probably contributes significantly to the therapeutic responses in clinical trials.

Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites.

When selection is strong and beneficial alleles have a single origin, local reductions in genetic diversity are expected. However, when beneficial alleles have multiple origins or were segregating in the population prior to a change in selection regime, the impact on genetic diversity may be less clear. We describe an example of such a "soft" selective sweep in the malaria parasite Plasmodium falciparum that involves adaptive genome rearrangements. Amplification in copy number of genome regions containing the pfmdr1 gene on chromosome 5 confer resistance to mefloquine and spread rapidly in the 1990s. Using flanking microsatellite data and real-time polymerase chain reaction determination of copy number, we show that 5-15 independent amplification events have occurred in parasites on the Thailand/Burma border. The amplified genome regions (amplicons) range in size from 14.7 to 49 kb and contain 2-11 genes, with 2-4 copies arranged in tandem. To examine the impact of drug selection on flanking variation, we genotyped 48 microsatellites on chromosome 5 in 326 parasites from a single Thai location. Diversity was reduced in a 170- to 250-kb (10-15 cM) region of chromosomes containing multiple copies of pfmdr1, consistent with hitchhiking resulting from the rapid recent spread of selected chromosomes. However, diversity immediately flanking pfmdr1 is reduced by only 42% on chromosomes bearing multiple amplicons relative to chromosomes carrying a single copy. We highlight 2 features of these results: 1) All amplicon break points occur in monomeric A/T tracts (9-45 bp). Given the abundance of these tracts in P. falciparum, we expect that duplications will occur frequently at multiple genomic locations and have been underestimated as drivers of phenotypic evolution in this pathogen. 2) The signature left by the spread of amplified genome segments is broad, but results in only limited reduction in diversity. If such "soft" sweeps are common in nature, statistical methods based on diversity reduction may be inefficient at detecting evidence for selection in genome-wide marker screens. This may be particularly likely when mutation rate is high, as appears to be the case for gene duplications, and in pathogen populations where effective population sizes are typically very large.