Investigation on the Involvement of Glutathione (GSH) System in the Mechanism of Antimalarial Action and its Possible Role in Conferring Resistance of Plasmodium falciparum to Antimalarials

Abstract

The oxidant enzymes of Plasmodium falciparum such as glutathione reductase
(PfGR) and glutathione S-transferase (PfGST) may play important role in survival of malaria
parasites including their possible involvement in conferring antimalarial drug resistance. This
study was aimed to investigate the mutations of regulatory gene controlling glutathione level
in a total of 34 P. falciparum isolates collected during 2006-2009 from endemic areas of
multidrug resistance P. falciparum in Thailand. In vitro sensitivity testing of each P.
falciparum isolate to the four antimalarial drugs, chloroquine (CQ), quinine (QN),
mefloquine (MQ) and artesunate (ARS) was performed by using SYBR green modified
assay, and 50% inhibitory concentration (IC50) of each drug was determined. Real-time
quantitative PCR was performed in DNA extracted from all parasite isolates using IQ SYBR
green supermix. Allele-specific oligonucleotides from the gene encoding PfGR and PfGST
were selected based on the coding sequence alignment of 3D7 parasites. The amplification of
copy number of PfGR and PfGST gene were determined. Median (range) IC50 of CQ, QN,
MQ and ARS were 86.33 (39.18-189.86), 387.79 (75.92-870.80), 45.87 (9.26-101.56) and
2.26 (0.83-5.21) nM, respectively. All isolates were resistant to CQ. Median (range) IC50 of
the 24 QN-sensitive (70.6%) and 10 QN-resistant (29.4%) isolates were 262.63 (75.92-
491.43) and 688.19 (520-870) nM, respectively. Median (range) IC50 of the 16 MQ-sensitive
(47.1%) and 18 MQ-resistant (52.9%) isolates were 21.17 (9.26-32.75) and 67.82 (36.82-
101.56) nM, respectively. None had more than one copy number of either PfGR or PfGST
gene. No associations were observed between sensitivity of parasite isolates to the four
antimalarials, and amplification of both PfGR and PfGST genes. Larger number of parasite
isolates is required to increase power of the hypothesis testing in order to confirm this
finding.