THE TREATMENT OF PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTES WITH CHLOROQUINE LEADS TO ACCUMULATION OF FERRIPROTOPORPHYRIN IX BOUND
TO PARTICULAR PARASITE PROTEINS AND TO THE INHIBITION OF THE PARASITE'S 6-PHOSPHOGLUCONATE DEHYDROGENASE
FAMIN O.* & GINSBURG H.*
Summary :
Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 9 0 and 7 0 , enolase, elongation factor 1-a, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and
gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug.
Inhibition of both processes is deleterious to parasite survival.
Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.
K E Y W O R D S : malaria, Plasmodium fatciparum, chloroquine, ferriproto- porphyrin IX, protein binding, enzyme inhibition.
M O T S CLÉS : malaria, Plasmodium falciparum, chloroquine, ferriprotopor- phyrine IX, liaison aux protéines, inhibition d'enzymes.
Résumé : L'ACTION DE LA CHLOROQUINE SUR DES ÉRYTHROCYTES INFECTÉS PAR PLASMODIUM FALCIPARUM CONDUIT À UNE ACCUMULATION DE FERRIPROTOPORPHYRINE IX LIÉE À DES PROTÉINES PARASITAIRES ET A UNE INHIBITION DE LA 6-PHOSPHOGLUCONATE DÉHYDROGÉNASE PARASITAIRE
La ferriprotoporphyrine IX (FPIX) est un produit potentiellement toxique issue de la digestion de l'hémoglobine par les parasites
intraérythrocytaires de la malaria. FPIX est détoxifié par
biominéralisation ou par dégradation par le glutathion. Les deux processus sont inhibés par la drogue antipaludique, la chloroquine, menant à l'accumulation de FPIX dans les membranes de la cellule infectée et par conséquent à sa perméabilisation. Nous montrons dans ce travail que le traitement d'érythrocytes infectés par Plasmodium falciparum mène aussi à la liaison de FPIX à une sous- population de protéines parasitaires. Des enzymes parasitaires telles que l'aldolase, la pyrimidine nucléoside monophosphate kinase ou la pyrimidine 5'-nucléotidase sont inhibées par FPIX in vitro. Cependant, seule l'activité de &phosphogluconate déhydrogénase est réduite d'une manière significative dans les cellules traitées. D'autres protéines du cytosole parasitaire présentent de l'affinité pour FPIX. Le séquençage de ces protéines a permis d'identifier les protéines heat shock 70 et 90, l'énolase, le facteur délongation l-a, la
phosphoglycérate kinase, la glycérladhéyde 3-phosphate
déhydrogénase, la L-laclate déhydrogénase et la protéine specifique de la mise en place (onset specific) de la gamétocytogenèse.
L'implication possible de ces protéines dans le mode d'action anlipaludique de la chloroquine est discuté. Nous concluons que la liaison - induite par la drogue - de FPIX aux enzymes glycolytiques du parasite pourraient être responsable de l'inhibition démontrable de la glycolyse par chloroquine. L'inhibition de la 6-phosphogluconate déhydrogénase pourrait expliquer la réduction de l'activité de la voie de l'hexose monophosphate par la drogue. L'inhibition des deux processus est pernicieuse à la survie du parasite. La liaison de FPIX à d'autres protéines n'a probablement pas de conséquences concernant la mort rapide du parasite provoquée par chloroquine.
INTRODUCTION
I
ntraerythrocytic malaria parasites ingest a n d digest the c y t o s o l o f their host cells ( Y a y o n et al., 1 9 8 4 ; Francis et al., 1 9 9 7 ) . Ferriprotoporphyrin IX ( F P I X )Abbreviations used: A Q : a m o d i a q u i n e ; C Q : c h l o r o q u i n e ; F P I X : fer- r i p r o t o p o r p h y r i n I X ; G S H : g l u t a t h i o n e ; H M S : h e x o s e m o n o p h o s - p h a t e s h u n t ; M Q : m e f l o q u i n e ; Q : q u i n i n e .
* D e p a r t m e n t of B i o l o g i c a l C h e m i s t r y , I n s t i t u t e of Life S c i e n c e s , T h e H e b r e w U n i v e r s i t y of J e r u s a l e m , J e r u s a l e m 91904, Israel.
C o r r e s p o n d e n c e : Prof. H a g a i G i n s b u r g . Tel.: +972-2-658-5539 - F a x : +972-2-658-5440.
