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Parasitol Res
https://doi.org/10.1007/s00436-017-5647-z
ORIGINAL PAPER
Cellular mechanisms of action and resistance of Plasmodium
falciparum to artemisinin
Papichaya Phompradit 1 & Wanna Chaijaroenkul 1 & Kesara Na-Bangchang 1
Received: 3 July 2017 / Accepted: 9 October 2017
# Springer-Verlag GmbH Germany 2017
Abstract The recent reports of high failure rates and decline
in in vitro sensitivity of Plasmodium falciparum to
artemisinin-based combination therapies (ACTs) suggest the
possibility of clinical artemisinin resistance along the ThaiCambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of
P. falciparum to artesunate (stage specific activity, interaction
with hemozoin, and anti-oxidant levels) in the two paired
P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the
time of recrudescence. In addition, the link of these cellular
mechanisms to the polymorphisms of the candidate
artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1,
and K13 propeller) was also investigated. Morphological
change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12–48 h of exposure to
artesunate (at IC90). A marked decrease in parasite viability
was found in the recrudesced isolates of both MSF046 and
MSD060. The extent of the reduction (% change of baseline)
in total glutathione concentrations was significantly lower in
recrudesced (32.1 and 1.7%) compared with primary (45.5
and 53.7%) isolates of both MSF046 and MSF060. The extent
of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with
the primary isolate (99.5%). For MSF060 on the other hand,
increase in hemozoin content was found in the recrudesced
isolate and the extent of such increase was significantly higher
* Kesara Na-Bangchang
kesaratmu@yahoo.com
1
Thammasat University Center of Excellence in Pharmacology and
Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn
International College of Medicine, Thammasat University, Rangsit
Campus, Pathumthani 12121, Thailand
in recrudesced (93.1%) than the primary isolate (87.5%).
Polymorphism of K13 (N458Y) together with pfmdr1 copy
number correlated well with sensitivity of both isolates to
artesunate. Results of this preliminary study suggests possible
role of glutathione-dependent detoxification system as well as
heme degradation as cellular mechanisms of action and resistance of artemisinins.
Keywords Plasmodium falciparum . Artemisinin-based
combination (ACT) . Artemisinin resistance . Hemozoin .
Glutathione
Abbreviations
ACT
Artemisinin-based combination therapy
GSSG Standard oxidized form of glutathione glutathione
GSH
Glutathione
RBC
Red blood cells
RSA
Ring-stage survival assay
Introduction
Malaria-endemic areas of Thailand are located along the forested borders with Myanmar to the west, Cambodia to the east,
and Malaysia to the south. To combat the threat of resistance of
Plasmodium falciparum to monotherapeutic drugs and to improve treatment outcome, the World Health Organization
(WHO) recommends artemisinin-based combination therapies
(ACTs) as the first-line treatment for uncomplicated
P. falciparum malaria in all endemic areas. The treatment regimens generally provide satisfactory clinical treatment outcome in most areas. Nevertheless, artemisinin-resistant
P. falciparum has now been reported from the four countries
of the Greater Mekong subregion, namely, Cambodia,
Parasitol Res
Myanmar, Thailand, and Viet Nam (WHO 2012). In
Cambodia’s Pailin province, resistance was demonstrated to
both components of multiple ACT regimens (Dondorp et al.
2009), and special provision for directly observed therapy
(DOT) using a non-ACT regimen atovaquone-proguanil has
been put in place (WHO 2012). The first artemisinin resistance
in Cambodia was confirmed in a study conducted during
2006–2007 (Noedl et al. 2010). Two patients who received
artesunate monotherapy showed prolonged parasite clearance
time (PCT: 95 and 133 h) in the presence of adequate concentrations of the active plasma metabolite dihydroartemisinin
[mean (±SD) plasma concentrations of 4268 ± 2418 and
2953 ± 1989 nmol/L at 1.5 and 2 h, respectively]. In vitro
sensitivity assay showed significantly higher IC50 (concentration that inhibits parasite growth by 50%) of the parasite isolates collected from those patients to dihydroartemisinin than
those from cured patients [median (range): 14.2 (14.00–14.42)
vs. 3.34 (2.90–3.85) nmoL/L] (Noedl et al. 2010). Emergence
of artemisinins resistance appears to spread to the western border of Thailand along Thai-Myanmar border (Na-Bangchang
et al. 2010). Increase in treatment failure rates of artesunatemefloquine combination therapy has been documented particularly in Tak province along the Thai-Myanmar border.
