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RESEARCH ARTICLE
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Molecular analysis of Pfmrp1, Pfnhe1, Pfmc-2tm and Pfgch1 in
Plasmodium falciparum isolates from Mae sot, Tak province
Pimwan Thongdee1,2, Jiraporn Kuesap2, Kesara Na-Bangchang1,3*
1
Graduate Program in Bioclinical Sciences, Chulabhorn International College of
Medicine, Thammasat University, Pathumthan ,12121 Thailand
2
Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences,
Thammasat University, Pathumthani,12121 Thailand
3
Center of Excellence in Pharmacology and Molecular Biology of Malaria and
Cholangiocarcinoma, Thammasat University, Pathumthani,12121 Thailand
*Corresponding author
Abstract
The sensitivity of ten Plasmodium falciparum isolates collected from Mae
Sot distrct, Tak province in 2012, to chloroquine, quinine and artemisinin was
investigated in association with polymorphisms of the parasite resistant genes, i.e.,
Plasmodium falciparum multidrug resistance protein 1 (pfmrp1: SNP at amino acid
positions 191 and 437) and Plasmodium falciparum sodium hydrogen exchanger 1
(pfnhe1: at the microsatellite in ms4760 locus) including Plasmodium falciparum
maurer’s cleft two transmembrane proteins (pfmc-2tm: new SNPs) and Plasmodium
falciparum GTP cyclohydrolase 1 (pfgch1: new SNPs). Results revealed nonsynonymous mutations at the position 437 of pfmrp1 of which the amino acids
were DNNND repeat. For pfnhe1, the amino acids were NHNDNHNNDDD repeat.
For pfmc-2tm, non-synonymous mutations at the positions 189, 190, 192, 194, 196
and 197. All isolates were pfgch1 wild type. These genetic markers should be
further validated for monitoring of antimalarial drug resistance.
Keywords: Antimalarial drug resistance, Plasmodium falciparum multidrug
resistance protein 1, Plasmodium falciparum sodium hydrogen exchanger 1,
Plasmodium falciparum maurer’s cleft two transmembrane proteins, Plasmodium
falciparum GTP cyclohydrolase 1
Address correspondence and reprint request to: Kesara Na-Bangchang 99 Moo 18 Klongnung,
Klongluang, Pathumthani12121, Thailand. Tel.: +66 2 5644440 79x1803; fax: +66 2 5644398.
E-mail address: kesaratmu@yahoo.com (K. Na-Bangchang).
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Introduction
Malaria is one major public health problem in tropical countries. Globally,
an estimated 3.3 billion people in 97 countries and territories are at risk of being
infected with malaria and developing disease, and 1.2 billion are at high risk1.
Resistance to antimalarials has been documented for P. falciparum, P. malariae
and P. vivax. In P. falciparum, resistance has been observed in all currently used
antimalarials (amodiaquine, chloroquine, mefloquine, quinine, and sulfadoxinepyrimethamine) and, more recently, in artemisinin derivatives. The geographical
distributions and rates of spread have varied considerably2. In Thailand,
chloroquine were found the first reported of antimalarial drug resistance in 19613.
In addition, sulfadoxine–pyrimethamine (Fansidar™) and mefloquine were
developed antimalarial drug resistance recently4.
In Thailand, resistance to antimalarials has been found such as chloroquine,
sulfadoxine-pyrimethamine and low quinine efficacy, while chloroquine remains
effective to treatment P.vivax infection2. Around Thai-Myanmar border ; Tak,
Mae-hongson, Kanchanaburi and Thai-Cambodia border ; Chanthaburi, Trat. These
areas have been sign of antimalarial drug resistance that major problem must be
monitoring and following.
Pfmrp belongs to the C subfamily of ABC transporters also known as
multidrug resistance associated proteins that, as revealed in other systems, can
transport glutathione, glucuronate, as well as glucuronide and sulfate conjugated
compound5. Pfmrp localizes to the parasite plasma membrane and to membranebound vesicles in asexual stages6. Pfmrp knock-out parasite lines revealed
increased intracellular glutathione accumulation and increased susceptibility to
several antimalarial drugs, including chloroquine, quinine and artemisinin6.
