Project description:Control of malaria is threatened by emerging parasite resistance to artemisinin drug (ART) therapies. The molecular details of how Plasmodium malaria parasites response to ART and how this relates to resistance is not clear. To determine how parasites respond to ART by altering gene expression, we performed a transcriptomic study of dihydroartemisinin (DHA) response in P. falciparum K1 strain and in P. berghei ANKA strain. Microarray data from DHA-treated P. falciparum trophozoite stage parasites were compared with data from other ART treatments. Genes with consistent changes in expression were identified, which includes notably down-regulation of cytosolic ribosomal protein genes. RNA-seq data revealed a similar pattern of transcriptomic change, although the pattern was much clearer in that more than one-third of P. falciparum trophozoite genes are differentially expressed with greater statistical support for down-regulation of ribosomal protein genes. The poor overlap of differentially-expressed genes between microarray and RNA-seq and less-well defined patterns for the former suggests that the accuracy of microarray is limited by technological bias. The trophozoite response to DHA is overall “ring-like” and less “trophozoite-like”, which is consistent with previous findings that Plasmodium can enter a quiescent ring-like state to resist ART. RNA-seq data from DHA-treated P. falciparum rings reveal a more muted response, although there is considerable overlap of differentially expressed genes with DHA-treated trophozoites. In contrast, P. falciparum schizonts are unresponsive to DHA, suggesting that the protective response acts mainly to arrest parasite development through the G2/M checkpoint. The transcriptional response of P. berghei to DHA treatment in vivo in infected mice is strikingly similar to the P. falciparum in vitro ring and trophozoite responses, in which ribosomal protein genes are notably down-regulated. These results suggest Plasmodium species respond to DHA in the same way. This knowledge could be applied to outwit the parasite to deliver more effective artemisinin therapies, and maybe hinder the development of drug resistance.

Project description:Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programs around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline based drugs is becoming critical. So far only few resistance markers have been identified and verified from which only two ABC transmembrane transporters namely PfMDR1 and PfCRT have been experimentally verified. Another P. falciparum ABC transporter, the multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identify a parasite clone that is derived from the 3D7 P. falciparum strain and which shows increased resistance to chloroquine and mefloquine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5M-bM-^@M-^Y upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription that result in increased levels of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of such genetic polymorphisms to underlie drug resistance phenotypes. Presented here are the data from microarray-based genome-wide transcriptomic and genomic studies of the drug-sensitive and drug-resistant 3D7 clones 11C/wt and 6A/mut. 2 P. falciparum lab clones derived from 3D7 strain were harvested during the intra-erythrocytic cycle at 8hr intervals over 48 hours to obtain a total of 6 time point samples per clone. RNA from a total of 12 samples were extracted. Synthesis of target DNA was carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211 and used in replicate microarray hybridizations against a common RNA reference pool of 3D7 strain.

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Plasmodium parasites mount an arrest response against dihydroartemisinin [Microarray]

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