Studies conducted during 2008–2009 with the same regimen
in the same area showed a marked decline of cure rates during
42 day follow-up from 99.2 to 72.58% (Congpuong et al.
2010). In the light of accumulating reports of ACT treatment
failure, study on mechanism of action and resistance of
artemisinins is essential. The aim of the study was to investigate cellular mechanisms of action and resistance of
P. falciparum to artesunate (stage specific activity, interaction
with hemozoin, and anti-oxidant levels). In addition, the link of
these cellular mechanisms to the polymorphisms of the candidate artemisnin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1,
and K13 propeller) was also investigated.
Materials and methods
Chemicals
Artesunate, saponin from Quillaja bark, sodium chloride,
sodiumhydro-gencarbonate (NaHCO3), D-sorbital, Triton
X-100 were purchased from Sigma-Aldrich (Missouri,
USA). RPMI 1640, gentamicin was purchased from
Invitrogen(California,USA).HEPES(N-2-hydroxyethyl-piperazine-N-2 ethanesulfonic acid) and sodium chloride
(NaCl) were purchased from MERCK (Darmstadt,
Germany). EDTA (ethylenediamine-tetraacetic acid), glucose, and magnesium chloride were purchased from USB
Affymetrix (California, USA). Giemsa stain solution and gas
mixture were purchased from Biotech (Bangkok, Thailand)
and LABGAZ (Bangkok, Thailand), respectively. Total
glutathione detection kit (catalog no. ADI-900-160) was purchasedfromEnzoLifeSciences(NewYork,USA).GroupBor
ABhumanseraandgroupOhumanerythrocyteswereobtained
from healthy donors aftergiven informed consents.
Plasmodium falciparum isolates
The study was conducted in 2009 at the Mae Tao clinic for
migrant workers, Tak Province, Thailand (area along ThaiMyanmar border). Ethical approval of the study protocol
was obtained from the Ethics Committee of the Ministry of
Public Health of Thailand. Written informed consents were
obtained from all patients before study participation.
Two pairs of P. falciparum isolates (MSF046 and MSF060)
collected from Burmese patients with acute uncomplicated
falciparum malaria before and at the time of parasite reappearance after treatment with a 3-day artesunate-mefloquine
combination (25 mg/kg body weight mefloquine and 12 mg/kg
bodyweight artesunate). Results of the in vitro sensitivity assay
in conjunction with plasma drug concentrations
(dihydroartemisinin, and mefloquine) from our previous study
confirmed resistance of both isolates to mefloquine (NaBangchang et al. 2013). The IC50 and IC90 (concentration that
inhibits parasite growth by 50 and 90%, respectively) of
artesunate in primary vs. recrudesced MSF046 isolates were
2.8 vs. 5.8 nM and 12 vs. 20 nM, respectively. The corresponding IC50 and IC90 for the primary vs. recrudesced MSF060
isolates were 3.7 vs. 4.6 nM and 11 vs. 12 nM, respectively.
Both were adapted to culture according to the method of
Trager and Jensen with modification (Trager and Jensen
1976). Susceptibility of P. falciparum isolates to artesunate
(0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 nM) was investigated using SYBR Green I assay (Bennett et al. 2004;
Smilkstein et al. 2004). The experiments were repeated three
times, triplicate for each experiment. The IC50 and IC90 values
were determined from log-dose response analysis using the
CalcuSyn™computer program (Biosoft, Cambridge, UK).
Plasmodium falciparum genotyping of the three polymorphic
genes for merozoite surface antigen 1 (msp1), merozoite surface antigen 2 (msp2), and glutamate-rich protein (glurp) was
performed in paired samples obtained before treatment and at
the time of parasite re-appearance to distinguish re-infection
from recrudescence (Ariey et al. 1999; Wooden et al. 1992,
1993). The 3D7 P. falciparum was used as a reference clone
(absence of previous exposure to artemisinins).