E-mail: h a g a i @ v m s . h u j i . a c . i l
is a t o x i c product o f this p r o c e s s . T h e erythrocyte c y t o s o l c o m p r i s e s e s s e n t i a l l y h e m o g l o b i n , a n d t h e parasite must detoxify F P I X . T h i s is a c h i e v e d b o t h b y its biomineralization into h e m o z o i n ( G o l d b e r g & Slater, 1 9 9 2 ) and b y its degradation by glutathione ( G S H ) after it has left the food v a c u o l e into -the parasite c y t o s o l ( A t a m n a & G i n s b u r g , 1 9 9 5 ; G i n s b u r g et al., 1 9 9 8 ) . C h l o r o q u i n e ( C Q ) inhibits b o t h p r o c e s s e s . R e c e n t fin- dings indicate that treatment o f Plasmodium falci- parum-infected e r y t h r o c y t e s with C Q i n c r e a s e s the c o n t e n t o f F P I X in t h e m e m b r a n e fraction o f t h e cells (Ginsburg et al., 1998; Zhang et al., 1 9 9 9 ) . W h e r e a s this i n c r e a s e c o u l d lead to the p e r m e a b i l i z a t i o n o f the m e m b r a n e and cell death, as attested b y the c o r r e l a -
Parasite, 2003, 10, 39-50
Mémoire 39
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2003101p39
FAMIN O. & GINSBURG H.
tion b e t w e e n m e m b r a n e - a s s o c i a t e d FPIX and parasite killing, the p h a r m a c o l o g i c a l action o f CQ is thought to b e pleiotropic. H e n c e , other targets in the cell may b e affected by free or CQ-associated FPIX and thus b e either a primary o r an adjunct target for drug action.
D u e to its amphipathic nature, FPIX can bind non-spe- cifically to proteins. Several erythrocyte e n z y m e s have b e e n s h o w n to b e inhibited by FPIX ( Z e r e z et al.,
1987): aldolase, a k e y e n z y m e o f glycolysis that pro
vides m e t a b o l i c energy to the parasite; 6-phosphoglu- c o n a t e d e h y d r o g e n a s e , a k e y e n z y m e in the h e x o s e m o n o p h o s p h a t e shunt that provides reducing equiva
lents to counteract oxidative stress; adenylate kinase, instrumental in purine metabolism; pyrimidine n u c l e o side m o n o p h o s p h a t e kinase, involved in pyrimidine n u c l e o s i d e synthesis; and pyrimidine 5'-nucleotidase which functions in the degradation o f pyrimidine and purine nucleotides. T h e inhibitory constants o f FPIX o n the activity o f all t h e s e e n z y m e s w e r e reported to b e = 1 µM.
T h e present investigation aims to elucidate the effects o f FPIX o n t h e s e e n z y m e s in the parasite, to detect additional parasite proteins that bind FPIX and to assess if indeed inhibition o f the function o f these pro
teins c o u l d contribute to the antimalarial m o d e o f action o f CQ.
MATERIALS AND METHODS
MATERIALS
F
resh O+ or A+ b l o o d and human 0+ or A+ plasma w e r e kindly d o n a t e d by the Hadassah Hospital B l o o d B a n k . RPMI-1640 w a s p u r c h a s e d from B i o l o g i c a l Industries, K i b b u t z B e t H a e m e k , Israel.Hemin-agarose b e a d s , ATP, AMP, NADP. NADH. fruc- tose-2,6-bisphosphate (K salt), 6-phosphogluconic acid, pyruvate kinase-lactate d e h y d r o g e n a s e mixture and all e n z y m e s used in this investigation w e r e purchased from Sigma Chemical Co. C h l o r o q u i n e diphosphate w a s o b t a i n e d from Serva. Amodiaquine dihydrochlo- ride and q u i n i n e hydrochloride w e r e p u r c h a s e d from Sigma Chemical Co. Mefloquine w a s g e n e r o u s l y pro
vided by A.F. C o w m a n . Hemin was obtained from Porphyrin Products, Logan, UT. G E L C O D E blue stain was purchased from PIERCE (lot no: 9 8 1 2 2 1 1 6 9 ) . All other c h e m i c a l s w e r e o f the best available grade.
PARASITE CULTIVATION
T h e FCR3 strain o f P. falciparum w a s cultivated as pre
viously d e s c r i b e d ( Z h a n g et al., 1 9 9 9 ) in RPMI-1640 m e d i u m s u p p l e m e n t e d with 10 mM g l u c o s e , 25 mM N a H C 03, 2 5 mM H e p e s and 10 % h u m a n heat-inacti
vated plasma. Cultures w e r e synchronized by the sor
bitol t e c h n i q u e (Lambros & Vanderberg, 1 9 7 9 ) using the less toxic alanine. Parasites w e r e fractionated to dif
ferent stages using the Percoll-alanine gradient centri- fugation protocol (Kutner et a l . , 1 9 8 5 ) .
