Emergency of chloroquine resistance (CQR) in Plasmodium vivax, the most widely distributed human malaria parasite, is treating to render this cheap and widely used first-line antimalarial drug, ineffective. Resistance to primaquine has also been reported in P.vivax, indicating that this parasite is developing a multidrug resistance (MDR) phenotype. MDR and CQR in P.falciparum, the most deadly species, have been associated with the pfmdr1 and pfcrt genes. Pfmdr1 codes for an energy-dependent membrane transport protein. Mutations, over expression and increase in copy number of this gene have all been associated to mefloquine resistance, though not completely, with the CQR phenotype.  To initiate studies on MDR in P.vivax, we previously characterized the orthologous gene, pvmdr1, in P.vivax. Strikingly, there were no mutations associated with CQR, suggesting that a different mechanism should be involved in P.vivax resistance phenotype.  In P.falciparum the gene responsible for CQR, was identified as pfcrt, in which specific mutations are directly associated with the resistant phenotype. Pfcrt codes for an integral membrane transporter protein localized at the parasite`s acidic digestive vacuole and has been assigned to the drug/metabolite transporter supermfamily. Nevertheless, the pfcrt orthologous gene in P.vivax, pvcg10 has no mutations associated to CQR,  suggesting a different mechanism of resistance. Functional studies through reverse genetics of pvcg10 and pvmdr1 are difficult to conduct because of the present inviability of continuously growing P. vivax in vitro.  To overcome this difficulty, we have generated a transgenic line of the P.falciparum chloroquine sensitive (CQS) clone 3D7 over expressing pvmdr1, in order to determine whether the over expression of this transgene alters sensitivity patterns of the recipient 3D7 to antimalarics.  Eight clonal transgenic lines of 3D7 over expressing pvmdr1 and presenting plasmids integrated in genomic DNA were obtained. Drug sensitivity assays of these clones are currently being conducted. Two new vectors expressing higher levels of pvcg10 and pvmdr1 under the control of the HSP86 promoter of P.falciparum are being currently transfected in both CQR (3D7) and CQS (Dd2) clones in order to confirm our hypothesis.
(*correspondence hernando@icb.usp.br)



Acknowledgments: Pamela Orjuela is a PhD student from CNPq (No 141572/2004). We thank Dr. Carmen Fernandez Becerra and Mr. Marcio Massao Yamamoto for helpful discussions. We would also like thank Mr. Apuã C.M. Paquola for the statistical analysis of the limiting dilution assay. The lab of HAP receives support from FAPESP (01-09401-0) and CNPq (ID 302572/2002-3).