Morphological change and the viability of Plasmodium
falciparum following exposure to artesunate
Parasite preparation and drug exposure
Parasite culture was synchronized using 5% sorbitol to obtain
ring stage parasites (Lambros and Vanderberg 1979). The
Parasitol Res
infected packed RBC was diluted with fresh uninfected RBCs
and RPMI 1640 medium to obtain parasite suspension at 1%
parasitemia and 2% hematocrit. The parasite was exposed to
artesunate at the IC90 in a final volume of 5 mL in a 25 cm2
tissue culture flask (Corning, Massachusetts, USA). The culture flask was flushed with a gas mixture containing 5% O2,
5% CO2, and 90% N2 for 30 s and incubated at 37 °C for 6 h.
The infected packed RBC was washed and resuspended in
drug-free culture medium and further incubated for an additional 42 h period. Non-exposed parasite served as control.
washed three times (through centrifugation at 16,000×g for
1 min) with saline solution (1 mL: 130 mM NaCl, 25 mM
HEPES, 5 mM KCl, 20 mM glucose, and 1 mM MgCl2:
pH 7.1) The pellet was lysed with ice-cold methanol
(100 μL) and the lysate was further centrifuged at 17,000×g
for 5 min to remove insoluble cell fragments. The final supernatant was mixed with saline solution (100 μL: 130 mM
NaCl, 25 mM HEPES, 5 mM KCl, 20 mM glucose, and
1 mM MgCl2: pH 7.1) and stored at −80 °C until analysis.
Determination of glutathione concentration
Morphological change and parasite viability following
artesunate exposure
Thin blood film of the P. falciparum-infected packed RBC
(artesunate exposed and non-exposed) was prepared every
6 h after drug exposure until 48 h and stained with Giemsa.
The morphology and viability of the parasite was examined
microscopically (5–10 X oil power fields). A total of 200 alive
and dead parasites were counted and the viability of individual
isolate was estimated from the ratio of the number of alive and
the number of all parasites and multiplied by 100 [(number of
alive parasites/200) × 100]. The experiment was repeated
three times.
Glutathione concentrations in Plasmodium falciparum
isolates following artesunate exposure
Parasite preparation and drug exposure
The primary and recrudesced P. falciparum isolates MSF046
and MSF060) and 3D7 P. falciparum clone were used in the
experiment (three experiments, triplicate each). The parasite
was cultured and synchronized as described above. Following
24–30 h post-invasion, the trophozoite stage parasite was collected and parasitemia (%) was determined. The infected
packed RBC was diluted with fresh uninfected RBC and
RPMI-1640 medium to obtain parasite suspension at 2%
parasitemia.
Parasite suspension (200 μL of 2% parasitemia) was exposed to 700 nM artesunate (Witkowski et al. 2013) in culture
medium (10 mL) in a 25 cm2 tissue culture flask (Corning,
Massachusetts, USA). The culture flask was flushed with gas
mixture containing 5% O2, 5% CO2, and 90% N2 for 30 s and
incubated at 37 °C for 2 h. The infected packed RBC was
washed and cell pellet was collected. Non-exposed parasite
served as control.
Parasite preparation and glutathione extraction
Infected RBC pellet was suspended in saponin (0.05% w/v,
1 mL in RPMI 1640 medium). The cell pellet was immediately separated through centrifugation at 3000×g for 8 min, and
Total glutathione concentration in cell supernatant was measured using total glutathione detection kit (catalog no.ADI900-160, Enzo Life Sciences, New York, USA). The assay
was performed according to the manufacturer’s procedure.
In brief, the standard oxidized form of glutathione (GSSG)
at the concentration range 12.5–100 pmoles was prepared in
triplicate (rows A-E of the microtitration plate) by diluting
GSSG (4 μM) in assay buffer (1×, 50 μL). Aliquots of parasite
samples (50 μL each) were added in columns 4 to 12 in the
microtitration plate (triplicate). The reaction was initiated by
the addition of fresh Reaction Mix (50 μL). The absorbance
was immediately measured at the wavelength of 405 nm at
1 min intervals over a 10 min period (Varioskan Flash machine, Thermo Fisher Scientific, Massachusetts, USA).
The average of triplicate absorbance readings was
subtracted by background absorbance. The standard curve
was plotted between absorbance and GSSG concentrations.
