Syafruddin D

Hasanuddin University Repository

ELSEVIER Molecular and Biochemical Parasitology 84 (1997) 137 141 
MOLECULAR 
AND 
BIOCHEMICAL 
PARASITOLOGY 
Short communication 
The sensitivity of Plasmodium protein synthesis to prokaryotic 
ribosomal inhibitors 
Ariati S. Budimulja a, Syafruddin a, Pramuan Tapchaisri b, Prapon Wilairat c, 
Sangkot Marzuki a,* 
aEijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia 
bDepartment of Mierobiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 
°Department ofBiochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand 
Received 24 June 1996; revised 18 October 1996; accepted 18 October 1996 
Keywords: Plasmodium falciparum; Protein synthesis; Cycloheximide; Chloramphenicol; Tetracycline; Doxycycline 
The prokaryotic protein synthesis inhibitors te- 
tracycline and doxycycline are used in the treat- 
ment of malaria, as a way to overcome the 
problem of drug resistance to more conventional 
antimalarial drugs. It has been suggested that the 
antimalarial activity is mediated through the inhi- 
bition of the protein synthetic machinery of the 
mitochondria [1], as suggested by its sensitivity in 
a wide range of eukaryotic organisms to inhibitors 
of the 70S prokaryotic ribosomes; these inhibitors 
do not normally inhibit the main protein synthe- 
sizing machinery in the cytosol [2,3]. 
Several recent findings, however, have 
prompted us to examine the sensitivity of the 
Plasmodium protein synthesis to these inhibitors 
and to question the mechanism of action of the 
* Corresponding author. Tel.: + 62 21 3148694; fax: + 62 
21 3147982; e-mail: smarzuki@eijkman.go.id 
prokaryotic protein synthesis inhibitors as anti- 
malarial drugs. Most notable is the observation 
that the malaria parasite has a 35 kb circular 
extrachromosomal DNA of plastid origin [4] 
which has led to interesting questions with regard 
to the Plasmodiurn place in the evolutionary tree, 
in particular in view of an earlier suggestion that 
the ribosomes of protozoa might not in all cases 
fit into the dichotomous prokaryote-eukaryote 
(70S vs. 80S) classification [5]. The appropriate- 
ness of the dichotomous classification in general 
has been questioned [6,7], and the ribosomes of 
protozoa suggested to occupy an intermediate 
position. 
Differing from previous tudies [8-12], we have 
measured carefully the initial rate of the protein 
synthetic activity in the presence of a wide range 
of antibiotic concentrations, allowing the con- 
struction of proper dose-dependent inhibition 
0166-6851/97/$17.00 Copyright © 1997 Elsevier Science B,V. All rights reserved 
PIIS0166-6851(96)02781-8 
138 A.S. Budimulja et al./ Molecular and Biochemical Parasitology 84 (1997) 137-141 
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Fig. l. Sensitivity ofP.faleiparum (O), S. cerevisiae ([~) and E. coli (~) protein synthesis to inhibitors of the 70S and 80S ribosomes. 
