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Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.

Kinga Modrzynska K, Creasey A, Loewe L, Cezard T, Trindade Borges S, Martinelli A, Rodrigues L, Cravo P, Blaxter M, Carter R, Hunt P - BMC Genomics (2012)

Bottom Line: The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution.The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Immunology and Infection Research, University of Edinburgh, UK.

ABSTRACT

Background: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results: Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions: Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

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Structure and sequence conservation of P. chabaudi AAT1 and PCHAS_031370 and their orthologues. Secondary structure predictions of AAT1 (A) and PCHAS_031370 (C) proteins reveal 10 and 12 TM-helix proteins, respectively. The mutations discovered within CQ-R and CQ-hiR P. chabaudi parasites (AS-3CQ and AS-30CQ, respectively) are highlighted (magenta). The alignments of Plasmodium spp. protein fragments (B, D) indicate the positions of mutations in, or close to, conserved regions.
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Figure 5: Structure and sequence conservation of P. chabaudi AAT1 and PCHAS_031370 and their orthologues. Secondary structure predictions of AAT1 (A) and PCHAS_031370 (C) proteins reveal 10 and 12 TM-helix proteins, respectively. The mutations discovered within CQ-R and CQ-hiR P. chabaudi parasites (AS-3CQ and AS-30CQ, respectively) are highlighted (magenta). The alignments of Plasmodium spp. protein fragments (B, D) indicate the positions of mutations in, or close to, conserved regions.

Mentions: The A173E aat1 mutation shares some properties with the determinant (K76T pfcrt) of CQ-R in P. falciparum. For example, like pfcrt, aat1 is predicted to encode a 10-transmembrane (TM) helix transporter (Figure 5A) and its P. falciparum orthologue (PFF1430c) is targeted to the membrane of the DV (D. Fidock, P. Moura, pers comm.). The wild-type function of pfcrt is uncertain but amino acid transport has been suggested [36,37]. Both K76T and A173E mutations result in negative charge shifts. Residue 173 in aat1 is at the start of a highly conserved region (Figure 5B) close to the start of the first TM-helix (TM1): in pfCRT, residue 76 lies at the start of TM1, predicted to be internal to the DV where CQ is thought to act. These data suggest that AAT1 and CRT may share some structure/function relationships impacting on their physiological function in the absence and/or presence of CQ.


Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.

Kinga Modrzynska K, Creasey A, Loewe L, Cezard T, Trindade Borges S, Martinelli A, Rodrigues L, Cravo P, Blaxter M, Carter R, Hunt P - BMC Genomics (2012)

Structure and sequence conservation of P. chabaudi AAT1 and PCHAS_031370 and their orthologues. Secondary structure predictions of AAT1 (A) and PCHAS_031370 (C) proteins reveal 10 and 12 TM-helix proteins, respectively. The mutations discovered within CQ-R and CQ-hiR P. chabaudi parasites (AS-3CQ and AS-30CQ, respectively) are highlighted (magenta). The alignments of Plasmodium spp. protein fragments (B, D) indicate the positions of mutations in, or close to, conserved regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3362770&req=5

Figure 5: Structure and sequence conservation of P. chabaudi AAT1 and PCHAS_031370 and their orthologues. Secondary structure predictions of AAT1 (A) and PCHAS_031370 (C) proteins reveal 10 and 12 TM-helix proteins, respectively. The mutations discovered within CQ-R and CQ-hiR P. chabaudi parasites (AS-3CQ and AS-30CQ, respectively) are highlighted (magenta). The alignments of Plasmodium spp. protein fragments (B, D) indicate the positions of mutations in, or close to, conserved regions.
Mentions: The A173E aat1 mutation shares some properties with the determinant (K76T pfcrt) of CQ-R in P. falciparum. For example, like pfcrt, aat1 is predicted to encode a 10-transmembrane (TM) helix transporter (Figure 5A) and its P. falciparum orthologue (PFF1430c) is targeted to the membrane of the DV (D. Fidock, P. Moura, pers comm.). The wild-type function of pfcrt is uncertain but amino acid transport has been suggested [36,37]. Both K76T and A173E mutations result in negative charge shifts. Residue 173 in aat1 is at the start of a highly conserved region (Figure 5B) close to the start of the first TM-helix (TM1): in pfCRT, residue 76 lies at the start of TM1, predicted to be internal to the DV where CQ is thought to act. These data suggest that AAT1 and CRT may share some structure/function relationships impacting on their physiological function in the absence and/or presence of CQ.

Bottom Line: The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution.The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Immunology and Infection Research, University of Edinburgh, UK.

ABSTRACT

Background: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results: Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions: Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

Show MeSH
Related in: MedlinePlus