Abstract
Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent emergence in South America, it is important to study genetic determinants of resistance in the African context using African parasites. Through in vitro evolution of Senegalese parasites, we had previously generated the artemisinin-resistant parasites Pikine_R and Thiès_R and established pfcoronin mutations to be sufficient to confer artemisinin resistance in the standard ring-stage survival assay (RSA). In the current study, we used genetic analysis of revertants to demonstrate pfcoronin to be the major driver of elevated RSA in the artemisinin-resistant parasites Pikine_R and Thiès_R evolved in vitro. We interrogated the role of a second gene PF3D7_1433800, which also had mutations in both the Pikine_R and Thiès_R selected lines, but found no evidence of a contribution to reduced susceptibility in the RSA survival assay. Nevertheless, our genetic analysis demonstrates that parasite genetic background is important in the level of pfcoronin mediated RSA survival, and therefore we cannot rule out a role for PF3D7_1433800 in other genetic backgrounds. Finally, we tested the potential synergy between the mutations of pfcoronin and pfkelch13 through the generation of single and double mutants in the Pikine genetic background and found that the contribution of pfcoronin to reduced susceptibility is masked by the presence of pfkelch13. This phenomenon was also observed in the 3D7 background, suggesting that pfcoronin may mediate its effects via the same pathway as pfkelch13. Investigating the biology of proteins containing the beta-propeller domain could further elucidate the different pathways that the parasite could use to attain resistance.
Highlights
There were 228 million cases of malaria and over 400,000 malaria deaths worldwide in 2018 [1]
Given the independent emergence of artemisinin resistance associated with pfkelch13 mutations in Southeast Asia and South America, it is important to study genetic determinants in the African context
We report that pfcoronin mutations are necessary for the level of artemisinin resistance observed in both Pikine_R and Thiès_R
Summary
There were 228 million cases of malaria and over 400,000 malaria deaths worldwide in 2018 [1]. Artemisinin drug resistance has emerged in Southeast Asia [2] that is attributed to mutations in the propeller domain of the Kelch protein of P. falciparum (PfKelch). The C580Y mutation in particular, which is approaching fixation in Southeast Asia [3], has recently been reported to have independently emerged in South America [4] and Papua New Guinea [5], threatening the success achieved by ACTs. ACTs largely remain effective in the African continent, despite recent reports of pfkelch mutations in Rwanda [6] and Tanzania [7]. Given that the PfKelch C580Y mutation has been linked to a slowdown of the endocytic machinery for hemoglobin uptake [11], it has yet to be determined if all the different genetic pathways of resistance converge in reducing the uptake of hemoglobin
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