P. falciparum K13 mutations present varying degrees of artemisinin resistance and reduced fitness in African parasites

  1. Barbara H. Stokes
  2. Kelly Rubiano
  3. Satish K. Dhingra
  4. Sachel Mok
  5. Judith Straimer
  6. Nina F. Gnädig
  7. Jade R. Bath
  8. Ioanna Deni
  9. Kurt E. Ward
  10. Josefine Striepen
  11. Tomas Yeo
  12. Leila S. Ross
  13. Eric Legrand
  14. Frédéric Ariey
  15. Clark H. Cunningham
  16. Issa M. Souleymane
  17. Adama Gansané
  18. Romaric Nzoumbou-Boko
  19. Claudette Ndayikunda
  20. Abdunoor M. Kabanywanyi
  21. Aline Uwimana
  22. Samuel J. Smith
  23. Olimatou Kolley
  24. Mathieu Ndounga
  25. Marian Warsame
  26. Rithea Leang
  27. François Nosten
  28. Timothy J.C. Anderson
  29. Philip J. Rosenthal
  30. Didier Ménard
  31. David A. Fidock

  • Curated by eLife

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    Evaluation Summary:

    This study is of interest to the broad malaria research community and especially those who work on drug resistance. The authors provide a summary of their surveys of African and Southeast Asian Plasmodium falciparum parasites for the Kelch 13 gene, a marker of artemisinin resistance. The contribution of several K13 mutations to artemisinin resistance is investigated in different genetic backgrounds and confirms the lack of a barrier for the potential emergence of artemisinin resistance in African parasites. These findings are of prime importance in the context of public health perspective on managing the risk of resistance appearing in Africa.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites, driven by K13 mutations, has led to widespread antimalarial treatment failure in Southeast Asia. In Africa, our genotyping of 3,299 isolates confirms the emergence of the K13 R561H variant in Rwanda and reveals the continuing dominance of wild-type K13 across 11 countries. We show that this mutation, along with M579I and C580Y, confers varying degrees of in vitro ART resistance in African parasites. C580Y and M579I cause substantial fitness costs, which may counter-select against their dissemination in high-transmission settings. We also define the impact of multiple K13 mutations on ART resistance and fitness in multiple Southeast Asian strains. ART susceptibility is unaltered upon editing point mutations in ferrodoxin or mdr2, earlier resistance markers. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

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  1. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The authors have studied mutations in the K13 gene that is linked to Artemisinin resistance in a range of African parasites. They show that these mutations can confer resistance in a in vitro survival assay but that they are often linked to reduced fitness. The authors also show that different parasites have less of an impact on fitness when the K13 mutations are introduced in line with the suggestion that the overall genetic background is critical for transmission of K13 mutations. The paper also shows evidence that genes potentially contributing to the genetic background are not involved.

    The overall work involves a significant amount of work that to generate a wide range of different parasite lines that allow a detailed assessment of how different mutations interact with the genetic background of the parasite. This provides a significant amount of new insights. A key conclusion the authors draw from this work relates to the relationship between fitness and resistance and by inference on why artemisinin resistance has occurred in SE Asia. While this indeed would be a striking conclusion I think the data at this stage is not strong enough to make this claim. The claim is mainly based on Figure 3 E and F as well as 5 C and D. While indeed, initially it looks like RSA has much less of a survival impact in Dd2 there is some concern that the data is generated using different baselines (isogenic WT parasite in Figure 3 and Dd2eGFP in Figure 5 D). This is noteworthy as in Figure 5C the Dd2wt parasite is used and the fitness cost appears to be different.

    Please see our reply below to Reviewer 1 Comment #2.

    A striking finding is that the UG659C560Y line appears to have a relatively small fitness cost - especially if looked at for the whole 40 generations rather than the somewhat arbitrarily picked 38 days. This data could suggest that there are parasites in Africa that have the capacity to acquire resistance with minimal cost to fitness.

    We thank the Reviewer for this suggestion and have now recalculated our fitness data using a 36-day period, which we have adopted as a standardized timeline and which allows us to compare across all prior and newly acquired fitness assays. We note that this is already relatively lengthy compared to a number of other reports in the literature. For example, Baragana et al. (2015, Nature) measured competitive growth rates over a 14-day period. Gabryszweski et al. (2016, Mol Biol Evol) used 20-day assays. Siddiqui et al. (2020, mBio) used longer 48-day assays. We agree with the Reviewer that our data suggest that some African strains can achieve in vitro ART resistance with a minimal cost to fitness. In support of this, our new data presented in the revised Figure 3 provide evidence for the R561H mutation having little to no fitness cost in 3D7 parasites that are closely related to Rwandan isolates (see our response above to Comment #2 from the Editors).

    As pointed out above, we now include new fitness data on the R561H variant in African parasites, based on competition assays with an eGFP reporter line. To standardize our fitness data, we now have analyzed our data to day 36 across assays, as follows:

    Methods lines 538-539: “Cultures were maintained in 12-well plates and monitored every four days over a period of 36 days (18 generations) by harvesting at each time point a fraction of each co-culture for saponin lysis.”