P. falciparum (FCR-3 strain), cultured in vitro [19], was harvested at the parasitemia level of ~ 10%, predominantly of early and 
late trophozoite stage. Infected red blood cells were resuspended in methionine-free RPMI-1640 medium (Gibco BRL, Selectamine 
Kit) supplemented with 25 mM HEPES buffer and 2 g/L NaHCO 3 (final volume 1 ml) to a final concentration of 1 x 10 7 
parasites/ml, containing cycloheximide, metine, chloramphenicol, tetracycline or doxycyeline at the indicated concentrations. After 
preincubation for 5 min at 37°C in an atmosphere of 5% CO2 and 20% 02, with gentle shaking in a gyratory shaker, 5 #1 of the 
Expre35S35S protein labeling mix (Du Pont; 50 mCi/tube, specific activity 1175 Ci/mmol) was added. At intervals (0, 10, 20, 30, 40, 
50 and 60 min), 100 #1 aliquots were taken and immediately added into microfuge tubes containing ice cold RPMI-1640 medium 
with excess methionine (1 mg/ml). Parasites were released using 0.2% (w/v) saponin [20], collected by centrifugation at 1150 x g at 
room temperature and resuspended in 1 ml of 7% trichloroacetic acid containing 1 mg/ml methionine. Protein precipitate was 
transferred onto nitrocellulose membranes (Bio-Dot Mierofiltration Apparatus, Bio-Rad), and washed three times with 7% 
trichloroaeetic a id. The nitrocellulose membranes were air-dried and impregnated with solid scintillator (Microbeta TM 1450-441 
MeltiLex, Wallac, Finland), for the determination f the radioactivity (1450 Microbeta TM PLUS, Wallac, Finland). The initial rates 
of protein synthesis were determined from the linear part of the time course. Results are expressed as the percent of the rate of 
incorporation i the absence of inhibitor (ranging from 1300 to 2000 dpm/min per 10 -7  cells), and are the mean + S.D. of 3-6 
independent experiments. The inhibition curves of S. cerevisiae (BSC 483/la strain) and E. coli (JM 101) were examined essentially 
as for P. falciparum except hat a yeast nitrogen base (0.67%) medium (DIFCO Laboratories) upplemented with 5 g/L D-glucose 
and 5 mg/L of all amino acids besides methionine, was used for the protein synthesis of S. cerevisiae (1 mg cell dry weight/ml), and 
methionine assay medium (DIFCO Laboratories) for E. coli (2 x 107 cells/ml). 
curves. Int raerythrocyt ic  parasites incorporated  
[35S]methionine into tr ichloroacetic ac id- insoluble 
mater ia l  l inearly for at least 60 min  (data not  
shown). Its sensitivity to the 80S r ibosomes in- 
hibitor ,  cycloheximide, could be demonst ra ted  
clearly, and  a typical drug inh ib i t ion curve was 
observed (Fig. l a) with an ICso of  a round 5 x 
10 -7  M. An  a lmost  similar inh ib i t ion  curve was 
observed when the prote in  synthet ic activity of  the 
yeast S. cerevisiae was examined.  In contrast ,  
cycloheximide did not  inh ib i t  the incorporat ion  of  
[35S]methionine by the prokaryot ic  Escher ichia 
coli  ceils, even at very high concentrat ions  (up to 
10 -3  M). A similar inh ib i tory  effect was also 
obta ined for emetine (ICso 10 7 M), another  in- 
h ib i tor  of  the 80S r ibosomes (data not  shown). 
A.S. Budimulja et al. / Molecular and Biochemical Parasitoh)gy 84 (1997) 137-141 139 
When the protein synthesis activity of the Plas- 
modium parasite was similarly tested against he 
70S ribosomal inhibitors (chloramphenicol, tetra- 
cycline and doxycycline), the activity was found to 
be inhibited also in a dose dependent manner 
(Fig. lb-d). However, in contrast to that ob- 
served for E. coli (Fig. lb-d)  the inhibition curves 
did not follow a typical sigmoidal drug inhibition 
pattern and thus the ICs0 could not be determined 
precisely (between 7 x 10 .6 and 2 x 10 .5 M for 
chloramphenicol, 10 - 6 _ 10 - 4 M for tetracycline 
and around 10 4 M for doxycycline). Rather, the 
curves suggest he possibility of biphasic inhibi- 
tion. This inhibition is not an artefact, as the 
protein synthetic activity of a typical eukaryote, 
the yeast S. cerevisiae, is not affected at much 
higher concentrations of the inhibitors. 
Our results are consistent with the suggestion 
that the Plasmodium ribosomes do not fit into the 
prokaryote-eukaryote (70S vs. 80S) classification. 
