INFECTIOUS DISEASES
Understanding artemisinin resistance Genetic studies provide clues to how malaria parasites become drug resistant By Carol Hopkins Sibley
T
he drug resistance specter looms over most infectious diseases. Malaria provides a particularly urgent example of increasing resistance and treatment failure. In the past decade, artemisinin combination therapies (ACTs) have contributed to impressive reductions in malaria morbidity and mortality (1). However, in 2009, Dondorp et al. found that when patients in western Cambodia infected with Plasmodium falciparum (the deadliest form of malaria) were treated with ACTs, they took longer than normal to clear their parasites. It is the artemisinin component that normally clears parasites quickly, and the authors therefore concluded that this component of the ACT was compromised (2). Slow-clearing parasites were also found in western Thailand and other parts of Cambodia (see the first figure) (3, 4). Two reports in this issue, by Mok et al. (page 431) (5) and Straimer et al. (page 428) (6), apply specialized techniques to better understand the mechanisms that underlie this resistance to artemisinins. It is difficult and time-consuming to measure the speed of parasite clearance in patients. Collaborative efforts were therefore launched to define genetic changes that might quickly identify the resistant parasites. Studies focused on parasites from Cambodia found a number of broad genomic regions that seemed like good candidates for such molecular markers of resistance. Building on the work of an international consortium (7), Ariey et al. identified Kelch 13 (K13) as a gene strongly correlated with the slow-clearance phenotype in parasites from western Cambodia (8). A molecular marker of resistance is usually defined by a simple, consistent pattern of genetic changes called single nucleotide polymorphisms (SNPs), but K13 is different. Studies in western Cambodia found that each slow-clearing parasite contained one of 15 different mutations that change a single amino acid in the “propeller region” of the K13 protein. As the geographic range of studies widened, more alleles have been discovered, further increasing the inventory of new mutations in the Mekong region (9, Worldwide Antimalarial Resistance Network, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. E-mail: [email protected]
Slow-clearing parasites in Southeast Asia Myanmar (Burma)
Laos Thailand
Cambodia Vietnam
Spread of resistance in Southeast Asia. In the past 5 years, many different drug-resistant malaria parasites have emerged in Southeast Asia. Two articles in this issue (5, 6) provide specialized approaches to define more fully the molecular basis of artemisinin resistance in malaria parasites. Data from (8, 9, 10).
10). Even more worrisome, new populations of resistant parasites have emerged independently in Myanmar (10). It appears that the slow-clearing phenotype is spreading in the Mekong region, not because all parasites carry a single, highly resistant K13 mutant but mainly because a wide variety of K13 mutants are emerging independently. Resistance to earlier antimalarial drugs spread from the Mekong region to Africa (11). To investigate whether artemisinin resistance had spread already, two groups assessed ~2000 parasite isolates from 25 sites across Africa (see the second figure) (12, 13). They identified 20 additional K13 alleles. Most were rare (1 to 3% of the population); a few were shared among distant African sites, but most were observed in only one location. This observation raised an important question: Does artemisinin resistance already have a toehold in Africa, or are other changes in parasites also required? The two reports in this issue help to clarify some aspects of this muddy situation. Straimer et al. used zinc-finger nucleases to change single SNPs and thereby create or revert K13 propeller mutants in cultured
SCIENCE sciencemag.org
malaria parasites. They adapted a recently developed laboratory method (7) to test the artemisinin sensitivity of the modified parasites and found that when a resistant K13 mutation was repaired to wild type in Cambodian parasites, artemisinin resistance was lost. Conversely, conversion of a wild-type K13 gene to a propeller mutant rendered drug-sensitive lines more resistant to artemisinin treatment. This approach provides a laboratory test to determine whether K13 mutants like those seen in Africa can alone confer artemisinin resistance in a parasite. In other organisms, the Kelch proteins often act as interaction hubs that bind other proteins involved in a network of stress responses (8). Different K13 propeller mutants confer slow clearance on Mekong parasites (8–10), but supporting changes in stress responses might also be needed to render the parasites fully resistant to artemisinin. If so, the slow-clearing parasites should share other parts of their genome, even when they carry different K13 alleles. In fact, earlier genome-wide analyses found multiple genetic regions common to the slow-clearing parasites, which did not all carry the same K13 23 JANUARY 2015 • VOL 347 ISSUE 6220
Published by AAAS
373
INSIGHTS | P E R S P E C T I V E S
K13 propeller mutations in Africa Mauritania Niger
Mali
Senegal
Eritrea
Chad
Guinea
B
Sudan
Gambia Guinea Bissau
a kin ur
Benin Nigeria
Ethiopia
Sierra Leone
Central African Republic
Liberia Togo Ghana Ivory Coast
Cameroon Uganda Congo
Equatorial Guinea Gabon
Kenya
Congo Rwanda Burundi
Tanzania
am
biq
ue
Malawi
Angola
oz
Zambia M
On the path to resistance? African parasites carry some of the same mutations found in Southeast Asia, as well as novel ones, although artemisinin-resistant parasites have not yet been found there. Data from (12, 13).
