Project description:Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium parasites that cause malaria are urgently needed. Here, we show that azithromycin—a clinically used macrolide antibiotic that targets the bacterium-like ribosome of the malaria parasites apicoplast organelle and causes a slow-killing ‘delayed death’ phenotype—can also rapidly kill parasites throughout the asexual blood-stages of the lifecycle via a ‘quick-killing’ mechanism of action. Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency that is directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin for in vitro treatment windows less than 48 hours. Analogues were also effective against the zoonotic malaria parasite P. knowlesi, and against both multi-drug and artemisinin resistant P. falciparum lines. Metabolomic profiles of azithromycin analogue treated parasites were similar to those of chloroquine treated parasites, suggesting that the quick-killing mechanism of action may in part be localised to the parasite food vacuole. However, metabolomic signatures associated with mitochondrial disruption were also present. In addition, unlike chloroquine, azithromycin and analogues were active across blood stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on quick-killing activity, which suggests that apicoplast-targeting, delayed-death activity can either be preserved or removed independently of quick-killing. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Therefore, development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed death mechanism of action in a single, multifactorial chemotype.

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 âring-likeâ and less âtrophozoite-likeâ, 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. Two condition drug-treatment experiment, Dihydroartemisinin vs. Vehicle control treatment with matched reference untreated controls. Biological replicates: 5 independently grown and harvested experimental culture replicates. One replicate of treatment/reference time-point per array.

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:Artemisinin (ART)-resistant Plasmodium falciparum (Pf) threatens global malaria control. Resistance, driven by mutations in Pfk13, is multifaceted but quiescence plays a central role. Epigenetic regulation may contribute, given that only a percentage of parasites survive a pulse of the active drug metabolite dihydroartemisinin (DHA). The identities or roles of these epigenetic factors, however, have yet to be identified. tRNA modifications are a conserved epigenetic translational control mechanism, whereby cellular stress leads to modification reprogramming and codon-biased translation. Here we use liquid chromatography-mass spectrometry to profile tRNA modifications in ring-stage ART-sensitive (ART-S) Dd2 and ART-resistant (ART-R) Dd2PfK13_R539T before and after drug pulse. ART-R parasites differentially reprogram their tRNA modification profiles in response to DHA, specifically by mcm5s2U hypomodification of tRNAs. Proteomic and subsequent codon usage analysis revealed that the ART-R parasite proteome displays codon bias, uncovering a new layer of proteomic regulation in drug-resistant parasites. A subset of these proteins were not transcriptionally regulated, suggesting codon-bias translation. Upregulated proteins were enriched for LysAAA, HisCAT and AspGAT and downregulated proteins were enriched for their cognate codons (LysAAG, HisCAC and AspGAC). PfK13 and its interacting partner BIP were among the codon-controlled upregulated proteins. Interestingly, mcm5s2U occurs on the U34 of LysAAA/AAG codons to regulate translational fidelity, providing a mechanistic link between tRNA modification and proteomic data. Transcriptomics revealed enrichment of wobble-base tRNA modification in ART-R parasites post-ART treatment. An anhydrotetracycline-regulated conditional knockdown (cKD) of the terminal s2U methyltransferase, PfMnmA, displayed increased ART survival, signifying that hypomodification plays a critical role in the ART-R parasite response to DHA. cKD parasites also had altered responses to proteotoxic and mitochondrial antimalarials, uncovering overlaps between epigenetic stress response pathways. This work describes a novel epigenetic pathway via tRNA s2U reprogramming that ART-R parasites may use to help survive ART-induced stress.

			Action	DRS
	MTBLS6580	Other
	FILES	Other
	a_MTBLS6580_LC-MS_negative_hilic_metabolite_profiling-1.txt	Txt
	a_MTBLS6580_LC-MS_negative_hilic_metabolite_profiling-2.txt	Txt
	a_MTBLS6580_LC-MS_positive_hilic_metabolite_profiling-1.txt	Txt

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A conserved metabolic signature associated with response to fast-acting antimalarial agents

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