Resistance to quinine involves multiple transporters, in particular mutations
in the pfcrt and pfmdr1 genes previously associated with resistance to other
quinoline drugs. A quantitative trait loci (QTL) analysis confirmed these remarks
and recommended an additional role of a newly discovered locus on chromosome
13 interacting with other loci, further highlighting the complex network of gene
interactions involved in the acquisition of a quinine resistance phenotype. Among
100 predicted genes, this locus harbors a homolog of the Na+/H+ exchanger
(pfnhe1), with a polymorphic coding microsatellite (ms4760) associated with
quinine susceptibility7. Recently, new genetic markers of ACT resistance,
Plasmodium falciparum GTP cyclohydrolase I (pfgch1) and Plasmodium
falciparum maurer’s cleft two transmembrane proteins (pfmc-2tm) have been
discovered8.
Materials and Methods
Analysis of genetic polymorphisms of pfmrp1, pfnhe1, pfmc-2tm and
pfgch1 genes of Plasmodium falciparum isolates
The analysis of genetic polymorphisms of pfmrp1, pfnhe1, pfgch1 and
pfmc-2tm genes of Plasmodium falciparum were performed in 10 isolates collected
from Mae Sot, Thailand.
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1. Amplification of pfmrp1 single-nucleotide polymorphisms (SNPs)
PCR amplification followed by sequencing was used to detect SNPs in
pfmrp at positions 191 and 437. Primer pfmrp-501F and primer pfmrp-1409R
(Table 1) were used for amplification and sequencing. The PCR cycling conditions
were as followed: denaturation at 94°C for 2 min, followed by 40 cycles of 94°C
for 30 sec, 52°C for 30 sec, 72°C for 1 min and then 72°C for 15 min. All PCR
products were verified on 1% agarose gels. The amplified fragments were
performed to recover or concentrate DNA fragments (50bp-10kb) from an agarose
gel or any other enzymatic reaction by using a Gel/PCR DNA Fragments
Extraction Kit (Gene aid, New Taipei, Taiwan). The purified DNA was sent to 1st
BASE DNA Sequencing Company (Selangor Darul Ehsan, Malaysia) for analysis
sequence.
2. Amplification of pfnhe-1 microsatellite profiles
A sequence containing the ms4760 microsatellite locus was amplified using
pfnhe-3802F and pfnhe-4322R primers (Table 1). The PCR cycling conditions were
as followed: denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30
sec, 52°C for 30 sec, 72°C for 1 min and then 72°C for 15 min. All PCR products
were verified on 1% agarose gels. The amplified fragments were performed to
recover or concentrate DNA fragments (50bp-10kb) from an agarose gel or any
other enzymatic reaction by using a Gel/PCR DNA Fragments Extraction Kit
(Gene aid, New Taipei, Taiwan). The purified DNA was sent to 1st BASE DNA
Sequencing Company (Selangor Darul Ehsan, Malaysia) for sequence analysis.
3. Amplification of pfmc-2tm
The pfmc-2tm gene was amplified and sequenced by using pfmc-2tm oligos
and pfmc-2tm internal oligos primers (Table 1). The PCR cycling conditions were
as followed: denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30
sec, 52°C for 30 sec, 72°C for 1 min and then 72°C for 15 min. All PCR products
were verified on 1% agarose gels. The amplified fragments were performed to
recover or concentrate DNA fragments (50bp-10kb) from an agarose gel or any
other enzymatic reaction by using a Gel/PCR DNA Fragments Extraction Kit
(Gene aid, New Taipei, Taiwan). The purified DNA was sent to 1st BASE DNA
Sequencing Company (Selangor Darul Ehsan, Malaysia) for sequence analysis.
4. Amplification of pfgch1
The pfgch1 gene was amplified and sequenced by using pfgch1-oligos and
pfgch1-internal oligos primers (Table 1). The PCR cycling conditions were as
followed: denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30 sec,
52°C for 30 sec, 72°C for 1 min and then 72°C for 15 min. All PCR products were
verified on 1% agarose gels. The amplified fragments were performed to recover or
concentrate DNA fragments (50bp-10kb) from an agarose gel or any other
enzymatic reaction by using a Gel/PCR DNA Fragments Extraction Kit (Gene aid,
New Taipei, Taiwan). The purified DNA was sent to 1st BASE DNA Sequencing
Company (Selangor Darul Ehsan, Malaysia) for sequence analysis.