allele [see references in (8)]. The results of Straimer et al. offer direct support for this “permissive genome” idea. The authors used the new method to test the same Mekong-derived K13 alleles in cultured parasites from various geographic regions. They found that Mekong-derived parasites survived artemisinin treatment better than those from Africa, even when both were engineered to carry the same allele of K13. Mok et al. address the question of whether other genes are also required to allow K13 mutant parasites to survive artemisinin exposure. Malaria parasites are unusual because their genes are transcribed in a set temporal pattern during the 72-hour cell cycle of P. falciparum. Using that metric, the authors identified three different patterns of transcription; these patterns correlated with progression through the cell cycle. One transcription pattern correlated with slow clearance and with delayed development early in the growth cycle. Given that artemisinins are metabolized in a few hours, this early delay could protect the resistant parasites during that brief, critical period (14). The authors then measured the mRNA expression of genes during this early stage, comparing individual parasites from different geographic regions, and again related expression levels to their clearance phenotype. Genes involved in the oxidative stress response were prominent among those differentially expressed in slow- and fast-clearing parasites, providing strong evidence that 374
genes in these networks are involved in the artemisinin response. These findings suggest that the diverse mutations that have been observed in K13 may each diminish the capacity of the protein to function as a binding partner for proteins in the stress response network (8). Changes in expression of those stress proteins could compensate for the reduced K13 function and protect the parasites against artemisinin challenge. Generally, drug resistance is tracked by identifying a few SNPs that uniquely identify resistant pathogens. The diversity of K13 mutant alleles found in slow-clearing parasites does not conform to that simple model. Even more puzzling, the slow-clearing phenotype is spreading in the Mekong region, not because a highly resistant parasite is spreading under selection but mainly due to the independent emergence of a wide variety of K13 mutants. Even more alleles of K13 have emerged in African parasites, but it is not clear whether a K13 mutation alone is enough to render an African parasite artemisinin-resistant or whether other genetic changes are also required. The knowledge that—as Straimer et al. show—a parasite carrying a K13 propeller mutant is protected against artemisinin in the laboratory allows novel K13 alleles identified only by molecular methods to be tested in that assay. If African parasites lack “permissive genomes,” the presence of a K13 mutant allele alone may not (yet) signal ar-
temisinin resistance. African and Southeast Asian parasite genomes differ markedly, and identifying differences in the families of genes that respond to oxidative stress could thus help to find the full complement of genes required to confer artemisinin resistance. Artemisinin resistance in the Mekong is now well established (9, 10). Eliminating those resistant parasites before they spread more widely is crucial (3). The insights in the two reports in this issue (4, 5) provide a basis for a clearer definition of a molecular signature. These markers can then be used to detect new foci of artemisinin-resistant parasites at the earliest possible time, and allow appropriate responses to be mobilized. ■ REFERENCES
1. World Health Organization, World Malaria Report, www.who.int/malaria/publications/world_malaria_ report_2014/en; accessed 11 Dec. 2014. 2. A. M. Dondorp et al., N. Engl. J. Med. 361, 455 (2009). 3. A. M. Dondorp, P. Ringwald, Trends Parasitol. 29, 359 (2013). 4. A. P. Phyo et al., Lancet 379, 1960 (2012). 5. S. Mok et al., Science 347, 431 (2015). 6. J. Straimer et al., Science 347, 428 (2015). 7. B. Witkowski et al., Lancet Infect. Dis. 13, 1043 (2013). 8. F. Ariey et al., Nature 505, 50 (2014). 9. E. A. Ashley et al., N. Engl. J. Med. 371, 411 (2014). 10. S. Takala-Harrison et al., J. Infect. Dis. 10.1093/infdis/ jiu491 (2014). 11. C. Roper et al., Science 305, 1124 (2004). 12. S. M. Taylor et al., J. Infect. Dis. 10.1093/infdis/jiu467 (2014). 13. E. Kamau et al., J. Infect. Dis. 10.1093/infdis/jiu608 (2014). 14. N. Klonis et al., Proc. Natl. Acad. Sci. U.S.A. 110, 5157 (2013).
