Identification and bioinformatic characterization of a multidrug resistance associated protein (ABCC) gene in Plasmodium berghei.
BACKGROUND
The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.


METHODS
The pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages.


RESULTS
The pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.


CONCLUSIONS
In this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.
González-Pons
María
M
Department of Microbiology, University of Puerto Rico School of Medicine, PO Box 365067, San Juan, PR 00936-5067, USA. maria.gonzalez9@upr.edu
Szeto
Ada C
AC
González-Méndez
Ricardo
R
Serrano
Adelfa E
AE
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D015139:Blotting, Southern; D002738:Chloroquine / Q000494:pharmacology*; D002874:Chromosome Mapping; D019295:Computational Biology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D008288:Malaria / Q000469:parasitology; D051379:Mice; D008969:Molecular Sequence Data; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D016366:Open Reading Frames; D010962:Plasmodium berghei / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics*; D016053:RNA, Protozoan / Q000737:chemistry; D016415:Sequence Alignment
D018528:ATP-Binding Cassette Transporters; D000962:Antimalarials; D002738:Chloroquine; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; D016053:RNA, Protozoan; C000715087:ABCC2 protein, human; C059987:mdr gene protein, Plasmodium