Mitochondrial heterogeneity in human malarial parasite Plasmodium falciparum

Abstract

Mitochondria of the human malarial parasite Plasmodium falciparum in sexual blood stages (or gametocytes) had been structurally different from those of asexual blood stages of their life cycle in human host. We report here the existence of mitochondrial heterogeneity based on their characteristics of ultrastructural morphology in the asexual and sexual blood stages of P. falciparum from in vitro continuous cultures. Mitochondria in the sexual stage-parasites were more numerous and contained a greater density of cristae than the organelles in the asexual stage-parasites. It was demonstrated that there were apparent variations in size and appearance of the mitochondria between the male and female parasites of the sexual gametocytic stages. Mitochondrial oxygen consumption of the sexual stage-parasites was relatively low, and it was not different from the asexual blood stage-parasites. However, both stages of the parasites' growth and their oxygen consumption were found to be sensitive to atovaquone, cyanide and 5-fluoroorotate which were inhibitors of mitochondrial electron transport system and pyrimidine biosynthetic pathway, respectively. Therefore, the role of mitochondrial organelles with different morphological properties in the asexual and sexual stages of parasite's development remains to be elucidated.

Full text

Mitochondrial Heter ogeneity in Human
Malarial Parasite
Plasmodium falciparum
Sudaratana R. Krungkrai a, Pr eecha Leangaramgul b, Sanya Kudan b, Phisit
Prapunwattana b and Jerapan Krungkrai b,*
aDepartment of Biochemistry, Faculty of Science, Rangsit University, Patumthani 12000, Thailand.
bDepartment of Biochemistry, Faculty of Medicine, Chulalongkorn University, Rama IV Road,
Bangkok 10330, Thailand.
* Corresponding author: E-mail: fmedijkk@md2.md.chula.ac.th
Received 11 Feb 1999
ABSTRACT Mitochondria of the human malarial parasite Plasmodium falciparum in sexual blood stages
(or gametocytes) had been structurally different from those of asexual blood stages of their life cycle in
human host. We report here the existence of mitochondrial heterogeneity based on their characteristics
of ultrastructural morphology in the asexual and sexual blood stages of P. falciparum from in vitro
continuous cultures. Mitochondria in the sexual stage-parasites were more numerous and contained a
greater density of cristae than the organelles in the asexual stage-parasites. It was demonstrated that
there were apparent variations in size and appearance of the mitochondria between the male and female
parasites of the sexual gametocytic stages. Mitochondrial oxygen consumption of the sexual stage-parasites
was relatively low, and it was not different from the asexual blood stage-parasites. However, both stages
of the parasites’ growth and their oxygen consumption were found to be sensitive to atovaquone, cyanide
and 5-fluoroorotate which were inhibitors of mitochondrial electron transport system and pyrimidine bio-
synthetic pathway, respectively. Therefore, the role of mitochondrial organelles with different morpho-
logical properties in the asexual and sexual stages of parasite’s development remains to be elucidated.
KEYWORDS: malaria, Plasmodium falciparum, gametocyte, mitochondria.
ScienceAsia 25 (1999) : 77-83
INTRODUCTION
During the asexual blood stage the human
malarial parasite, Plasmodium falciparum, grows and
matures within the erythrocyte of the human host.
The absence of cristate structure in mitochondrial
organelle at this parasite stage has been demonstrated
from electron microscopic studies.1-5 Energy
requirement is provided by metabolizing glucose
primarily by anaerobic glycolysis.6,7 It has been
clearly evident from biochemical and enzymatic
studies that the asexual stage parasite has
mitochondrial electron transport system and oxygen-
requiring system that is necessary for parasite’s
growth and multiplication.3-10 In addition to the
development of the asexual blood stages, P. falciparum
contains sexual blood stages in the human host
necessary for survival and transmission into the
mosquito vector to complete its life cycle. Relatively
little is known concerning cristate structure and
biochemical function of the mitochondrial organelle
in the sexual blood stage of parasite development,2,11,12
although it develops within the erythrocytes of
human host.
In this communication, the ultrastructural
characteristics of mitochondrial organelles based on
the electron microscopic studies of asexual and
sexual blood stages of P. falciparum grown in vitro
were investigated. The biochemical properties of the
sexual stage parasites were compared to the asexual
blood stage parasites.
MATERIALS AND M ETHODS
Malarial parasites
Human malarial parasite P.falciparum (T9, NP10
and KT3 isolates) was cultivated from the frozen
sample in sorbitol-glycerol cryoprotectant13 by the
candle jar method of Trager and Jensen14, using 5%
hematocrit of human erythrocytes group ‘O’ in the
RPMI 1640 medium supplemented with 25 mM
N-2-hydroxylethylpiperazine-N’-2-ethanesulfonic
acid (Hepes), 32 mM NaHCO3 and 10% human
serum group ‘O’. Cultures started at low parasitemia
(~1-2%) were changed with medium twice daily until
the cultures had ~30% parasitemia and then parasites
were harvested. Synchrony of the culture to get
mainly trophozoite stages was performed by the
sorbitol procedure of Lambros and Vanderberg.15 For
the sexual blood (gametocytic) stages, either NP10
or KT3 isolate was used as gametocyte-producing
strain and then induced according to Ifediba and
Vanderberg.16 Approximately 4% parasitemia of
mixed stages gametocytes were routinely obtained
ESEARCH ARTICLE
R