10.1126/science.aaa4102
sciencemag.org SCIENCE
23 JANUARY 2015 • VOL 347 ISSUE 6220
Published by AAAS
Understanding artemisinin resistance Genetic studies provide clues to how malaria parasites become drug resistant By Carol Hopkins Sibley
T
he drug resistance specter looms over most infectious diseases. Malaria provides a particularly urgent example of increasing resistance and treatment failure. In the past decade, artemisinin combination therapies (ACTs) have contributed to impressive reductions in malaria morbidity and mortality (1). However, in 2009, Dondorp et al. found that when patients in western Cambodia infected with Plasmodium falciparum (the deadliest form of malaria) were treated with ACTs, they took longer than normal to clear their parasites. It is the artemisinin component that normally clears parasites quickly, and the authors therefore concluded that this component of the ACT was compromised (2). Slow-clearing parasites were also found in western Thailand and other parts of Cambodia (see the first figure) (3, 4). Two reports in this issue, by Mok et al. (page 431) (5) and Straimer et al. (page 428) (6), apply specialized techniques to better understand the mechanisms that underlie this resistance to artemisinins. It is difficult and time-consuming to measure the speed of parasite clearance in patients. Collaborative efforts were therefore launched to define genetic changes that might quickly identify the resistant parasites. Studies focused on parasites from Cambodia found a number of broad genomic regions that seemed like good candidates for such molecular markers of resistance. Building on the work of an international consortium (7), Ariey et al. identified Kelch 13 (K13) as a gene strongly correlated with the slow-clearance phenotype in parasites from western Cambodia (8). A molecular marker of resistance is usually defined by a simple, consistent pattern of genetic changes called single nucleotide polymorphisms (SNPs), but K13 is different. Studies in western Cambodia found that each slow-clearing parasite contained one of 15 different mutations that change a single amino acid in the “propeller region” of the K13 protein. As the geographic range of studies widened, more alleles have been discovered, further increasing the inventory of new mutations in the Mekong region (9, Worldwide Antimalarial Resistance Network, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. E-mail: [email protected]
Slow-clearing parasites in Southeast Asia Myanmar (Burma)
Laos Thailand
Cambodia Vietnam
Spread of resistance in Southeast Asia. In the past 5 years, many different drug-resistant malaria parasites have emerged in Southeast Asia. Two articles in this issue (5, 6) provide specialized approaches to define more fully the molecular basis of artemisinin resistance in malaria parasites. Data from (8, 9, 10).
10). Even more worrisome, new populations of resistant parasites have emerged independently in Myanmar (10). It appears that the slow-clearing phenotype is spreading in the Mekong region, not because all parasites carry a single, highly resistant K13 mutant but mainly because a wide variety of K13 mutants are emerging independently. Resistance to earlier antimalarial drugs spread from the Mekong region to Africa (11). To investigate whether artemisinin resistance had spread already, two groups assessed ~2000 parasite isolates from 25 sites across Africa (see the second figure) (12, 13). They identified 20 additional K13 alleles. Most were rare (1 to 3% of the population); a few were shared among distant African sites, but most were observed in only one location. This observation raised an important question: Does artemisinin resistance already have a toehold in Africa, or are other changes in parasites also required? The two reports in this issue help to clarify some aspects of this muddy situation. Straimer et al. used zinc-finger nucleases to change single SNPs and thereby create or revert K13 propeller mutants in cultured
SCIENCE sciencemag.org
malaria parasites. They adapted a recently developed laboratory method (7) to test the artemisinin sensitivity of the modified parasites and found that when a resistant K13 mutation was repaired to wild type in Cambodian parasites, artemisinin resistance was lost. Conversely, conversion of a wild-type K13 gene to a propeller mutant rendered drug-sensitive lines more resistant to artemisinin treatment. This approach provides a laboratory test to determine whether K13 mutants like those seen in Africa can alone confer artemisinin resistance in a parasite. In other organisms, the Kelch proteins often act as interaction hubs that bind other proteins involved in a network of stress responses (8). Different K13 propeller mutants confer slow clearance on Mekong parasites (8–10), but supporting changes in stress responses might also be needed to render the parasites fully resistant to artemisinin. If so, the slow-clearing parasites should share other parts of their genome, even when they carry different K13 alleles. In fact, earlier genome-wide analyses found multiple genetic regions common to the slow-clearing parasites, which did not all carry the same K13 23 JANUARY 2015 • VOL 347 ISSUE 6220
Published by AAAS
373
INSIGHTS | P E R S P E C T I V E S
K13 propeller mutations in Africa Mauritania Niger
Mali
Senegal
Eritrea
Chad
Guinea
B
Sudan
Gambia Guinea Bissau
a kin ur
Benin Nigeria
Ethiopia
Sierra Leone
Central African Republic
Liberia Togo Ghana Ivory Coast
Cameroon Uganda Congo
Equatorial Guinea Gabon
Kenya
Congo Rwanda Burundi
Tanzania
am
biq
ue
Malawi
Angola
oz
Zambia M
On the path to resistance? African parasites carry some of the same mutations found in Southeast Asia, as well as novel ones, although artemisinin-resistant parasites have not yet been found there. Data from (12, 13).