Concentrations of GSSG in cell supernatant were obtained
from the regression equation of the standard curve.
Hemozoin content in Plasmodium falciparum isolates
following artesunate exposure
Preparation of parasite inocculum and drug exposure
The primary and recrudesced P. falciparum isolates (MSF046
and MSF060) and 3D7 P. falciparum clone were used in the
experiment (three experiments, triplicate each). Parasite culture was synchronized using 5% sorbitol as previously described. Following 24–30 h post-invasion, the trophozoite
stage parasite was collected and parasitemia was determined.
The infected packed RBC was diluted with fresh uninfected
RBC and the culture medium to obtain parasite suspension at
3% parasitemia.
The parasite (1% parasitemia and 1% hematocrit) was exposed to artesunate (700 nM in 10 mL culture medium final
volume) and chloroquine (1 μM in 10 mL culture medium
final volume) in a 25 cm2 tissue culture flask (Corning,
Massachusetts, USA). The flask was flushed with a gas mixture containing 5% O2, 5% CO2, and 90% N2 for 30 s.
Following incubation at 37 °C for 2 h, the infected RBC
Parasitol Res
was washed with the culture medium and the pellet was collected. Non-exposed parasite served as control.
at the Institut Pasteur in Cambodia, Phnom Penh, Cambodia
(Menard et al. 2016).
Parasite isolate preparation and hemozoin extraction
Statistical analysis
The infected RBC was resuspended in saponin (0.05% w/v) in
phosphate buffer saline (PBS: 1 mL) (Amersco, Ohio, USA)
and cell pellet was separated through centrifugation at
13000×g for 2 min. Parasite pellet was washed three times
with PBS (1 mL), and the cells were disrupted by sonication
(21% amplitude, 20 s) in 2% SDS (1 mL) (Bio-rad, California,
USA). The pellet was collected through centrifugation at
13,000×g for 15 min. The process was repeated twice to ensure complete cell break. Cell pellet was washed 7–8 times
with 2% SDS (1 mL) through centrifugation at 13,000×g for
2 min and resuspended in 1 mL solution consisting of 1 mM
Tris-HCL (pH 8.0), 0.5% SDS, and 1 mM CaCl2 containing
2 mg/mL of proteinase K. Following an overnight incubation
at 37 °C, cell pellet was separated through centrifugation at
13,000×g for 15 min and washed three times with 2% SDS
(1 mL) through centrifugation at 13000×g for 2 min. The
pellet was incubated at 25 °C in 1 mL of 6 M Urea (Bio-rad,
California, USA) for 3 h with agitation and finally washed 3–5
times with 2% SDS and subsequently distilled water (1 mL)
through centrifugation at 13,000×g for 2 min. The resulting
pellet (hemozoin) was resuspended in distilled water and
stored at 4 °C until analysis.
Statistical analysis was performed using SPSS software version 18. Quantitative data are presented as mean (± SD).
Comparison of quantitative variables between the two paired
samples (primary and recrudesced) was performed using
paired t test. Statistical significance level was set at α = .05.
Determination of hemozoin concentration
Hemozoin concentration in cell pellet was determined by
depolymerizing heme polymer in 20 mM NaOH in 2% SDS
(1 mL) and incubated at 25 °C for 2 h. The absorbance was
immediately measured at the wavelength of 400 nm (Chen
et al. 2001). Hemozoin concentration was estimated against
the standard curve of commercial heme (Sigma, Missouri,
USA) prepared at 1.0, 0.5, 0.25, 0.125, and 0.063 μg/mL.