EFFECT OF DRUG TREATMENT
ON THE FERRIPROTOPORPHYRIN IX LEVEL IN PARASITE CYTOPLASM
Synchronized cultures w e r e s e e d e d at the ring stage and a l l o w e d to g r o w for a n o t h e r 2 0 h o u r until most parasites reached the trophozoite stage. Parasitemia and cell n u m b e r w e r e determined, a n d cells w e r e cultured for three-four hours in a b s e n c e or the presence o f 10 µM drug. Thereafter, cultures w e r e w a s h e d twice in wash medium (culture m e d i u m without plasma, 3 7 ° C ) to r e m o v e the drug, and cells w e r e used for the deter
mination o f FPIX levels in the cytoplasm o f the para
site, for the identification o f parasite FPIX- binding pro
teins and for m e a s u r e m e n t o f the activity o f e n z y m e s k n o w n to b e inhibited by FPIX, as described b e l o w .
PREPARATION OF FREE PARASITES FROM CULTURES Trophozoite-infected erythrocytes w e r e e n r i c h e d by Percoll-alanine gradient centrifugation to > 9 0 % para
sitemia. Parasites w e r e released from infected R B C by saponin lysis ( 0 . 0 0 3 % saponin ( w / v ) in P B S ) followed with repeated w a s h e s in P B S buffer ( 1 , 5 0 0 x g for five minutes) until n o h e m o g l o b i n could b e detected in the wash solution ( Z h a n g et al., 1 9 9 9 ) . T h e free parasites w e r e resuspended in an equal v o l u m e o f buffer (0.1 M KC1 in 2 0 mM Na-phosphate, pH 7 . 4 ) .
T H E ISOLATION OF PARASITE PROTEINS FROM INFECTED CELLS
Free parasites w e r e disrupted by five cycles o f free
zing in liquid nitrogen and thawing at 3 7 ° C. T h e cell debris and h e m o z o i n w e r e spun d o w n by centrifuga
tion at 10,000 xg for 15 minutes. T h e lysate w a s dis
solved in twice c o n c e n t r a t e d s a m p l e buffer containing 2 % SDS, 10 % glycerol in 6 3 mM Tris-Cl buffer pH 6.8 ( v o l u m e ratio 1:1).
STAINING OF F P I X ON S D S GELS
Samples o f parasite cytoplasm w e r e run o n SDS-PAGE ( 1 0 % p o l y a c i y l a m i d e ) omitting dithiotreitol from the running buffer in order to avoid destruction o f FPIX.
G e l s w e r e fixed for 2 0 minutes with 12.5 % trichlo
roacetic acid and w a s h e d for 2 0 minutes in d o u b l e dis
tilled water. G e l s w e r e then stained with a mixture o f 0.1 % ( w / v ) o-dianisidine chloride and 2 0 mM hydrogen p e r o x i d e in 4 0 mM citric acid until a p p e a r a n c e o f stained FPIX (Francis & B e c k e r , 1 9 8 4 ) .
For quantitative assessment o f h e m e content, s a m p l e s containing various a m o u n t s o f FPIX b o u n d to BSA
40
Mémoire
P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0FÉRRIPROTOPORPHIN I X BINDING TO MALARIA PARASITE PROTEINS
w e r e run in parallel with parasite samples. After stai
ning, the gels w e r e s c a n n e d (UMAX 1 2 2 0 p s c a n n e r ) , a calibration curve w a s c o n s t r u c t e d from the areas o f the FPIX-BSA bands and the integral o f the bands from the parasite s a m p l e w a s u s e d to calculate the a m o u n t o f F P I X in the parasite s a m p l e s normalized to the n u m b e r o f parasites present in the sample.
ISOLATION O F
FPIX
B I N D I N G PROTEINS FROM CELL LYSATES AND THEIR SEQUENCINGParasite cytosol w a s prepared as d e s c r i b e d a b o v e in 0.1 M KC1, 2 0 mM Na-phosphate buffer pH 7.4, and i n c u b a t e d with h e m i n - a g a r o s e b e a d s ( h e m i n c o n c e n tration 5 pM) for t w o hours in the same buffer at 37° C.
