Artemisinin resistance in rodent malaria--mutation in the AP2 adaptor μ-chain suggests involvement of endocytosis and membrane protein trafficking

Abstract

Background: The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi.

Methods: Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared.

Results: An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the μ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene.

Conclusion: An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.

Figure 1

The AS lineage of drug resistant parasites, featuring artemisinin (ART) phenotypes and ubp-1 genotypes. Parasites were selected by passage in presence of drugs shown [29-33]. Artemisinin resistance (phenotype 1) appears during selection by chloroquine [25,26] and is mediated by either of two mutations in the ubp-1 gene (PCHAS_020720) [25,27]. AS-ART was generated during selection by artemisinin [29]. Its increased resistance to artemisinin (phenotype 2) and its genotype is the subject of the present study. *, parasites used in the present study; wt, wild-type; kg bw, kilograms body weight.

Figure 2

Artemisinin responses of Plasmodium chabaudi AS-3CQ, AS-30CQ and AS-ART. Mean% parasitaemia ± standard error of groups of three mice. A, untreated controls; B, 1 × 10exp6 parasites on day 0 and treated with 200mg ART/kg^-1 day^-1 days 1–3; C, 1 × 10exp7 parasites on day 0 and treated with 200mg ART/ kg^-1 day^-1 days 1–5. AS-sens, blue; AS-30CQ, red; AS-ART, green.

Figure 3

Conservation of AP2 μ-chain amino acid sequence. Conservation between P. chabaudi (PCHAS_143590) sequence and A) P. falciparum (PF3D7_1218300) or (B) rat (P84092 UniProtKB/Swiss-Prot) sequences. The P. chabaudi mutated residue I568 and the corresponding P. falciparum residue I592 (A) and Rat V401 (B) are highlighted (purple). Note Plasmodium spp.-specific sequence (~90 residues, relative to rat) lying between positions 154–244 (P. chabaudi).

Figure 4

I568T mutation in AP2 μ-chain interacts with YXXΦ motif on cargo protein. A. Rat (P84092) V401 is homologous to P. chabaudi AP2 μ-chain I568, and contributes to the hydrophobic pocket that binds L9 residue of bound peptide (DEEYGYECL) in structure 2PR9. B. Homology model for P. chabaudi wild-type homologue shows similar structure with I568 corresponding to rat V401. C. Homology model for P. falciparum orthologue shows similar structure, with I592 corresponding to rat V401 and P. chabaudi I568. D. Homology model for P. chabaudi mutant - 568T is predicted to reduce the hydrophobic character of the binding pocket in which peptide L9 binds. Note increased polar character associated with threonine hydroxyl group in D relative to B.