Investigation of the molecular mechanism of drug resistance with specific reference to the pfcrt gene in plasmodium falciparum

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Investigation of the molecular mechanism of drug resistance with specific reference to the pfcrt gene in plasmodium falciparum

 

Author: Chung, Yik-to
Title: Investigation of the molecular mechanism of drug resistance with specific reference to the pfcrt gene in plasmodium falciparum
Degree: Ph.D.
Year: 2011
Subject: Plasmodium falciparum.
Chloroquine.
Drug resistance.
Hong Kong Polytechnic University -- Dissertations
Malaria.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xi, 113 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2462548
URI: http://theses.lib.polyu.edu.hk/handle/200/6299
Abstract: Malaria is a serious disease threatening the tropical area. The parasites developed resistance to the first line drug chloroquine (CQ), causing more than 250 million cases and 1 million deaths every year. The Plasmodium falciparum chloroquine resistance transporter (PfCRT) was identified as the determinant that mediates CQ efflux. The protein consists of 10 putative transmembrane domains (TMD) and point mutations at different positions were reported in field isolates. In this thesis, the S163R mutation discovered by drug selection experiments in vivo was further studied by [³H]-CQ accumulation assay in vitro using heterologous expression system developed in our research group. PfCRT mutants were generated by site-directed mutagenesis and expressed in Pichia pastoris. Substituted cysteine accessibility analysis (SCAM) and cross-linking analysis was performed on TMD4 which harbors the residue 163. Our data confirms that positive charge on residue 163 is capable of restoring CQ sensitivity to PfCRT mutants. An aqueous-facing surface of TMD4 is revealed with S163, I166 and F170 lining a potential CQ binding crevice. Positive charge on the lumen-facing residues significantly reduces the drug transport. Residue 163 is also spatially close to TMD1 which harbors the well-known CQ resistance modulating residue K76T. The two TMDs are believed to function in chorus to form the drug translocation path.

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