    Figure 3 Legend lines 920-921: “K13 mutant clones were co-cultured at 1:1 starting ratios with isogenic K13 wild-type controls over a period of 36 days.”

    The selective sweep to C560Y in SE Asia is something that has been known for a while. It is striking that it has been selected as based on the data presented here P563L has a similar fitness and RSA profile. The authors could explore this further.

    The Reviewer highlights the important point that RSA values and fitness were comparable for C580Y and P553L, yet only the former swept across Southeast Asia. This would argue for additional factors that contribute to the successful dissemination of C580Y. These could include favorable genetic backgrounds that help propagate C580Y mutant parasites, or increased transmission rates, relative to P553L. To date, reasons for C580Y’s success beyond its moderate resistance and relatively minor fitness cost have not been firmly established. One possibility might be related to piperaquine pressure that selected for amplification in plasmepsins II and III as well as novel mutations in PfCRT, which emerged in parasites harboring K13 C580Y and which have been shown to spread as a series of genetically closely related sublineages (referred to as KEL1/PLA1; Hamilton et al. 2019, Lancet Infect Dis; Imwong et al. 2020, Lancet Infect Dis). These points are discussed as follows:

    Discussion lines 361-369: “Our studies into the impact of K13 mutations on in vitro growth in Asian Dd2 parasites provide evidence that that the C580Y mutation generally exerts less of a fitness cost relative to other K13 variants, as measured in K13-edited parasites co-cultured with an eGFP reporter line. A notable exception was P553L, which compared with C580Y was similarly fitness neutral and showed similar RSA values. P553L has nonetheless proven far less successful in its regional dissemination compared with C580Y (Menard et al., 2016). These data suggest that additional factors have contributed to the success of C580Y in sweeping across SE Asia. These might include specific genetic backgrounds that have favored the dissemination of C580Y parasites, possibly resulting in enhanced transmission potential (Witmer et al., 2020), or ACT use that favored the selection of partner drug resistance in these parasite backgrounds (van der Pluijm et al., 2019).”

    Overall, the main conclusion that there are K13 mutations that can confirm resistance to Art in the context of African parasites is clearly presented and convincing and this highlights the risk that exists for public health officials in African nations. What would be interesting from a readers perspective is how likely it is that this loss of fitness hurdle is going to be overcome in Africa and whether the risk of resistance development will increase as transmission rates drop.

    We appreciate this suggestion from the Reviewer. Our revised manuscript now addresses this topic as follows:

    Discussion lines 393-399: “It is nonetheless possible that secondary determinants will allow some African strains to offset fitness costs associated with mutant K13, or otherwise augment K13-mediated ART resistance. Identifying such determinants could be possible using genome-wide association studies or genetic crosses between ART-resistant and sensitive African parasites in the human liver-chimeric mouse model of P. falciparum infection (Vaughan et al., 2015; Amambua-Ngwa et al., 2019). Reduced transmission rates in areas of Africa where malaria is declining, leading to lower levels of immunity, may also benefit the emergence and dissemination of mutant K13 (Conrad and Rosenthal, 2019).”

    Reviewer #2 (Public Review):

    In this paper, the investigators performed two large-scale surveys of the propeller domain mutations in the K13 gene, a marker of artemisinin (ART) resistance, in African (3299 samples) and Cambodian (3327 samples) Plasmodium falciparum populations. In the African parasite population, they identified the K13 R561H variant in Rwanda, while parasites from other areas had the wild-type K13. In Cambodia, however, they documented a hard genetic sweep of C580Y mutation that occurred rapidly. They generated the C580Y and M579I mutations in four different parasite strains with different genetic backgrounds and found that these mutations conferred varying degrees of in vitro ART resistance. They further edited the SE Asian parasite strains Dd2 and Cam3.II with 7 K13 mutations and found that all the propeller domain mutations conferred ART resistance in the Dd2 parasite, whereas three of the mutations did so in the Cam3.II background. The R561H and C580Y mutations were also evaluated in several parasites collected from Thailand. In vitro growth competition analysis showed that K13 mutations caused substantial fitness costs in the African parasite background, but much less fitness costs in the SE Asian parasites. This study demonstrated the potential emergence of ART resistance in African parasite populations and offered insights into the importance of the parasite's genetic background in the emergence of ART resistance.

    We thank the Reviewer for this thorough summary and favorable assessment of our work.

    Reviewer #3 (Public Review):

    Stokes et al address the question: Why have mutations in the K13 gene spread rapidly across South East Asia and led to widespread treatment failure with artemisinin-based antimalarials? In contrast, why do K13 mutations remain quite rare in Africa, and artemisinin-based antimalarials remain effective?

    The work combines a number of different studies on different parasites of different origins. Gene editing has been used to assess the effects of K13 mutations in different parasite backgrounds, leading to a very complex view of the competing factors of level of resistance conferred and fitness cost.