allele [see references in (8)]. The results of Straimer et al. offer direct support for this “permissive genome” idea. The authors used the new method to test the same Mekong-derived K13 alleles in cultured parasites from various geographic regions. They found that Mekong-derived parasites survived artemisinin treatment better than those from Africa, even when both were engineered to carry the same allele of K13. Mok et al. address the question of whether other genes are also required to allow K13 mutant parasites to survive artemisinin exposure. Malaria parasites are unusual because their genes are transcribed in a set temporal pattern during the 72-hour cell cycle of P. falciparum. Using that metric, the authors identified three different patterns of transcription; these patterns correlated with progression through the cell cycle. One transcription pattern correlated with slow clearance and with delayed development early in the growth cycle. Given that artemisinins are metabolized in a few hours, this early delay could protect the resistant parasites during that brief, critical period (14). The authors then measured the mRNA expression of genes during this early stage, comparing individual parasites from different geographic regions, and again related expression levels to their clearance phenotype. Genes involved in the oxidative stress response were prominent among those differentially expressed in slow- and fast-clearing parasites, providing strong evidence that 374
genes in these networks are involved in the artemisinin response. These findings suggest that the diverse mutations that have been observed in K13 may each diminish the capacity of the protein to function as a binding partner for proteins in the stress response network (8). Changes in expression of those stress proteins could compensate for the reduced K13 function and protect the parasites against artemisinin challenge. Generally, drug resistance is tracked by identifying a few SNPs that uniquely identify resistant pathogens. The diversity of K13 mutant alleles found in slow-clearing parasites does not conform to that simple model. Even more puzzling, the slow-clearing phenotype is spreading in the Mekong region, not because a highly resistant parasite is spreading under selection but mainly due to the independent emergence of a wide variety of K13 mutants. Even more alleles of K13 have emerged in African parasites, but it is not clear whether a K13 mutation alone is enough to render an African parasite artemisinin-resistant or whether other genetic changes are also required. The knowledge that—as Straimer et al. show—a parasite carrying a K13 propeller mutant is protected against artemisinin in the laboratory allows novel K13 alleles identified only by molecular methods to be tested in that assay. If African parasites lack “permissive genomes,” the presence of a K13 mutant allele alone may not (yet) signal ar-
temisinin resistance. African and Southeast Asian parasite genomes differ markedly, and identifying differences in the families of genes that respond to oxidative stress could thus help to find the full complement of genes required to confer artemisinin resistance. Artemisinin resistance in the Mekong is now well established (9, 10). Eliminating those resistant parasites before they spread more widely is crucial (3). The insights in the two reports in this issue (4, 5) provide a basis for a clearer definition of a molecular signature. These markers can then be used to detect new foci of artemisinin-resistant parasites at the earliest possible time, and allow appropriate responses to be mobilized. ■ REFERENCES
1. World Health Organization, World Malaria Report, www.who.int/malaria/publications/world_malaria_ report_2014/en; accessed 11 Dec. 2014. 2. A. M. Dondorp et al., N. Engl. J. Med. 361, 455 (2009). 3. A. M. Dondorp, P. Ringwald, Trends Parasitol. 29, 359 (2013). 4. A. P. Phyo et al., Lancet 379, 1960 (2012). 5. S. Mok et al., Science 347, 431 (2015). 6. J. Straimer et al., Science 347, 428 (2015). 7. B. Witkowski et al., Lancet Infect. Dis. 13, 1043 (2013). 8. F. Ariey et al., Nature 505, 50 (2014). 9. E. A. Ashley et al., N. Engl. J. Med. 371, 411 (2014). 10. S. Takala-Harrison et al., J. Infect. Dis. 10.1093/infdis/ jiu491 (2014). 11. C. Roper et al., Science 305, 1124 (2004). 12. S. M. Taylor et al., J. Infect. Dis. 10.1093/infdis/jiu467 (2014). 13. E. Kamau et al., J. Infect. Dis. 10.1093/infdis/jiu608 (2014). 14. N. Klonis et al., Proc. Natl. Acad. Sci. U.S.A. 110, 5157 (2013).
10.1126/science.aaa4102
sciencemag.org SCIENCE
23 JANUARY 2015 • VOL 347 ISSUE 6220
Published by AAAS