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5. Data analysis
Sequences were aligned using the Gap4 program, version 4.10 available
from
http://www.mrclmb.cam.ac.uk/pubseq/manual/gap4_windows_2.html.
Investigation the relatedness of the sequences a Clustal C program was used to
create synonymous SNPs in pfmrp1, pfnhe1, pfmc-2tm and pfgch1 (ANGIS,
http://www.angis.org.au). Unique DNA sequences described in this paper have
been deposited in the GenBank.
Table1 Primers for amplification of the pfmrp1, pfnhe19, pfmc-2tm and pfgch1
genes
PCR
(position)
Gene
Primer
Sequence (5’-3’)
pfmrp-501F
pfmrp-1409R
TTTCAAAGTATTCAGTGGGT
GGCATAATAATTGATGTAAA
ms4760
pfnhe-3802F
microsatellite pfnhe-4322R
locus
TTATTAAATGAATATAAAGA
TTTTTTATCATTACTAAAGA
191,437
pfmrp1
pfnhe1
pfmc2tm
965, 1210
469, 618
pfgch1
pfmc-2tm oligos* AAACACCATCTTTTACCTTTTGAA
pfmc-2tm
AGCATCGTGCTCTTTAACTCC
internal oligos*
pfgch1-oligos*
pfgch1-internal
oligos*
ATTTGGATTGTTTCAACGACCT
CATGTCAGGAAAATAACGAGCA
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*new designed primer
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Results were compared pfmrp1 gene (Acc No. NC_004325) from GenBank.
Pfmrp1 gene are 7109 base pair (bp) that translate to 2369 amino acid. The results
of genetic diversity of pfmrp1 gene of P.falciparum malaria 10 samples in
Thailand. The diagnostic performance of nested-PCR of pfmrp1 gene by used
primer were desiged specific to gene. The results showed amino acid were
Histidine (H191). We found non-synonymous mutations at position 437. Amino
acid were changed from Serine (S) to Alanine (A) at position 437 (S437A) 9
sample (90%).
Results
Analysis of genetic polymorphisms of pfmrp1, pfnhe1, pfmc-2tm and
pfgch1 genes of Plasmodium falciparum isolates
1. Study genetic diversity of pfmrp1 gene of P.falciparum malaria
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2. Study genetic diversity of pfnhe1 gene of P.falciparum malaria
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Results were compared pfnhe1 gene (Acc No. GQ845118, GQ845119,
GQ465284) from GenBank. Pfnhe1 gene are 318, 312, 403 base pair (bp) that
translate to 106, 104, 134 amino acid. The results of genetic diversity of pfnhe1
gene of P.falciparum malaria 10 samples in Thailand. The diagnostic performance
of nested-PCR of pfnhe1 gene by used primer were desiged specific to gene. The
results showed amino acid were DNNND repeat and NHNDNHNNDDD repeat in
Table 2
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Table2 Frequency mutations of pfnhe1 ms4760 haplotypes in P.falciparum malaria
in Thailand
Mutations of pfnhe1 gene
No.DNNND repeat No. NHNDNHNNDDD repeat
No. genotype profile
2
1
ms-6 = 1(10%)
3
1
ms-7 = 9(90%)
3. Study genetic diversity of pfmc-2tm gene of P.falciparum malaria
Results were compared pfmc-2tm gene (Acc No. NC_000910) from
GenBank. pfmc-2tm gene are 1018 base pair (bp) that translate to 339 amino acid.