Investigation of polymorphisms of the candidate
molecular markers of artemisinin drug resistance
The polymorphisms of pfcrt, pfmdr1, pfatp6, and pfmrp1
genes of the P. falciparum MSF046 and MSF060 paired isolates (primary and recrudesced) were performed using nestedpolymerase chain reaction (nested-PCR) and the mutations of
both genes at different amino acid codons were detected by
restriction fragment length polymorphism (RFLP) according
to the previously described methods (Djimde et al. 2001,
Ferreira et al. 2006, Fidock et al. 2000). The results have
previously been reported (Phompradit et al. 2014). Sequence
polymorphism of the recently proposed marker of artemisinin
resistance K13 propeller in both paired isolates was performed
Results
Morphological change and the viability of Plasmodium
falciparum following artesunate exposure
Following exposure to artesunate at the IC90 of P. falciparum
isolates (MSF046: 12 vs. 20 nM; MSF060: 11 vs. 12 nM)
collected from patients before treatment and at the time of
parasite reappearance, the parasites remained at the ring stage
during the first 6 h in both the exposed and control parasites
(Figs. 1 and 2). At 48 h, the exposed parasites were arrested in
the ring stage with abnormal appearance. The control parasite
(non-exposed) on the other hand developed into trophozoite
and schizont stages. Pyknotic cells (dead parasites) were detected at highest number following 24 h exposure to
aretsunate. There was no difference in morphological development between the non-exposed and artesunate exposed primary and recrudesced isolates.
The viability of both paired isolates was examined following
exposure to artesunate for 6, 12, 24, and 48 h in comparison
with the non-exposed parasites. For MSF046, the viability of
primary isolate at 6, 12, 24, and 48 h of exposure were 99.1,
98.2, 63.9, and 9.9% of the non-exposed isolate, respectively.
The corresponding values for the recrudesced isolate were 99.2,
98.4, 91.5, and 16.2%, respectively (Fig. 3a). For MSF060, the
viability of primary isolates at 6, 12, 24, and 48 h were 98.6,
93.4, 76.2, and 9.4% of the non-exposed isolate, respectively.
The corresponding values for the recrudesced isolate were 98.5,
96, 85.6, and 15.9%, respectively (Fig. 3b). Significant difference in parasite viability between the primary and recrudesced
isolates was detected at 24 h of artesunate exposure [p = 0.026
(MSF046) and p = 0.026 (MSF060)].
Glutathione concentrations in Plasmodium falciparum
isolates following artesunate exposure
Total glutathione concentrations (median values) in
P. falciparum MSF046 and MSF060 primary and recrudesced
isolates including the 3D7 P. falciparum clone following exposure to 700 nM artesunate (2 h) are presented in Table 1. The
extent of reduction (% change of non-exposed control) of total
Parasitol Res
Fig. 1 Giemsa-stained thin blood films of the recrudesced MSF046
P. falciparum isolate following exposure to artesunate (IC90 ) in
comparison with the non-exposed control parasite during the period of
0 to 48 h. Red arrows indicate the change in morphology of the exposed
parasites. Similar morphological change was observed with the primary
isolate
Fig. 2 Giemsa-stained thin blood films of the recrudesced MSF060
P. falciparum isolate exposed to artesunate (IC90) in comparison with
the non-exposed control during the period of 0 to 48 h. Red arrows
indicate the change in morphology of the exposed parasites. Similar morphological change was observed with the primary isolate
glutathione contents in the recrudesced isolates was significantly lower than the primary isolates (recrudesced vs. primary
isolates of MSF046 and MSF060: − 32.1 and − 1.7 vs. − 45.5
and − 53.7%; p = 0.043). The reduction in glutathione concentration in 3D7 clone after artesunate exposure was 36.4%.
including the 3D7 P. falciparum clone following exposure
to 700 nM artesunate and 1 μM chloroquine (2 h) are presented in Table 2. The extent of the reduction in hemozoin
content (compared with baseline) in all isolates varied from
93 to 98% and 77–99% for chloroquine and artesunate, respectively. For MSF046, the extent of reduction was significantly higher in the recrudesced isolate compared with the
primary isolate (76.8 vs. 99.5%, p = 0.027). For MSF060 on
the other hand, increase in hemozoin content was found in
the recrudesced isolate and the extent of such increase was
significantly higher in recrudesced than the primary isolate
(93.1 vs. 87.5%, p = 0.024).
Hemozoin content in Plasmodium falciparum isolates
following artesunate exposure
Hemozoin contents (median values) in P. falciparum
MSF046 and MSF060 primary and recrudesced isolates
Parasitol Res
MSF046 and MSF060. An increase in pfmdr1 copy number
was found in the recrudesced isolates of both MSF046 and
MSF060. Interestingly, the mutation of K13 at codon 458 was
found in both paired isolates of MSF060 but only detected in
the recrudesced isolate of MSF046.
