A Systems-Based Analysis of Plasmodium vivax Lifecycle Transcription from Human to Mosquito

Abstract

BackgroundUp to 40% of the world's population is at risk for Plasmodium vivax malaria, a disease that imposes a major public health and economic burden on endemic countries. Because P. vivax produces latent liver forms, eradication of P. vivax malaria is more challenging than it is for P. falciparum. Genetic analysis of P. vivax is exceptionally difficult due to limitations of in vitro culture. To overcome the barriers to traditional molecular biology in P. vivax, we examined parasite transcriptional changes in samples from infected patients and mosquitoes in order to characterize gene function, define regulatory sequences and reveal new potential vaccine candidate genes.Principal FindingsWe observed dramatic changes in transcript levels for various genes at different lifecycle stages, indicating that development is partially regulated through modulation of mRNA levels. Our data show that genes involved in common biological processes or molecular machinery are co-expressed. We identified DNA sequence motifs upstream of co-expressed genes that are conserved across Plasmodium species that are likely binding sites of proteins that regulate stage-specific transcription. Despite their capacity to form hypnozoites we found that P. vivax sporozoites show stage-specific expression of the same genes needed for hepatocyte invasion and liver stage development in other Plasmodium species. We show that many of the predicted exported proteins and members of multigene families show highly coordinated transcription as well.ConclusionsWe conclude that high-quality gene expression data can be readily obtained directly from patient samples and that many of the same uncharacterized genes that are upregulated in different P. vivax lifecycle stages are also upregulated in similar stages in other Plasmodium species. We also provide numerous examples of how systems biology is a powerful method for determining the likely function of genes in pathogens that are neglected due to experimental intractability.

Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes

“…We next sought to cross-validate the SNPs predicted by sequencing using a high-density P. vivax tiling microarray (14). Microarrays are less expensive and simpler to use than whole-genome sequencing and thus may represent a valuable alternative approach to direct sequencing for assessing diversity in field isolates.…”

“…Although mutations in the chloroquine resistance transporter (pvcrt, PVX_087980) are not thought to be associated with chloroquine resistance (23,42,43), increased expression of pvcrt has been found in chloroquineresistant P. vivax (44). In addition, the level of dihydrofolate reductase (pvdhfr, PVX_089950) expression is higher in some P. vivax isolates relative to P. falciparum (14). Thus, transcription may play a role in drug resistance in P. vivax.…”

“…Additional sequencing of worldwide isolates of P. vivax has been approved at sequencing centers (12), but the necessity of maintaining parasites in infected primates greatly limits the number of samples available. Although the availability of the genome sequence provides new opportunities to discover drug and vaccine targets and to perform gene expression (13,14) and proteomic studies, the lack of worldwide genetic diversity data has hampered population studies. At present, many P. vivax studies still rely on a small set of polymorphic antigens such as circumsporozoite protein, merozoite surface proteins, and apical membrane antigen (AMA-1) to assess diversity (15)(16)(17)(18)(19)(20).…”

“…Here, we report whole-genome sequencing results with 30× coverage of a P. vivax isolate obtained directly ex vivo without further propagation in vitro or in monkeys. In addition to wholegenome sequencing, the parasite isolate was analyzed using a whole-genome tiling microarray (14,24). Our studies provide thousands of cross-validated polymorphisms in this Peruvian isolate of P. vivax, which are applicable to analysis of genetic diversity in P. vivax and to the identification of highly variable genes, such as a P. vivax multidrug resistance protein (pvmrp1, PVX_097025), which may be under immune or drug pressure.…”

PFE0565w, a Plasmodium falciparum Protein Expressed in Salivary Gland Sporozoites

“…There is evidence in the literature that supports this hypothesis, as PFE0565w has been placed into a category of genes that is highly up-regulated in salivary gland sporozoites compared with other life cycle stages (called a Sporozoite Conserved Orthologous Transcript, SCOT), and their gene products are predicted to be important in the early establishment of exoerythrocytic stages. 46 Taken together, PFE0565w could be another candidate for a pre-erythrocytic stage vaccine because it does not share identity with any known human protein. …”