The results of genetic diversity of pfmc-2tm gene of P.falciparum malaria 10
samples in Thailand. The diagnostic performance of nested-PCR of pfmc-2tm gene
by used primer were desiged specific to gene. The results showed amino acid in
Table 3. We found non-synonymous mutations at position 189, 190, 192, 194, 196
and 197. Amino acid were changed from Phenylalanine (F) to Tyrosine (Y) at
position 189 (F189Y) 1 sample (11.1%), Isoleucine (I) to Serine (S) at position 190
(I190S) 3 sample (33.3%), Leucine (L) to Serine (S) at position 192 (L190S) 2
sample (22.2%), Phenylalanine (F) to Asparagine (N) at position 194 (F194N) 1
sample (11.1%), Isoleucine (I) to Asparagine (N) at position 196 (I196N) 2 sample
(22.2%), Histidine (H) to Tyrosine (Y) at position 197 (H197Y) 8 sample (88.9%),
respectively. In addition, the other sample could not analysed.
Table3 Frequency mutations of pfmc-2tm gene in P.falciparum malaria in Thailand
Mutations of pfmc-2tm gene
189
F=8(88.9%)
Y=1(11.1%)
190
I=6(66.7%)
S=3(33.3%)
192
L=7(77.8%)
S=2(22.2%)
194
F=8(88.9%)
N=1(11.1%)
196
I=7(77.8%)
N=2(22.2%)
197
H=1(11.1%)
Y=8(88.9%)
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192
4. Study genetic diversity of pfgch1 gene of P.falciparum malaria
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197
Results were compared pfgch1 gene (Acc No. NC_004316) from GenBank.
Pfgch1 gene are 1520 base pair (bp) that translate to 506 amino acid. The results of
genetic diversity of pfgch1 gene of P.falciparum malaria 10 samples in Thailand.
The diagnostic performance of nested-PCR of pfgch1 gene by used primer were
desiged specific to gene. The results showed all of them were wild type.
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Discussion
ACTs remain the most effective treatment for uncomplicated P. falciparum
malaria. Artemisinin derivatives are very effective in rapidly reducing the parasite
biomass, and, when combined with an effective partner medicine, they are likely to
clear all parasites successfully4. Recently, the reported of antimalarial drug
resistance was found in many countries. Artemether drug resistance was found in
Nigeria10. Quinine drug resistance were found in Thailand11and Kenya12.
Surprisingly, Artemisinin-based combination drug resistance was found in Africa13.
In Thailand, ACTs currently in clinical use has been documented4. Nonetheless, it
is afraid that ACTs will lose their efficacy owing to their widespread use. The
present study was to examine genetic polymorphisms of pfmrp1, pfnhe1, pfmc-2tm
and pfgch1 genes of Plasmodium falciparum isolates. The study at Mae sot, Tak
provinces, this area have been sign of antimalarial drug resistance that important
problem must be monitoring and following.
Pfmrp1 contains to the parasite plasma membrane and to membrane-bound
vesicles in asexual stages increased susceptibility to several antimalarial drugs,
including chloroquine, quinine and artemisinin. The results showed amino acid
were Histidine (H191) similar to previous results in Thailand14. Whereas amino
acid were changed from Serine (S) to Alanine (A) at position 437 (S437A) 9
samples (90%) similar to Cambodian14. Mutation of pfnhe1 related to quinine drug
resistance in falciparum malaria15. The results showed genotype profile of ms-6 1%
that similar to Vietnam15 and ms-7 9% same as previous results in Thailand14. And
the new genetic markers of ACT resistance as pfgch1 were found non-synonymous
mutations at position 189, 190, 192, 194, 196 and 197 and mutation in pfmc-2tm
was no result. However, this is a preliminary report. The conclusion for 10 strains
might not be suitable to judgment. Confirmation is this finding is required with a
larger sample size.
Acknowledgements
The study was supported by The Commission on Higher Education,
Ministry of Education of Thailand, The National Research University Project of
Thailand (NRU), Office of Higher Education Commission, Thammasat University
(Center of Excellence in Pharmacology and Molecular Biology of Malaria and
Cholangiocarcinoma), The Royal Golden Jubilee Ph.D. Programme, Thailand
Research Fund, Thammasat University Joint Fund, and Graduated Student Grant to
P. Thongdee (Grant no. PHD/0365/2552), Thammasat University. The authors
gratefully acknowledge the financial support provided by Thammasat University
Research Fund under the TU Research Scholar, Contract No. 076/2557.
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