Discussion
Fig. 3 The viability (%) of MSF046 (a) and MSF060 (b) primary and
recrudesced P. falciparum isolates at 0, 6, 12, 24, and 48 h following 6 h
exposure to artesunate at the IC90
The polymorphisms of the candidate molecular markers
of artemisinin drug resistance
The polymorphisms of pfcrt, pfmdr1, pfatp6, pfmrp1, and K13
genes in the paired isolates (primary and recrudesced) of
MSF046 and MSF060 are presented in Table 3. The genetic
patterns of pfcrt, pfmdr1, and pfatp6 polymorphisms were
comparable in both the two parasite-paired isolates. The
pfmrp1 mutations at codon 191, 437, and 876 (YAV) was
detected in the primary and recrudesced isolates of both
No parasite morphological change was observed in the two
pairs of the primary and recrudesced P. falciparum isolates
during 6 h of exposure to artesunate (at IC90). Following exposure to artesunate, the survived parasites obviously arrested
themselves at ring stage during 12 until 48 h of exposure.
This, however, was not observed in the non-exposed parasites.
Moreover, significant difference in the viability of the primary
and recrudesced isolates was observed at 24 h of artesunate
exposure. Significantly higher number of viable parasites was
found in the recrudesced compared with primary isolates
(MSF046: 91.5% vs 76.2; MSF060: 85.6% vs 63.9, respectively). Results support the previous report that prolonged
parasite clearance time in vivo is one of the important clinical
treatment outcome parameters observed in resistant parasites
(Dondorp et al. 2009). It was proposed that the loss of
artemisinin sensitivity in the ring-stage parasite might explain
the observed slow parasite clearance times in western
Cambodia (Dondorp et al. 2009). A mathematical model estimated using detailed pharmacokinetic and parasite clearance
data from uncomplicated falciparum malaria patients treated
with artesunate from Pailin (western Cambodia) and Wang
Pha (northwestern Thailand) suggests a significant decline in
efficacy of artesunate on ring stage parasites in Pailin
(Dondorp et al. 2009). The reduced susceptibility of
P. falciparum to artemisinin appears to be associated with an
altered in vitro phenotype of ring stage. A 17-fold higher
survival rate of dihydroartemisinin-exposed ring-stage parasites was found in isolates from Pailin compared with that
from Ratanakiri (median survival rate: 13.5 and 0.8%,
Table 1 Total glutathione concentrations ( median values of triplicate experiments) in P. falciparum MSF046 and MSF060 primary and recrudesced
isolates including the 3D7 P. falciparum clone following exposure to 700 nM artesunate for 2 h. Data are presented as mean (±SD) values of three
experiments (triplicate each)
Median total glutathione concentration (pmole)
Primary isolate
Recrudesced isolate
Non-exposed
Artesunate-exposed
% change
Non-exposed
Artesunate-exposed
% change
MSF046
25.7 ± 0.5
14.0 ± 0.2
45.5 ± 1.2a
MSF060
3D7
39.1 ± 0.2
22.5 ± 0.6
18.1 ± 0.1
14.3 ± 0.2
53.7 ± 0.3b
36.4 ± 0.7
40.5 ± 0.2
23.2 ± 0.2
–
27.5 ± 0.3
22.8 ± 0.3
–
32.1 ± 1.0a
1.7 ± 0.9b
a
Significant difference in % total glutathione concentration change between primary and recrudesced isolates (p = 0.028; paired t test)
b
Significant difference in % total glutathione concentration change between primary and recrudesced isolates (p = 0.043; paired t test)
Parasitol Res
Table 2 Hemozoin concentrations (median values of triplicate experiments) in P. falciparum MSF046 and MSF060 primary and recrudesced isolates
including the 3D7 P. falciparum clone following exposure to 700 nM artesunate and 1 μM chloroquine for 2 h. Data are presented as mean (± SD) values
of three experiments (triplicate each)
Parasite
Median hemozoin concentration (ng/ml)
Primary isolate
Nonexposed
MSF046 216.0 ± 2.6
Recrudesced isolate
Chloroquineexposed
5.0 ± 0.2
MSF060 263.0 ± 0.3 13.6 ± 0.5
3D7
174.0 ± 0.8 12.4 ± 0.4
% change Artesunateexposed
% change
97.7 ± 0.1
99.5 ± 0.1a 250.0 ± 2.1 16.8 ± 0.6
87.5 ± 0.1b 213.0 ± 0.4 13.2 ± 0.3
93.6 ± 0.2 –
–
1.0 ± 0.1
94.8 ± 0.2 33.0 ± 0.3
92.9 ± 0.2 11.2 ± 0.4
Nonexposed
Chloroquineexposed
% change Artesunateexposed
% change
93.3 ± 0.3 58.1 ± 1.0
93.8 ± 0.2 14.8 ± 0.3
–
76.8 ± 0.2a
93.1 ± 0.1b
a
Significant difference in % hemozoin concentration change between primary and recrudesced isolates after artesunate exposure (p = 0.027; paired t test)
b
Significant difference in % hemozoin concentration change between primary and recrudesced isolates after artesunate exposure (p = 0.024; paired t test)
respectively). On the other hand, survival rates of both the
exposed and non-exposed mature stages were comparable
(0.6 and 0.7%, respectively) (Witkowski et al. 2013). These
results support the potential application of the ring-stage survival assay (RSA) as an in vitro tool for detecting ACT resistance (Kite et al. 2016).