78 ScienceAsia 25 (1999)
on day 10-15 of cultivation after adding normal fresh
erythrocytes. The gametocytic stages were purified
using Percoll step-wise gradient centrifugation
according to Knight and Sinden.17 Parasites were
freed of their host erythrocytes by incubating in an
equal volume of 0.05% (for sexual gametocytic
stages) or 0.15% saponin (for asexual blood stages)
in the RPMI 1640 medium at 37°C for 20 min, and
washed at least 3 times before experiments.
Micr oscopic examination of P. falcipar um morphology
Transmission electron microscopy (TEM) of the
infected erythrocytes at the asexual and sexual
gametocytic stages was performed according to the
method of Sinden.11 The processed samples were
examined with a JEOL-100SX transmission electron
microscope at the Center of Scientific and Techno-
logical Research Equipment of Chulalongkorn
University, Bangkok. Light microscopy (LM) of the
infected erythrocytes was examined on methanol
fixed and Giemsa’s stained parasites by a Nikon
labophot-2 microscope.
Measur ement of oxygen consumption by P. falcipar um
The rates of oxygen consumption for a consistent
number of host cell-free P. falciparum at both stages
of parasite development were measured in a modified
medium4 containing 75 mM sucrose, 225 nM
mannitol, 5 mM MgCl2, 5 mM KH2PO4, 1 mM
ethylene glycol bis(β-aminoethyl)-N-N-tetraacetic
acid, 5 mM Hepes, pH 7.4, by using a Clark-type
oxygen electrode and YSI oxygen monitor according
to the method of Robinson and Cooper.18 The oxygen
uptake in a chamber with volume of 3 ml was
followed for 3-5 min and recorded at 37°C with a
temperature-controlled circulator. Mitochondrial
inhibitors at desired concentrations were tested
against oxygen consumption of the two stages of the
parasites by injecting into the reacting chamber. The
rates of oxygen consumption by these parasites were
then followed for the next 3-5 min. The 50% effective
concentration (EC50) was defined as the concen-
tration of the inhibitor causing 50% inhibition of the
parasite oxygen consumption, compared to the control
parasite oxygen consumption without inhibitor.
In vitr o antimalarial test
Antimalarial activity against asexual stages of P.
falciparum in vitro (blood schizontocidal activity) was
quantified by measuring % parasitemia in a four-day
culture in the presence of the tested compounds at
various concentrations.19 All compounds were tested
in triplicate at each concentration used. The 50%
inhibitory concentration (IC50) was defined as the
concentration of the compound causing 50%
inhibition of the parasite growth in a 4-day culture,
compared with the compound-free control of the
parasite culture.
Gametocytocidal activity against P. falciparum in
vitro was tested by measuring % parasitemia in a 24-
well microculture plate in the presence of various
concentrations of tested compounds for 2 days
starting at day 5 of cultures after provision of fresh
erythrocytes. At day 7 and again at day 8, all wells
received fresh medium without the compound. Thin
blood films were prepared at day 9. This represents
a four-day period of gametocyte development in
microculture during the antimalarial test, the first
two days under pressure of test compound. The test
method and IC50 value calculation were essentially
from Bhasin and Trager.20
RESULTS AND D ISCUSSION
The in vitro cultures of T9, NP10 and KT3 isolates
(all multidrug-resistant parasites) of P. falciparum
were taken from frozen samples in sorbitol-glycerol
cryoprotectant, and continuously induced for
production of the sexual gametocytic stages for over
2 years. It was noted here that the establishment of
new cultures (or subcultures) by dilution with fresh
erythrocytes markedly reduced gametocyte
production even in the presence of inducing
conditions as described by Ifediba and Vanderberg16,
and this might result in loss of gametocytic stages in
those cultures. T9 parasite was found to be no longer
a gametocyte-producing strain. This may result from
a developmental defect during maturation that has
arisen during the long-term cultivation of the asexual
stages in vitro (~ four-year culture from 1990-1994),
as previously described in the different isolates of P.
falciparum by Guinet et al.21
NP10 and KT3 parasites had been cultured for
more than 2 years and they could be induced for
gametocytogenesis to give parasitemia of the sexual
stages as high as ~4%. The gametocytes were purified
from the asexual stage parasites by the established
method of Percoll step-wise gradient centrifugation.
The purified gametocytes used for TEM and oxygen
consumption are demonstrated in low magnification
of Giemsa’s stained parasites by LM in Fig 1. Mixed
populations of male and female gametocytes of stages
III-V existed in the parasites’ preparations. The
developmental stages of gametocytes were divided
into 5 stages according to the cytological classifica-
tion of Hawking et al.22

