Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages

Abstract

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.

On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity

“…Whilst the targets of MMV1557817 were confirmed using the above methods, it is also important to assess the ability of parasites to become resistant to the compound and the potential impact this has on other antimalarial drugs. Recent in vitro resistance selection and whole genome analysis undertaken to determine if resistance against MMV1557817 could be selected in Dd2 parasites identified a A460S mutation in one of its intended targets, Pf A-M17, as well as a N846I mutation in an AP-3 β subunit (PF3D7_0613500) and a M317I mutation in a non-essential conserved Plasmodium protein (PF3D7_1144400) (61). These three mutations were present in all clones obtained from multiple flasks, with parasites only displaying modest IC 50 shifts of between 1.5 – 2.9x, suggesting a low level of resistance (61).…”

“…Recent in vitro resistance selection and whole genome analysis undertaken to determine if resistance against MMV1557817 could be selected in Dd2 parasites identified a A460S mutation in one of its intended targets, Pf A-M17, as well as a N846I mutation in an AP-3 β subunit (PF3D7_0613500) and a M317I mutation in a non-essential conserved Plasmodium protein (PF3D7_1144400) (61). These three mutations were present in all clones obtained from multiple flasks, with parasites only displaying modest IC 50 shifts of between 1.5 – 2.9x, suggesting a low level of resistance (61). To confirm whether the A460S mutation was responsible for resistance, CRISPR/Cas9 technology was utilised to attempt to introduce this mutation into Pf A-M17 in Pf 3D7 wildtype parasites using a previously described donor plasmid method (62).…”

“…Interestingly, piperaquine resistant parasites were also sensitised to dual aminopeptidase inhibition by MMV1557817 (61). These parasites digest significantly less haemoglobin, highlighting the overall relationship of MMV1557817's activity with haemoglobin digestion, and suggesting inhibition of PfA-M1 and PfA-M17 could be useful in a number of different combination therapies.…”

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella

“…Our comprehensive results indicate that halofuginone damages biological pathways and induces autophagy in E. tenella. Previous studies also found that differential expression of regulated genes may lead to differences in drug-resistant and drug-sensitive strains (Antony et al, 2016;Ingham et al, 2020;Xie et al, 2020;Laing et al, 2022;Okombo et al, 2022;Zhang et al, 2022). Hence, it was expected that long-term selection would lead to transcriptional changes in different genes in the resistant strain.…”

Declining Frequency of Chloroquine-resistant Haplotype of <em>Plasmodium falciparum</em> (CVIET) in an Endemic Area of Southwest Nigeria 17 Years After Withdrawal of Chloroquine

“…Resistance to CQ historically is mainly facilitated by genetic changes within the coding region of the gene encoding a transporter on the parasite food vacuole membrane; the Plasmodium falciparum chloroquine resistance transporter (PfCRT) gene. This transporter is responsible for the influx/efflux of some antimalarial drugs to and from the food vacuole of the parasite [30][31][32]. Point mutations that translate to changes in amino acids at different positions have contributed to the parasites' competence to resist drugs such as chloroquine and structurally related antimalarial drugs [ [30,33,34].…”