Table 3 The polymorphisms of pfatp6, pfcrt, pfmdr1, pfmrp1, and K13
in the paired isolates (primary and recrudesced) of MSF046 and MSF060
P. falciparum
MSF046
MSF060
Primary Recrudesced Primary Recrudesced
pfatp6
pfcrt
R37K
R
R
ND
ND
G693D
S769 N
G
S
G
S
G
S
G
S
K76 T
A220S
Q271E
T
S
E
T
S
E
T
S
E
T
S
E
S
T
I
N
Y
C
N
D
6
Y
A
V
F
K
N
S
T
I
N
Y
C
N
D
8
Y
A
V
F
K
Y
S
T
I
N
Y
C
N
D
5
H
S
I
F
K
Y
S
T
I
N
Y
C
N
D
6
H
S
I
F
K
Y
A326S
I356T
R371I
pfmdr1 N86Y
F184Y
S1034C
N1042D
D1246Y
Copy number
pfmrp1 H191Y
S437A
I876V
F1390I
K1466R
K13
N458Y
Artemisinin and derivatives generate free radicals
(Butler et al. 1998) and iron plays an important role in their
mechanism of action and toxicity (Meshnick et al. 1993).
Glutathione (GSH)-dependent detoxification system is involved in the detoxification of a wide range of mutagens,
carcinogenic, and pharmacological active molecules, as
well as the production of by-products of oxidative stress
in P. falciparum (Muller 2015). GSH production in intraerythrocytic stages was shown to be significantly lower in
glutathione-deficient Plasmodium berghei with delayed
parasites growth (Padin-Irizarry et al. 2016). It is the most
sensitive indicator of oxidative stress in malaria-infected
erythrocytes both in the absence and presence of
artemisinins. In the presence of dihydroartemisinin, glutathione peroxidase and catalase activities, as well as GSH
levels in the erythrocytes, were significantly lower than
those of untreated erythrocytes (Ittarat et al. 2003). In line
with these observations, current results showed a significant decrease in total GSH concentrations in both parasite
isolates following artesunate exposure compared with the
non-exposed control. In addition, the extent of reduction in
GSH level in the primary isolates of both MSF046 and
MSF060 was shown to be higher than the recrudesced isolates following exposure to artesunate (− 2 to − 32% and
− 46 to − 54% for recrudesced and primary isolates, respectively). These findings suggest that GSH plays a considerable role in detoxification and protection of parasite
cells against the free radicals produced by artesunate. It is
possible that the resistant parasites can alter or modify the
GSH detoxification system to cope with artesunate action,
i.e., by increasing the activity of enzyme involved in GSH
synthesis. This results in a lower reduction of the remaining GSH level during drug treatment. In a previous study,
the GSH level was shown to be significantly higher in
arteether-resistant Plasmodium vinckei compared with
arteether-sensitive parasite. This high GSH level
corresponded with the increase in activities of enzymes
Parasitol Res
involved in detoxification system in the resistant parasites,
including glutathione reductase, glutathione-S transferase,
and glucose-6-phosphate dehydrogenase and the decrease
in the activity of superoxide dismutase was decreased
(Chandra et al. 2011).