ScienceAsia 25 (1999) 79
The mitochondrial organelles observed in P.
falciparum, described as double membranous
structure, were by no means unique between the
asexual and sexual development stages in that they
contain tubular-like cristate structures (Figs 2-5).
The mitochondrial organelles in the asexual blood
stage parasite (Fig 2) were never as numerous as
they were in the sexual gametocyte stage (Figs 4 and
5). Fig. 2 shows a clearly defined double membrane
structure of a mitochondrion in the trophozoite stage
of the asexual blood parasite. Like other protozoan
mitochondria, they are surrounded by a double
membrane, and the inner membrane gives rise to
the tubular cristae. The elongated organelle showed
a limited number of tubular cristae (Fig 3). All
mitochondrial organelles, so far examined, in the
asexual blood stages had no or little internal
membranous whorls rather than well-defined tubular
cristae. They are classified as ‘type I’ mitochondria.
In addition to the unique morphological
characteristics of ‘Type I’ mitochondria in the asexual
stage, the maturing female gametocytic stage parasite
(macrogametocyte) had numerous mitochondrial
organelles (more than 5) containing marked
proliferation and pronounced infolding of the inner
membrane giving rise to large numbers of tubular
cristae with clear intracristal space (Fig 4). They are
classified as ‘Type II’ mitochondria (Figs 6-9). All
macrogametocytes examined had both ‘Type I’ and
‘Type II’ mitochondria (Fig 6). Most mitochondrial
organelles in the macrogametocytes were ‘type II’
mitochondria. Higher magnification of these ‘‘Type
II’ organelles showed them to have different forms
in their shapes of the tubular cristae, for instance,
membranous whorls cristae (Fig 6), or well separated
tubular cristae (Fig 7), or finger-like cristae of closely
folded inner membrane (Figs 8 and 9). By
comparison with the maturing male gametocytic
stage (microgametocyte) (Fig 5), it was noted that
the developing male parasite had less numbers of
the organelles (less than 5), possessed higher
numbers of the cristae, and contained electron-dense
tubular cristae (Figs 10 and 11). They are classified
as ‘Type III’ mitochondria.
Based upon these findings, it is concluded that
1) the male gametocytic parasites have numerous
‘Type III’ mitochondria and their mitochondria are
less numerous than those of the female parasites, 2)
the female parasites contain mainly populations of
‘Type II’ mitochondria and a limited number of
‘Type I’ mitochondria, and 3) the asexual blood
parasite has a single mitochondrion associated with
‘Type I’ organelle (Table 1). Our results reported are
consistent with the observation23 in the other
apicomplexan blood parasite Haemogregarina
myoxocephali at different numbers of mitochondrial
organelles in various developmental stages of its life
cycle: the erythrocytic stage, ~1-2 organelles; the
sexual stage, ~4-6 organelles; and the sporozoites,
~8-10 organelles. The existence of such variations
in mitochondria of male and female gametocytic
stages, both in terms of their numbers and in the
density of cristae, suggests that these developing
sexual stages have high demand for energy
transduction and also metabolic activity differences
from the asexual stages. These active mitochondria
in the sexual stages may be necessary for their
survival during transmission into the mosquito
vector.
To see whether the mitochondria in sexual
gametocytic stages of P. falciparum were biochemically
active or not, mitochondrial oxygen consumption
of the parasites were performed with known
mitochondrial inhibitors. It was found that
mitochondrial oxygen consumption of cultured P.
falciparum from both stages were not different (Table
2). They had relatively low activity (~ 200 - 240
nmol/min/108 parasites), compared to the human
leukocytes which had an oxygen consumption of
1,090 nmol/min/108 cells (n=2). Cyanide, a known
inhibitor of mammalian electron transporting
complex IV (cytochrome c oxidase),24 had an
inhibitory effect against oxygen consumption of the
host cell-free parasites isolated from both asexual
and sexual stages (Table 2). At a concentration of
1.0x 10-3 M, cyanide inhibited 52% and 88% of the
oxygen consumption by the asexual and sexual stage
parasites, respectively. Cyanide has been shown to
exhibit low antimalarial effect against P. falciparum
growth in vitro with IC50 value in the micromolar
level.8,25 It has inhibitory effects against the purified
cytochrome c oxidase of both P. berghei4 and P.
falciparum.10
The parasite oxygen consumption of both stages
was found to be sensitive to atovaquone inhibition
Table 1. The comparison of type and number of
mitochondrial organelle between asexual and
sexual blood stages of
P. falciparum
.
Stages of parasite Mitochondrial characteristics
Type Number
Asexual stage I 1
Sexual stage
Female II >>I >5
Male III <5