    The authors put forward the hypothesis that fitness costs associated with K13 mutations select against their dissemination in the high malaria transmission settings in Africa. However, the complexity of the genetic backgrounds of the parasites makes it difficult to tease out the contributing factors.

    We agree that these are complex and multifactorial areas of investigation and appreciate the Reviewer’s summary.

  2. eLife

    Evaluation Summary:

    This study is of interest to the broad malaria research community and especially those who work on drug resistance. The authors provide a summary of their surveys of African and Southeast Asian Plasmodium falciparum parasites for the Kelch 13 gene, a marker of artemisinin resistance. The contribution of several K13 mutations to artemisinin resistance is investigated in different genetic backgrounds and confirms the lack of a barrier for the potential emergence of artemisinin resistance in African parasites. These findings are of prime importance in the context of public health perspective on managing the risk of resistance appearing in Africa.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The authors have studied mutations in the K13 gene that is linked to Artemisinin resistance in a range of African parasites. They show that these mutations can confer resistance in a in vitro survival assay but that they are often linked to reduced fitness. The authors also show that different parasites have less of an impact on fitness when the K13 mutations are introduced in line with the suggestion that the overall genetic background is critical for transmission of K13 mutations. The paper also shows evidence that genes potentially contributing to the genetic background are not involved.

    The overall work involves a significant amount of work that to generate a wide range of different parasite lines that allow a detailed assessment of how different mutations interact with the genetic background of the parasite. This provides a significant amount of new insights. A key conclusion the authors draw from this work relates to the relationship between fitness and resistance and by inference on why artemisinin resistance has occurred in SE Asia. While this indeed would be a striking conclusion I think the data at this stage is not strong enough to make this claim. The claim is mainly based on Figure 3 E and F as well as 5 C and D. While indeed, initially it looks like RSA has much less of a survival impact in Dd2 there is some concern that the data is generated using different baselines (isogenic WT parasite in Figure 3 and Dd2eGFP in Figure 5 D). This is noteworthy as in Figure 5C the Dd2wt parasite is used and the fitness cost appears to be different.

    A striking finding is that the UG659C560Y line appears to have a relatively small fitness cost - especially if looked at for the whole 40 generations rather than the somewhat arbitrarily picked 38 days. This data could suggest that there are parasites in Africa that have the capacity to acquire resistance with minimal cost to fitness.

    The selective sweep to C560Y in SE Asia is something that has been known for a while. It is striking that it has been selected as based on the data presented here P563L has a similar fitness and RSA profile. The authors could explore this further.
    Overall, the main conclusion that there are K13 mutations that can confirm resistance to Art in the context of African parasites is clearly presented and convincing and this highlights the risk that exists for public health officials in African nations. What would be interesting from a readers perspective is how likely it is that this loss of fitness hurdle is going to be overcome in Africa and whether the risk of resistance development will increase as transmission rates drop.

  4. eLife

    Reviewer #2 (Public Review):

    In this paper, the investigators performed two large-scale surveys of the propeller domain mutations in the K13 gene, a marker of artemisinin (ART) resistance, in African (3299 samples) and Cambodian (3327 samples) Plasmodium falciparum populations. In the African parasite population, they identified the K13 R561H variant in Rwanda, while parasites from other areas had the wild-type K13. In Cambodia, however, they documented a hard genetic sweep of C580Y mutation that occurred rapidly. They generated the C580Y and M579I mutations in four different parasite strains with different genetic backgrounds and found that these mutations conferred varying degrees of in vitro ART resistance. They further edited the SE Asian parasite strains Dd2 and Cam3.II with 7 K13 mutations and found that all the propeller domain mutations conferred ART resistance in the Dd2 parasite, whereas three of the mutations did so in the Cam3.II background. The R561H and C580Y mutations were also evaluated in several parasites collected from Thailand. In vitro growth competition analysis showed that K13 mutations caused substantial fitness costs in the African parasite background, but much less fitness costs in the SE Asian parasites. This study demonstrated the potential emergence of ART resistance in African parasite populations and offered insights into the importance of the parasite's genetic background in the emergence of ART resistance.

  5. eLife

    Reviewer #3 (Public Review):

    Stokes et al address the question: Why have mutations in the K13 gene spread rapidly across South East Asia and led to widespread treatment failure with artemisinin-based antimalarials? In contrast, why do K13 mutations remain quite rare in Africa, and artemisinin-based antimalarials remain effective?

    The work combines a number of different studies on different parasites of different origins. Gene editing has been used to assess the effects of K13 mutations in different parasite backgrounds, leading to a very complex view of the competing factors of level of resistance conferred and fitness cost.

    The authors put forward the hypothesis that fitness costs associated with K13 mutations select against their dissemination in the high malaria transmission settings in Africa. However, the complexity of the genetic backgrounds of the parasites makes it difficult to tease out the contributing factors.

  6. Version published to 10.1101/2021.01.27.428390v1 on bioRxiv