The observation of inhibitory activity of artesunate on
GSH-dependent detoxification system could be linked to the
function of a recently identified marker of artemisinin resistance, the K13 protein, which is associated with biochemical
pathway that may antagonize the pro-oxidant activity of
artemisinin leading to a decline in clinical efficacy of ACT
(Fairhurst and Dondorp 2016). We, therefore, further investigated the association between K13 polymorphism and resistance phenotype in both paired isolates. The mutation of K13
at N458Y was one of the non-synonymous mutations that
were observed in Southeast Asia (Menard et al. 2016). The
mutation was detected in both of the recrudesced isolates of
MSF046 and MSP060 after treatment with ACT (a 3-day
artesunate-mefloquine). For the MSF046 isolate, the mutation
corresponded with the increase in the IC50 of artesunate from
2.8 to 5.8 nM (twofold). On the other hand, the mutation was
detected in both paired isolates of MSF060 (artesunate IC50
3.7 and 4.6 nM in the primary and recrudesced isolate, respectively). This may suggest the innate resistance of the MSF060
isolate to artesunate. Apart from K13, an increase in pfmdr1
copy number was found in both recrudesced isolates.
Polymorphism of K13 (N458Y) together with pfmdr1 copy
number could be applied as reliable molecular markers for
monitoring of artemisinin resistance.
Hemoglobin degradation process in Plasmodium results in
the production of high amount of ferriprotoporphyrin IX (FP).
FP induces oxidative stress to the parasite leading to death.
The detoxification of FP by biomineralization to hemozoin is
an important mechanism for parasite survival (Hempelmann
2007). The automated molecular docking of artemisinin to
heme revealed that artemisinin approaches heme by pointing
O1 at the endoperoxide linkage toward the iron center
(Tonmunphean et al. 2001). Study in artemisinins-resistant
murine malaria showed that the resistance of Plasmodium to
artemisinin-based compounds depends on alterations of heme
metabolism, as well as a loss of hemozoin formation linked to
the down-expression of the recently identified Heme
Detoxification Protein (HDP) (Witkowski et al. 2012).
Artemisinins have been proposed to act similarly to quinolines
against Plasmodium via inhibition of hemozoin production
(Pandey et al. 1999). In the present study, once the trophozoite
stage parasite (MSF046) exposed to artesunate, a marked decrease in hemozoin concentrations was found in the primary
compared with recrudesced isolates (99.5 to 76.8%). On the
other hand, the extent of hemozoin reduction (compared with
baseline level) after chloroquine exposure was relatively
smaller for both isolates (MSF046: 97.7 to 93.3%; MSF060:
94.8 to 93.8%). This may imply at least in part, the action of
artesunate on the hemozoin production in malaria parasite.
However, a result of the inhibitory activity of artesunate on
this process might be due to a downstream effect on hemozoin
formation. The decrease in hemozoin formation following
treatment with the antimalarial atovaquone has been shown
not to be the direct effect, but a result of decreased hemoglobin
uptake into the digestive food vacuole, probably consequent
to its effects on the mitochondria (Combrinck et al. 2015).
With the limitation of the number of parasite isolates used in
the current study however, contradictory findings were found
in the MSF046 isolate where an increase in hemozoin content
was found in the recrudesced isolate (93.1%) compared with
primary isolate (87.5%). Further study in a larger number of
paired P. falciparum isolates is required to confirm this
observation.
Acknowledgements The study was supported by the Center of
Excellence in Pharmacology and Molecular Biology of Malaria and
Cholangiocarcinoma of Thammasat University, National Research
Council of Thailand (NRCT) and National Research University Project
of Thailand (NRU), Office of Higher Education Commission of Thailand.
We are grateful to Professor Steven A Ward and staff of Liverpool
Institute of Tropical Medicine, University of Liverpool, UK, for their
technical supports. We also thank Dr. Didier Menard, the Institut
Pasteur in Cambodia, Phnom Penh, Cambodia, for providing data on
the analysis of K13 polymorphism of the MSF046 and MSF060 isolates.
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