80 ScienceAsia 25 (1999)
Fig 1 Light micrograph of purified gametocytes from in vitro
cultures of P. falciparum. All preparations were methanol
fixed and stained with Giemsa’s stain. The bar represents
10 µM.
Fig 2 Transmission electron micrograph (TEM) of the asexual
trophozoite stage of P.falciparum. The mitochondrion shows
a clearly defined double membranes and an elogated form
which is prepared for binary fission. A membrane whorls-
like tubular crista resulting from an underdeveloped inner
membrane is marked by an arrowhead. The bar represents
1 µM. N, nucleus; M, mitochondria; P, crystalline pigment.
Fig 3 TEM of higher magnification of ‘Type I’ mitochondria in
the asexual trophozoite stage of P. falciparum. Only one
tubular cristate structure is demonstrated (arrowhead). The
bar represents 1 µM. M, mitochondria; P, crystalline
pigment.
Fig 5 TEM of maturing male gametocytic stage (microgame-
tocyte in stage IV) of P. falciparum. Few ‘Type III’
mitochondria are observed, Laveran’s ‘Bib’ is also visible
at the middle left. The bar represents 1µM. N, nucleus; M,
mitochondria; P, crystalline pigment.
Fig 4 TEM of maturing female gametocytic stage (macrogame-
tocyte in stage IV) of P. falciparum. Numerous ‘Type II’
mitochondria are observed. The bar represents 1 µM. M,
mitochondria; P, crystalline pigment.
Fig 6 A high magnification TEM of mitochondria in a maturing
female gametocyte. The lower (Type I) and the upper (Type
II) mitochondria are shown. The tubular cristae of ‘Type
II’ organelle is marked by an arrowhead. The bar represents
1 µM. M, mitochondria; P, crystalline pigment.

ScienceAsia 25 (1999) 81
Fig 7 TEM of ‘Type II’ mitochondria in a female gametocyte. Two
organelles are observed with high magnification. They
contain numerous tubular cristae, marked by arrowhead.
The bar represents 1 µM. M, mitochondria.
Fig 8 TEM of variant for ms of ‘Type II’ mitochondria in a female
gametocyte. Three organelles are observed with high
magification. Numerous tubular cristae resulting from the
extensively folding of the inner membrane are observed.
An arrowhead indicates the finger-like cristae. The bar
represents 1 µM. M, mitochondria; P, crystalline pigment.
Fig 9 TEM of another variant for ms of ‘Type II’ mitochondria in
a female gametocyte. The finger-like cristae is typically
found in the female gametocytes (arrowhead). The bar
represents 1 µM. M, mitochondria; P, crystalline pigment.
Fig 11 TEM of ‘‘Type III’mitochondria in a male gametocyte. An
apicoplast is observed with a multi-membranous organelle
containing electron-dense matrix and absence of internal
cristae. The bar represents 1 µM. M, mitochondria; P,
crystalline pigment. A, apicoplast.
Fig 10 TEM of ‘Type III’mitochondria in a male gametocyte.
Electron-dense and finely compact of the tubular cristae
is typically associated with this type of the organelle. The
numbers of cristae in ‘type III’ mitochondria are more than
those of ‘‘Type II’ mitochondria of the female gametocytes.
The bar represents 1 µM. An arrowhead indicates electron-
dense tubular cristae. M, mitochondria.