It has been suggested also that the ribosomes of 
Eirneria tenella, a protozoan parasite of domestic 
animals, have some prokaryotic characteristics 
[13] and its protein synthetic activity in vitro is 
sensitive to inhibition by tetracycline and chlo- 
ramphenicol. Results of several previous drug in- 
hibition studies are indeed in agreement with our 
suggestion. Sherman et al. [8] reported that chlo- 
ramphenicol at 4 x 10 3 M inhibited 
[14C]isoleucine incorporation by P. lophurae (cy- 
cloheximide gave 90% inhibition at 2 x 10 5 M). 
Poly(U)-directed synthesis of polyphenylalanine 
by isolated ribosomes of P. knowlesi was inhibited 
(> 95%) by 10 4 M chlortetracycline [10]. Fur- 
ther support came from the analysis of the se- 
quence of the Plasrnodium rRNAs for potential 
sites of interaction with antibiotics [14], which 
predict on the basis of equivalent nucleotides ob- 
served in sensitive or resistant strains of E. coli, 
that Plasrnodiurn falciparum ribosomes hould be 
sensitive to chloramphenicol. 
Some previous tudies arrived at conclusions in 
contrast o our results [8,9]. However, these stud- 
ies were carried out with prolonged incubation 
periods (from 7 to 27 h, with up to 3 h preincuba- 
tion prior to the addition of the radioactively 
labelled amino acid tracer). Such an experimental 
set up does not allow the proper measurement of
the rate of protein synthesis, as the rate of the 
amino acid incorporation is the manifestation of
the balance between protein synthesis and degra- 
dation; the latter presumably stimulated by the 
prolonged incubation in the presence of in- 
hibitors. 
What is the explanation for the untypical inhi- 
bition curves of the P. falciparum protein synthe- 
sis by the prokaryotic ribosomal inhibitors? One 
possibility is that the curves might in fact be the 
manifestation of mixed populations of ribosomes 
which have dissimilar sensitivity to the 70S ribo- 
somal inhibitors. There are indeed reports in the 
literature that support this possibility. Each hap- 
loid genome of P. falciparum contains six copies 
of rRNA gene sets [t5], expressed in a stage-spe- 
cific manner [16] during the parasite development 
in the mosquito vector and vertebrate host. The 
switching of ribosome types occurs during the 
development process, between the S (sporozoite; 
sexual), A (asexual) and O types of ribosomes. In 
the erythrocytic stage most of the ribosomes are 
of the A type. However, three copies of type A 
rRNA genes (and two copies of type S) have been 
found in P. falciparum [15]; uniquely, these rRNA 
genes are dispersed in as many chromosomes and 
evolved independently. An analysis of the struc- 
ture of the various large subunit rRNAs compris- 
ing the type A ribosomes might give indications of 
differences in drug sensitivity. 
Since the inhibition curves of the 70S ribosomal 
inhibitors suggested the possibility of a biphasic 
inhibition (for chloramphenicol the ICs0 are 10-7 
and 10 _3 M for the first and second phase of 
inhibition respectively), we have also examined 
whether the spectrum of protein products inhib- 
ited in the initial phase of inhibition is similar to 
that in the later phase. No qualitative differences 
could be observed in the pattern of protein syn- 
thesis products in the presence of various concen- 
trations of chloramphenicol when the [35S]labelled 
products were analysed by polyacrylamide gel 
electrophoresis in the presence of sodium dodecyl 
sulfate (SDS) (data not shown; similar results 
were obtained also with tetracycline and doxycy- 
cline). However, more subtle differences in the 
protein synthesis products would be undetectable 
by SDS-polyacrylamide g l electrophoresis (SDS- 
140 A.S. Budimulja et al. / Molecular and Biochemical Parasitology 84 (1997) 137-141 
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TIME (hour) 
Fig. 2. Effect of 80S and 70S ribosomal inhibitors on the in vitro growth of P. falciparum. The sensitivity of the parasite to 
cyclolaeximide, chloramphenicol, tetracycline and doxycycline was examined in vitro in 24 well plates. Each well contained (in 1 ml) 
the growth medium (RPMI supplemented with 5% human serum of the AB type, 25 mM HEPES and 2 g/L NaHCO3), RBC (10% 
haematocrit) and different concentrations of inhibitors as specified. The initial parasitemias were 0.3-1% of young trophozoites. 
