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.
Subject(s)
Amino Acid Substitution , Antimalarials/pharmacology , Artemisinins/pharmacology , Drug Resistance , Plasmodium chabaudi/drug effects , Plasmodium chabaudi/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , DNA Mutational Analysis , Genetic Markers , Humans , Mice , Mice, Inbred CBA , Molecular Sequence Data , Mutation, Missense , Plasmodium chabaudi/isolation & purification , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Protein Conformation , Protozoan Proteins/chemistryABSTRACT
BACKGROUND: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. METHODOLOGY/PRINCIPAL FINDINGS: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN)+mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN+MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN+MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. CONCLUSION: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as yet unidentified, are likely to be involved.
Subject(s)
Antimalarials/therapeutic use , Artemisinins/therapeutic use , Mefloquine/therapeutic use , Multidrug Resistance-Associated Proteins/metabolism , Plasmodium chabaudi/drug effects , Plasmodium chabaudi/metabolism , Protozoan Proteins/metabolism , Animals , Blotting, Western , Drug Resistance/genetics , Malaria/drug therapy , Malaria/parasitology , Mice , Multidrug Resistance-Associated Proteins/genetics , Plasmodium chabaudi/genetics , Polymerase Chain Reaction , Protozoan Proteins/genetics , Sequence Analysis, DNAABSTRACT
The genes encoding enzymes involved in glutathione (GSH) metabolism may modulate responses to antimalarial drugs, but the role of most of them in antimalarial drug resistance has scarcely been investigated. Using an in silico/PCR combined approach, we have isolated from Plasmodium chabaudi, full sequences of five Plasmodium falciparum gene orthologues involved in GSH metabolism: the gamma-glutamylcysteine synthetase (Pc-gammagcs), glutathione-synthetase (Pc-gs), glutathione peroxidase (Pc-gpx), glutathione reductase (Pc-gr) and glutathione-S-transferase (Pc-gst). DNA sequencing of these genes from drug sensitive parasites, P. chabaudi AS (0CQ), and ones isolated from parasite lines that show genetically stable resistance to chloroquine (CQ) at low, intermediate and high levels, AS (3CQ), AS (15CQ) and AS (30CQ), respectively, revealed no point mutations in the resistant parasites. We used these sequences to design internal oligonucleotide primers to compare relative mRNA amounts of these genes between all P. chabaudi clones, in untreated mice or following CQ treatment with sub-curative doses, by real-time PCR. Analysis of three independent experiments revealed that transcription levels of the Pc-gammagcs, Pc-gs, Pc-gpx, Pc-gr and Pc-gst genes were not changed between chloroquine sensitive and resistant parasite clones, and that treatment with chloroquine did not induce an alteration in the expression of these genes in sensitive or resistant parasites. We concluded that chloroquine resistance in this species is determined by a mechanism that is independent of these genes, and most likely, of GSH metabolism.
