Malaria parasites fine-tune mutations to resist drugs - Nature.com
About half a million people, most of them children living in Africa, are killed each year by malaria1. Management of malaria, particularly that caused by the highly virulent protozoan parasite Plasmodium falciparum, is challenged by the emergence of resistance to antimalarial drugs2. Writing in Nature, Kim et al.3 report the structure and molecular properties of a key protein that facilitates resistance, the P. falciparum chloroquine-resistance transporter (PfCRT). The structure reveals the consequences of finely tuned mutations of the amino-acid residues that line a crucial central cavity in PfCRT. These mutated residues allow resistant parasites to transport certain antimalarial drugs away from their site of action — and the effect of the mutations is different for closely related drugs.
Malaria parasites spend part of their life cycle inside human red blood cells. There, they use a specialized membrane-bound compartment known as the digestive vacuole to degrade the protein haemoglobin, thereby generating amino-acid building blocks for growth4. Haemoglobin digestion also produces a toxic side product called haem, which is exploited by antimalarials from the quinoline family of drugs (which includes chloroquine and piperaquine). These drugs bind to the released haem in the digestive vacuole and prevent the compound’s detoxification by the parasite — in effect, poisoning the parasite with its own metabolic debris4.
Chloroquine’s affordability, safety and efficacy made it the drug of choice for combating malaria, until widespread resistance developed in the 1980s. Piperaquine is a structurally related compound that retains activity against chloroquine-resistant parasites and is currently used in combination with another antimalarial drug, dihydroartemisinin. Unfortunately, resistance to piperaquine is now widespread in parts of southeast Asia5. Paradoxically, piperaquine-resistant parasites are often more sensitive to chloroquine than are piperaquine-sensitive parasites6.
Drug resistance can arise because of mutations either in the drug’s target or in biological machinery that transports the drug to or from the target. A landmark report7 in 2000 identified PfCRT as the main mediator of chloroquine resistance in P. falciparum. Resistance emerged independently at different locations around the world, but is always associated with a particular mutation in the transporter — the substitution of a lysine amino-acid residue by a threonine residue (a K76T mutation). K76T combines with other geographically specific PfCRT mutations to mediate resistance and improve the fitness of the mutant parasites6–8.
PfCRT is a member of the superfamily of proteins known as drug/metabolite transporters9 and is located in the membrane of the parasite’s digestive vacuole. Structural and biochemical studies of related proteins suggest that resistance to chloroquine arises as a result of the transporter passing chloroquine out of the digestive vacuole, thus removing it from its site of action8. Interestingly, piperaquine resistance emerges when parasites harbouring the K76T mutation acquire further mutations6.
Efforts to design new resistance-busting quinoline antimalarials have been stymied by a lack of information about the structures of mutated PfCRT. But in the past few years, advances in a technique called cryo-electron microscopy10 (cryo-EM) have revolutionized structural biology by enabling the direct imaging of membrane-embedded proteins, such as PfCRT, that are not amenable to study using X-ray crystallography. Kim et al. used single-particle cryo-EM to determine the structure of PfCRT in South American 7G8 parasites, which harbour mutations that confer high-level chloroquine resistance.
The authors first had to work out a protocol for efficiently expressing, purifying and reconstituting PfCRT into a membrane-like environment, to maintain a native conformation of the protein. PfCRT is relatively small (49 kilodaltons) compared with proteins that are typically studied using cryo-EM, and so Kim et al. prepared an antibody fragment (known as an antigen-binding fragment, or Fab) that binds to PfCRT, thereby forming a complex that has sufficient mass and stability to allow cryo-EM-based structural elucidation. This approach yielded a structure with 3.2-ångström resolution.
PfCRT was revealed to have ten transmembrane domains and a negatively charged central cavity. The cavity opens on to the digestive vacuole, but closes about halfway through the membrane. The Fab-bound cavity has an opening 25 Å in diameter, which is large enough to contain chloroquine (the maximum dimension of which is about 14 Å) and piperaquine (with a maximum dimension of about 21 Å). The structure shows that wild-type PfCRT has a positively charged lysine residue (K76) positioned in the cavity. This residue is thought to repel both chloroquine (which has two positive charges) and piperaquine (which has four positive charges), thereby trapping them in the digestive vacuole (Fig. 1a).
