4. Results and Discussion
4.4 Study 3: Multigenic architecture of piperaquine resistance trait in Plasmodium falciparum
4.4.4 Discussion
mutations were not identified earlier suggest that a proportion of piperaquine-resistant isolates to not involve mutations in pfcrt but rely on alternative pathways such as one involving plasmepsins duplications.
Alternatively, pfcrt mutations recently evolved and therefore were not captured in initial studies. Moreover, another possibility is that these pfcrt mutations need specific backgrounds to develop, such as one provided by plasmepsins duplication.
Temporal data suggests that the pfcrt mutations associated with piperaquine resistance arose on a genetic background of amplified plasmepsins and correlate with temporal de-amplification of pfmdr1 (Dhingra et al., 2019; Ross et al., 2018). Most of the pfcrt mutation are observed after 2010 becoming more prevalent between 2011-2013 and became the dominant allele by 2016 (Dhingra et al., 2019;
Ross et al., 2018). These mutations seem to have arisen independently in strains with Dd2 PfCRT background and mutant PfK13 (Ross et al., 2018). Curiously, high plasmepsin copy number (4 and more copies) were more frequent in 2012 and 2013, and by 2016 plasmepsin duplication (2-3 copies) was the predominant amplification status (Dhingra et al., 2019). Additionally, survival rates of piperaquine-treated field isolated adapted parasites increased, in vitro, over the years (Dhingra et al., 2019).
Together with our data, these findings suggest an initial selection of high number of plasmepsin locus amplifications with a simultaneous decrease in pfmdr1 copy number variations to accommodate the plasmepsin amplification events. These mutations might not cause a full resistant phenotype. Instead, these events likely generated a low grade piperaquine resistance that created a favorable P. falciparum genetic background for novel pfcrt mutations to arise in the context of piperaquine pressure. These parasites can have increased resistance to high concentrations of piperaquine, likely at a lower fitness cost.
The initial F145I, M343L and H97Y PfCRT mutations were shown to confer a high fitness cost and are being replaced by less resistant but fitter T93S and I218F mutations (Dhingra et al., 2019; Ross et al., 2018). These initial mutations H97Y, F145I and G535V were also shown to occur in parasites with 3 to 7 copies of pfpm2 (Ross et al., 2018). Moreover, in this study all piperaquine resistant PfCRT mutants had pfmdr1 single copy, despite the presence of field samples with multiple pfmdr1 copies, which further implies the pfmdr1 single copy as important to resistance and not just a consequence of mefloquine removal (Ross et al., 2018). Interestingly, in previous studies using pfcrt-edited parasites with a single plasmepsin copy, these parasites developed an enlarged and translucent digestive vacuole, while this phenotype was not observed for piperaquine resistant field parasites harboring PfCRT mutations combined with multiple copies of pfpm2 (Boonyalai et al., 2020; Ross et al., 2018). This suggests that
likely to be associated with a fitness cost (Dhingra et al., 2019). In vitro removal of PfCRT mutations coincides with improved growth rates and pfpm2 de-amplification, linking these two factors (Ross et al., 2018). Accordingly, in the field plasmepsin duplication still seems necessary as these parasites seem to retain at least plasmepsin locus duplication (Ross et al., 2018). Even though, pfcrt mutations in vitro seem sufficient to generate resistance and might dispense the plasmepsin duplications (Ross et al., 2018). As such, likely the initial expansion of mutations in PfCRT could have been propelled by plasmepsin duplications which could have a beneficial impact on the fitness cost caused by these mutations.
Recently, the structure of PfCRT was unraveled, and the piperaquine resistance associated mutation C350R that emerged in the 7G8 isoform and the mutations T93S, I218F, F145I, H97Y, G353V that emerged in the Dd2 isoform were located in the helices that line the central negatively charged cavity (J.
Kim et al., 2019). The mutation M343L, which confers low grade piperaquine resistance in the Dd2 isoform, was located deeper into the central cavity closer to the parasite cytosol (J. Kim et al., 2019).
