This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Malaria is one of the most important parasitic diseases worldwide. The evolution and spread of multidrug resistant strains of P. falciparum and P. vivax is a major health concern. Despite the advances in research, including the sequencing of the entire genomes of Anopheles gambiae, P. falciparum and P. yoelii, the molecular basis of the drug resistance phenomena in Plasmodia is not completely understood. The long-term goal of the proposed research is to elucidate mechanisms by which the malaria parasites become resistant to antimalarial drugs. The results of this research could lead to a better approach to drug design and disease control. Our working hypothesis is that several genes that belong to the ATP-binding cassette (ABC) superfamily of transporter proteins work in conjunction to confer drug resistance. A collection of drug sensitive and resistant lines of P. yoelii will be used as an in vivo rodent malaria model to investigate the mechanisms of drug resistance. Although a number of resistance mechanisms are possible, we will focus on two members of the ABC superfamily of transporter proteins that may mediate drug efftux in the parasite, the multidrug resistance associated protein gene (pymrp) and the ABCG homologue gene.
The specific aims are:- To characterize the P. yoelii multidrug resistance associated protein gene (pymrp) and to determine its role in malaria drug resistance. - To measure transcript levels of the pymrp gene in drug sensitive and resistant lines. - To determine the sub-cellular location and measure expression levels of the pyMRP protein. - To initiate transfection studies to address the function of the pyMRP protein by gene disruption.- To characterize a novel ABC gene, the P. yoelii ABCG homologue, and determine its role in malaria drug resistance. - To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses. - To analyze the P. yoelii ABCG homologue, from drug sensitive and resistant lines, for gene copy number, single nucleotide polymorphisms (SNP), transcript levels and chromosomal location. - To determine the sub-cellular location and measure expression levels of the P. yoelii ABCG protein.The rational for this research is that understanding the role of pymrp and the P. yoelii ABCG homologue gene in Plasmodium drug resistance could provide the basis for better drug design and potentially lead to reversal of drug resistance to the quinoline-containing antimalarial drugs. This is an innovative research because the function of the Plasmodium mrp and ABCG ortholog genes and their potential role in drug resistance remains to be elucidated. During the first year we expect to accomplish the following goals: 1) To measure transcript levels of the pymrp gene in drug sensitive and resistant lines; 2) To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses; and 3) To initiate the construction of the pyMRP recombinant protein.
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