In vitro fitness of chloroquineresistant Plasmodium falciparum parasites

1
In vitro fitness of chloroquine-resistant
Plasmodium falciparum parasites Standwell
Nkhoma, Malcolm Molyneux and Stephen Ward
BACKGROUND Recent work from Malawi, where
chloroquine (CQ) was replaced by
sulfadoxine-pyrimethamine as a first line
antimalarial, has shown a significant decline in
the prevalence of chloroquine-resistant parasites
in the field. This finding suggests that
CQ-resistance conferring mutations in the
Plasmodium falciparum chloroquine resistance
transporter (pfcrt) gene comes at a cost to
parasite fitness.   OBJECTIVE In this study, we
wanted to find out whether the fitness cost of
harbouring pfcrt mutations might be manifested by
impaired growth efficiency of asexual mutant
pfcrt parasites relative to that of wild type
parasites.
Detection of the K76T pfcrt genotype by the
PCR-RFLP assay We used the nested PCR-RFLP
strategy previously described by Djimdé et al
(2001) to determine the genotype of parasites
isolated from each competition culture. The
134-bp product, generated by PCR, was digested
with a restriction enzyme, Apo1. This enzyme
cleaves the product into 2 fragments of size 34bp
and 100bp in the presence of the wild type allele
(K76) and does not cut it in the presence of the
mutant allele (76T). In the presence of both
alleles, one band, corresponding to the wild type
allele appears at 100bp and the other,
corresponding to the mutant allele, appears at
134bp on a 2 agarose gel. Digestion products
were subjected to gel electrophoresis and
visualized by UV transillumination. Gel photos
were captured with the aid of the GelDoc-It
Imaging System (Ultra-Violet Products Ltd, UK).
RESULTS (i) HB3 versus TM6 At the beginning of
the experiment, the genotype present in each
competition culture was as expected according to
detection thresholds that we had established
previously. In cultures where the level of the
mutant allele was set below its detection level,
only the wild type genotype was detected (Lanes 1
and 2, Figure 1). In cultures where the
proportions of both alleles were set above their
detection levels, a mixed genotype was detected
(Lanes 3, 4, 5, 6 and 9, Figure 1). In a culture
where the proportion of the wild type genotype
was set below the detection threshold, only the
mutant genotype was detected (Lane 8, Figure 1).
After 2 days in culture, there were no noticeable
changes in the genotype of each competition
culture (data not shown). However, by day 4, the
proportion of resistant parasites (TM6) had risen
above detection in competition cultures where
their starting proportion was set below the
detection level of the resistant genotype (data
not shown). In addition, the proportion of
sensitive parasites (HB3) had fallen below
detection in some cultures in which both alleles
had been set above their detection thresholds.
After 26 days in culture, the wild type pfcrt
population had fallen below detection in all
competition cultures (Figure 2). In cultures that
had pure genotypes (100 HB3 and 100 TM6) at day
0, expected genotypes were consistently detected
throughout the period of investigation (Lanes 10
and 11, Figures 1 2). (ii) C2GCO3 versus
C3DD2 Similar results were obtained from
competition assays involving the transfectants,
which are genetically identical with the
exception of their pfcrt allele. At the start of
the experiment, the genotype present in each
competition culture was as expected according to
the previously defined thresholds (Figure 3).
However, by day 38, the wild type population
could not be detected in any of the competition
cultures except in the 100 pure wild type pfcrt
culture (Figure 4).  
MATERIALS METHODS Parasite clones Two sets of
parasite clones were used in competition
experiments HB3/TM6 and C2GCO3/C3DD2. HB3 is a
CQ-sensitive, wild type pfcrt clone from
Honduras. TM6 is an unrelated CQ-resistant strain
from Thailand and carries mutant pfcrt. C2GCO3
and C3DD2 are CQ-sensitive and CQ-resistant
transfectants respectively. The latter two
parasites are genetically identical with the
exception of their pfcrt genotype.   Parasite
cultivation Parasites were grown and maintained
in culture using a modification of the method of
Trager and Jensen (1976). Clones HB3 and TM6, and
their artificial mixtures were cultured in RPMI
1640 medium under an atmosphere of 4O2, 3CO2
and 93N2 at 370C. C2GCO3 and C3DD2 were grown in
a modified RPMI 1640 medium containing 2µg/ml of
blasticidin S-deaminase and 2nM of WR99210 to
enable them retain the transfected
DNA. Experimental design for growth competition
assays   Initially, we established the lowest
level of the chloroquine-sensitive, wild type
pfcrt allele (K76) and the chloroquine-resistant,
mutant pfcrt allele (76T) at which our
conventional polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) assay
would be able to detect them in a mixture of both
the wild type and mutant pfcrt parasites. Minimum
detection thresholds were found to be 20 and
10 for the wild type and mutant pfcrt alleles
respectively. We reasoned that if wild type pfcrt
parasites are more fit compared to the mutant
ones, then in a mixed infection carrying both
alleles, the following postulates must hold (i)
if the proportion of resistant parasites is set
below its detection threshold in competition
cultures, this level must remain below detection
level with the passage of sequential parasite
generations. (ii) if the proportion of resistant
parasites is set above its detection threshold,
then it must fall below detection after a series
of generations in culture due to competition from
wild type parasites. (iii) if the proportion of
wild type parasites is set below its detection
threshold in competition cultures where mutant
parasites are predominant, then it must rise
above detection level with the passage of
sequential parasite generations. Based on these
postulates, competition cultures were set up in
which the proportion of both the wild type and
mutant pfcrt parasites were varied (refer to
Table 1) while maintaining a constant starting
parasitaemia and haematocrit. HB3 was grown in
competition with TM6 whereas C2GCO3 was grown in
competition with C3DD2. Changes in parasite
genotype with the passage of time were detected
by extracting parasite DNA and analyzing the
genotype present by the standard PCR-RFLP assay.
Table 1 Protocol for competition experiments
between wild type (W) and mutant (M) pfcrt
parasites.
Figure 2 Agarose gel showing genotypes present
in HB3 vs TM6 competition cultures after 26 days.
Figure 1 Agarose gel showing genotypes present
in HB3 vs TM6 competition cultures at day 0.
Figure 3 Agarose gel showing genotypes present
in C2GCO3 vs C3DD2 competition cultures at day 0.
Figure 4 Agarose gel showing genotypes present
in C2GCO3 vs C3DD2 competition cultures after 38
days.
DISCUSSION Our data show that asexual
erythrocytic parasites carrying the mutant pfcrt
allele multiply more efficiently than those
harbouring the wild type genotype. This finding
suggests that CQ-resistant parasites have a
survival advantage over their CQ-sensitive
counterparts during the asexual erythrocytic
stage.   CONCLUSION Therefore, the loss of pfcrt
mutants in the field, as reported in Malawi, must
implicate a pfcrt-dependent loss of fitness at
some other point in the parasites life cycle.
This loss in fitness must outweigh the biological
advantage seen during the asexual erythrocytic
stage in order to generate negative selection
against pfcrt.