Contrary to other mutations, the M343L was shown to not confer a fitness cost when introduced into the Dd2 strain. However, it confers a fitness cost in a Cambodian adapted parasite with a Dd2-like pfcrt background (Ross et al., 2018). Location of the mutation is important for fitness cost, possibly due to alterations in solute transport, but additional factors including mutations in other genes are important for the overall fitness cost of the strain. PfCRT 7G8 isoform was shown to bind to piperaquine but not to transport, which was consistent with the sensitive role of the strain (J. Kim et al., 2019). The F145I and C350R mutation were shown to transport piperaquine when introduced into the 7G8 strain and associated with piperaquine resistance phenotype (J. Kim et al., 2019). Interestingly, these mutations have reduced chloroquine transport (J. Kim et al., 2019). On the contrary, the introduction of F145I mutation into the Dd2 strain did not increase piperaquine transport. Notably, in another study there was no significant difference in intracellular piperaquine concentration on the edited Dd2 piperaquine sensitive control line versus the Dd2 piperaquine resistant variants expressing the PfCRT mutations F145I, M343L, or G353V, suggesting that these mutations do not confer resistance by accumulating less piperaquine in the Dd2 background (Ross et al., 2018). Moreover, there was an increase in the intracellular accumulation of piperaquine resistant edited field isolates when removing PfCRT resistant conferring mutations (Ross et al., 2018). These reflect the genetic background impact in piperaquine resistant and highlight the complex relationship between PfCRT mutations and piperaquine resistance, which does not appear to be explained solely by changes in drug accumulation or efflux. Likewise, our results show that verapamil, a chemical blocker of PfCRT, in the Dd2 plasmepsin amplificated lines is enough to confer resistance to piperaquine.
As a blocker of PfCRT function, this result does not support generation of resistance by increased transport. There is possibility that more than one mechanism prevails to give resistance. One alternative mechanism involves piperaquine-mediated binding to and functional inhibition of certain PfCRT isoforms, recalling earlier reports of distinct drug binding sites in this transporter (Bellanca et al., 2014; Callaghan et al., 2015; Lekostaj et al., 2008; Richards et al., 2016). Moreover, due to the similar effect of verapamil and elacridar in the presence of plasmepsin duplication, in our study, another possibility is altered vacuolar function that negates the effect of high piperaquine concentrations. Due to the role of both piperaquine and plasmepsins on the hemoglobin degradation pathway it is likely that changes in this pathway affect piperaquine resistance and that PfCRT altered physiological function could lead to disruptions in this process. Further investigations in gene-edited parasites or heterologous expression systems will be important in delineating the relationship between PfCRT mutations, plasmepsins copy number, drug accumulation, and piperaquine resistance.
Piperaquine resistance is multifactorial, as seen by the disparity of resistant field samples and their genotypes, which can dispense either plasmepsins amplifications or pfcrt mutations. As such, conceivably in different genetic backgrounds to those used in this study, plasmepsins duplications could confer different degrees of resistance, and could potentiate other loci to mediate resistance in the field.
Additionally, other loci might be in the process of selection and fixation that could make PfCRT mutant parasites resistant while dispensing plasmepsin duplications. Continuous temporal monitoring of the dynamics of parasite populations in the areas of DHA-PPQ therapy usage is necessary to understand the evolution of resistant parasites and to prevent further fixation of resistant alleles that avoid fitness costs.
Deep sampling of contemporary isolates is of particular importance to detect emergence of novel alleles in this propense background and to avoid propagation of piperaquine resistance to neighboring areas.
Such fixation could lead to parasite populations to become permanently resistant even after removal of piperaquine based therapies. This could render new therapies based on piperaquine ineffective and compromise strategies of mass drug administration for eradication efforts.
Altogether, our results recapitulate in vitro a complementary mechanism between plasmepsins, pfmdr1, and pfcrt involved in piperaquine resistance, supporting the molecular epidemiological data in southeast Asia and furthering understanding of how piperaquine drug resistance evolves.