82 ScienceAsia 25 (1999)
(Tables 2 and 3). The antimalarial drug atovaquone
is a mitochondrial inhibitor of the parasite electron
transporting complex III (ubiquinol-cytochrome c
reductase).10,26-28 Furthermore, the atovaquone had
moderate gametocytocidal activity with IC50 of 5x10-8
M, compared to its potent blood schizontocidal
activity (IC50=5x10-10 M). It has been recently shown
that atovaquone is indeed a gametocytocidal drug.29
These lines of evidence would provide mitochondria
in the sexual gametocytic stages as a possible
chemotherapeutic target.
Our results suggest that the abundant mitochon-
dria in the sexual gametocytic stage parasites were
still in the underdeveloped forms but biochemically
active at least with regards to oxygen consumption.
The mitochondrial heterogeneity also suggests their
role in energy production. Mitochondrial ATP
synthetase inhibitors are reported to have
antimalarial activity against the asexual growth of P.
falciparum at micromolar concentrations.8,25,30
Existence of ATP synthetase activity which is respon-
sible for ATP production in the sexual gametocytic
stage remains to be elucidated.
The findings on the relatively low oxygen
consumption and reduced sensitivity to cyanide
(Tables 2 and 3) suggest that P. falciparum operates
either a branched chain respiratory pathway
containing a non-cytochrome electron transport
system with an alternative oxidase which is
insensitive to cyanide6,31 or a branched electron
transport chain consisting of a specialized
cytochrome system in which fumarate acts as an
electron acceptor and is reduced to succinate by an
NADH-dependent fumarate reductase.3 The latter
pathway has been described in the adult round worm
Ascaris suum showing that ATP is anaerobically
produced by substrate level phosphorylation in the
branched electron transport pathway.32
More interestingly, 5-fluoroorotate is reported to
be a potent inhibitor of pyrimidine biosynthetic
pathway of P.falciparum.19,33 It showed marked
inhibitory effect on the oxygen consumption by both
asexual and sexual stage parasites (Table 3),
suggesting that the parasite in the sexual stage has
linkage of the two metabolic pathways, pyrimidine
biosynthesis and mitochondrial electron transport
system, through dihydroorotate dehydrogenase. The
association between the pyrimidine pathway and
mitochondrial electron transport system in the
asexual stage parasite has been confirmed.5,9 It is then
concluded that P. falciparum in both developmental
stages have functional mitochondria that contribute
significantly to de novo synthesis of pyrimidine and
to the energy metabolism of the parasite.
Whether or not the mitochondrial heterogeneity
observed in the parasites have functional significance
must await study of their biochemistry and
physiology, for instance, enzymatic activities of the
mitochondrial electron transport chain in the sexual
stage parasite, differences in the mechanism of
energy metabolism, mitochondrial ATP synthetase,
and role of oxygen tension on gametocytes
circulating in human blood system.
ACKNOWLEDGEMENTS
We thank S Vettchagarun and D Burat for their
dedicated technical assistance with EM techniques
and some experiments on oxygen uptake. The
parasites T9 and KT3 isolates were kindly provided
by S Thaitong of Chulalongkorn University and
P Petmitr of Mahidol University, respectively. This
work was supported by the UNDP/World Bank/
WHO Special Programme for Research and Training
in Tropical Diseases. J Krungkrai is a career
development award recipient from the National
Science and Technology Development Agency of
Thailand.
Table 2. The mitochondrial oxygen consumption by
P.
falciparum
at asexual and sexual stages of
development.
Stages of parasite
Oxygen consumption a
(nmol/min/10 8 parasites)
Control +Atovaquone b+Cyanide c
Asexual stage 204±16 10±2 97±10
Sexual stage 242±20 126±14 30±4
aValues are means ±SD, taken from 3-4 separate experiments of the parasite
preparations.
bFinal concentration used was 1x10-6M atovaquone.
cFinal concentration used was 1x10-3M potassium cyanide.
Table 3. The 50% effective concentrations (EC50) of
mitochondrial electron transport system and
pyrimidine biosynthetic pathway inhibitors on
parasite oxygen consumption.
Stages of parasite EC50 (M)
Atovaquone Cyanide 5-Fluor oorotate
Asexual stage 5x10-8 9x10-4 1x10-7
Sexual stage 9x10-7 2x10-4 5x10-7

ScienceAsia 25 (1999) 83
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