Cultures were incubated at 37°C in an atmosphere of  5% CO 2. The growth medium was changed every day and the degree of 
parasitemia was determined at the time intervals indicated in the absence (©) or the presence of an inhibitor at the concentrations 
of 10 9 M (&), 10 -8  M (Y), 10 -7  M (!~), 10 .6  M (El), 10 5 M (A), 10 -4  M (V), 10 -3  M (~) ,  10 -2  M ( I ) .  
PAGE). A more definitive study on the functional 
significance of the above observation would re- 
quire the analysis of the products of protein syn- 
thesis by two dimensional gel electrophoresis. 
We could not obtain evidence on the sensitivity 
of the Plasmodium itochondrial protein synthe- 
sis to the prokaryotic protein synthesis inhibitors. 
First, cycloheximide and emet!ne, which in other 
organisms do not affect mitochondrial protein 
synthesis, were found to inhibit the total Plasmod- 
ium protein synthesis almost completely (Fig. la); 
there was only trace levels of cycloheximide- or 
emetine-insensitive [35S]methionine incorporation 
detectable, indicating the low activity of mito- 
chondrial protein synthesis (assuming that the 
Plasmodium itochondrial protein synthesis i  in- 
sensitive to inhibition by cycloheximide). Kiat- 
fuengfoo et al. [12] reported the continuing 
synthesis of two proteins (95 and 85 kDa) by P. 
falciparum in the presence of cycloheximide (10-4 
M) and suggested that these proteins are products 
of mitochondrial protein synthesis. While this 
could be the case, it should be noted that pro- 
longed inhibition of the cytoplasmic protein syn- 
thesis would eventually affect mitochondrial 
protein synthesis as well; all mitochondrial riboso- 
mal proteins and other factors of the protein 
synthetic machinery are synthesized in the cyto- 
plasm. The inhibition time in the above study [12] 
was 7 h, while in many organisms uch as the 
yeast S. cerevisiae and human, mitochondrial 
protein synthesis ceases within 1--2 h following 
the inhibition of cytoplasmic protein synthesis [3]. 
Results of our in vitro growth inhibition exper- 
iment (Fig. 2), which are in agreement with those 
of previous tudies [1,17,18], are consistent with a 
direct effect of the prokaryotic protein synthesis 
inhibitors on the cytoplasmic ribosomes, rather 
A.S. Budimulja et al. / Molecular and Biochemical Parasitology 84 (1997) 137-141 141 
than an effect through the inhibit ion of mitochon- 
drial protein synthesis. Thus, chloramphenicol, 
tetracycline and doxycycline were found to inhibit 
the in vitro growth of P. fa lc iparum within 24-72 
h of exposure, similar to the pattern of inhibit ion 
by the 80S ribosomal inhibitor, cycloheximide. 
Growth inhibit ion mediated by an effect on the 
mitochondrial  protein synthesis is characterized 
by a delayed inhibit ion pattern; the cellular turn- 
over of products of mitochondrial  protein synthe- 
sis is relatively low and thus these products which 
are essential for oxidative energy metabolism, 
have to be diluted first to a critical level by cell 
divisions before any effect on growth could be 
observed [3]. 
Acknowledgements 
This study is supported by grants from 
Krakatau Steel and PT Inti, through the Agency 
for Strategic Industries (Indonesia). Ariati S. 
Budimulja is a Ph.D. candidate at Mahidol  Uni- 
versity in Thai land and supported by a WHO/  
TDR-SEAMEO scholarship. 
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The sensitivity of Plasmodium protein synthesis to prokaryotic ribosomal inhibitors

The  sensitivity  of  Plasmodium  protein  synthesis  to  prokaryotic  ribosomal  inhibitors

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The sensitivity of Plasmodium protein synthesis to prokaryotic ribosomal inhibitors

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