Figure 1 | Mutations affect the transport of antimalarial drugs through the PfCRT protein. a, The drugs chloroquine and piperaquine target a membrane-bound organelle in the malaria parasite, called the digestive vacuole. Plasmodium falciparum chloroquine-resistance transporter (PfCRT) resides in the membrane of the digestive vacuole of the most virulent species of malaria parasite. A positively charged lysine amino-acid residue (K76) in wild-type PfCRT is thought to repel the positively charged drugs, preventing transport out of the vacuole (arrow) — which would lead to drug resistance. b, Kim et al.3 report the structure of PfCRT that contains a mutation known as K76T. The authors find that K76T alters the charge distribution of the lining of the PfCRT cavity. Both chloroquine and piperaquine bind to the mutated cavity, but only chloroquine passes through it (possibly because it has a lower positive charge than piperaquine), enabling resistance to chloroquine, but not to piperaquine. c, Further mutations in PfCRT (such as the C350R mutation) cause piperaquine to bind more weakly to the cavity than in b. This potentially underpins the drug’s ability to pass through PfCRT and leads to piperaquine resistance. In this scenario, the transport of chloroquine out of the digestive vacuole is less efficient than the transport of piperaquine.
Interestingly, Kim and colleagues’ biochemical experiments show that PfCRT from 7G8 parasites can bind both chloroquine and piperaquine, but transport only chloroquine; thus, parasites with the K76T mutation are resistant to chloroquine, but not piperaquine (Fig. 1b). By contrast, when the authors introduced further mutations to 7G8 that have been observed in South American11 and southeast Asian6 parasites in the past five years, piperaquine efflux increased — an effect that was associated with decreased sensitivity to piperaquine, but increased sensitivity to chloroquine (Fig. 1c).
Kim et al. used their cryo-EM structure to carry out molecular modelling and electrostatic analysis of PfCRT, to help explain why mutations can have opposite effects on the sensitivity of malaria parasites to chloroquine and piperaquine. The modelling suggests that mutations associated with piperaquine resistance can reduce the negative charge or alter the conformation of the PfCRT central cavity. This might prevent piperaquine from binding too tightly to the cavity and thus increase its transport out of the digestive vacuole. The authors propose that the distribution of surface charges in the cavity can be fine-tuned so that the initial binding of a drug to PfCRT, and its subsequent release for transport, is different for different drugs, thereby producing distinct effects on drug sensitivity. Taken together, Kim and colleagues’ findings show that P. falciparum is engaged in an ongoing balancing act, generating mutations that block the action of different drugs while maintaining optimal fitness of the parasite.
A limitation of the new structure is that PfCRT is locked in a conformation in which it is open to the digestive vacuole, as a result of Fab binding in its central cavity. Further studies will be required to work out how drug binding couples to the conformational rearrangements that permit drug transport.
On a practical level, it is particularly important to understand the mechanisms of resistance that are likely to arise in Africa, where more than 90% of cases of malaria caused by P. falciparum occur1. Compared with other parts of the world, there are fewer PfCRT mutations in Africa, where resistance to chloroquine is decreasing in many countries and where the combination of piperaquine and dihydroartemisinin remains highly effective12. We can look forward to further studies of PfCRT, including visualization of the drug-bound and open-to-cytoplasm structural conformations, which will further explain the effects of resistance mutations and might help to identify drugs that circumvent resistance. For now, we can appreciate the insights gained from Kim and colleagues’ beautiful marriage of structure, biochemistry, genetics and parasitology, and particularly from the first atomic-resolution structure of PfCRT — the fine-tuned, resistance-mediating machine of malaria parasites.
References
- 1.
World Health Organization. World Malaria Report 2018 (2018).
- 2.
Haldar, K., Bhattacharjee, S. & Safeukui, I. Nature Rev. Microbiol. 16, 156–170 (2018).
- 3.
Kim, L. et al. Nature https://ift.tt/2QYAWM6 (2019).
- 4.
Goldberg, D. E. Curr. Top. Microbiol. Immunol. 295, 275–291 (2005).
- 5.
van der Pluijm, R. W. et al. Lancet Infect. Dis. 19, 952–961 (2019).
- 6.
Ross, L. S. et al. Nature Commun. 9, 3314 (2018).
- 7.
Fidock, D. A. et al. Mol. Cell 6, 861–871 (2000).
- 8.
Summers, R. L. et al. Proc. Natl Acad. Sci. USA 111, E1759–1767 (2014).
- 9.
Martin, R. E. & Kirk, K. Mol. Biol. Evol. 21, 1938–1949 (2004).
- 10.
Nogales, E. & Scheres, S. H. Mol. Cell 58, 677–689 (2015).
- 11.
Pelleau, S. et al. Proc. Natl Acad. Sci. USA 112, 11672–11677 (2015).
- 12.
Conrad, M. D. & Rosenthal, P. J. Lancet Infect. Dis. 19, e338–e351 (2019).
https://ift.tt/2XQRrLm
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