Thereafter, the b e a d s w e r e s p u n d o w n and the super
natant was discarded. T h e b e a d s w e r e w a s h e d five times with buffer: 1.0 M KC1, 2 0 mM Na-phosphate pH 7.4. This c o n c e n t r a t i o n o f KCL w a s c h o s e n after it w a s ascertained that increasing [KCl] from 0.1 to 1.0 M did not result in selective elution o f protein bands, and that the majority o f proteins r e m a i n e d a s s o c i a t e d with the b e a d s after washing with 1.0 M KCl (data not s h o w n ) . T h e b o u n d proteins w e r e eluted from the b e a d s by incubation (for 15 minutes at n e a r boiling t e m p e r a t u r e ) with s a m p l e buffer: 2 % SDS, 10 % gly
cerol, 10 mM D T T , 6 3 mM TRIS-HCl, pH 6.8 ( b e a d s to buffer v o l u m e ratio 1:1). T h e s a m p l e s w e r e e l e c - t r o p h o r e s e d through an 8 % TRIS-glycine acrylamide gel. T h e gel was w a s h e d five times with D D W for five hours and stained with Gelcode® B l u e Stain (PIERCE, lot No. 9 8 1 2 2 1 1 6 9 ) . F o l l o w i n g destaining, the protein bands w e r e cut-out and reduced with D T T (5 m M ) and c a r b o x y m e t h y l a t e d “in gel” using 10 mM i o d o a c e t a - mide. T h e gel w a s then further destained in 5 0 % a c e - tonotrile with 1 0 0 mM a m m o n i u m b i c a r b o n a t e , dried and rehydrated with 1 0 0 mM a m m o n i u m b i c a r b o n a t e pH 7.4 containing modified trypsin ( P r o m e g a ) . After overnight incubation at 3 7 ° C with shaking, the resul
ting peptides w e r e eluted from the gel pieces with 6 0 % acetonitrile containing 0.1 % TFA and analyzed by LC- MS. T h e MS and MS/MS data from the run w e r e c o m pared to the simulated proteolysis and fragmentation o f the proteins in the "owl'' database using the Sequest software (J. Eng & J . Y a t e s Univ. o f W a s h i n g t o n ) .
MEASUREMENT O F ENZYME ACTIVITIES IN DIFFERENT COMPARTMENTS
O F PARASITE-INFECTED RBC
P. falciparum infected RBC ( t r o p h o z o i t e s t a g e ) w e r e isolated b y Percoll-alanine gradient to obtain > 9 0 % parasitemia. Parasites w e r e r e l e a s e d from host cell b y saponin lysis ( 0 . 0 0 3 % ( w / v ) saponin, v o l u m e ratio 1 / 2 0 ) followed by centrifugation at 4 , 5 0 0 0 xg for two minutes. T h e supernatant w a s c o l l e c t e d for the deter
mination o f e n z y m e activity in the host cell compart
ment. Free parasites w e r e w a s h e d five times in PBS buffer until n o h e m o g l o b i n ( a b s o r b a n c e at 4 1 2 n m ) could b e d e t e c t e d in the supernatant.
Free parasites w e r e disrupted by trituration (five times) through a 27 g a u g e n e e d l e at 4 ° C in 0 . 2 5 M sucrose, 20 mM Na-phosphate buffer, pH 7.4 (Bray et al., 1 9 9 9 ) . T h i s p r o c e d u r e p r e v e n t s t h e d i s r u p t i o n o f f o o d v a c u o l e s a n d the p o s s i b l e degradation o f cytosolic proteins by vacuolar proteases. T h e trituration mixture w a s centrifuged at 1 0 , 0 0 0 x g for 10 minutes and the supernatant w a s c o l l e c t e d for determination o f para
site e n z y m e activity.
For the determination o f e n z y m e activity in normal RBC, cells w e r e lysed in h y p o t o n i c 5 mM p h o s p h a t e buffer, pH 8 at 4 ° C.
Activity o f aldolase, 6 - p h o s p h o g l u c o n a t e dehydroge
n a s e and adenylate kinase was determined as des
cribed by B e u t l e r ( 1 9 8 4 ) . Activity o f pyrimidine-5'- n u c l e o s i d a s e was determined according to the method o f Zerez & T a n a k a ( 1 9 8 5 ) . Results w e r e normalized to protein c o n t e n t in lysates and to the cell n u m b e r in samples.
B I N D I N G O F
FPIX
T O PURE PROTEINS AND ENZYMEST h e following proteins were dissolved in PBS (25 µg/ml):
BSA, c y t o c h r o m e c oxidase, h e x o k i n a s e , alcohol dehy
drogenase, GSH reductase, triose phosphate isomerase, α-glycerophosphate d e h y d r o g e n a s e . Each protein or a mixture o f all proteins was incubated with 50 µM fre
shly p r e p a r e d FPIX for 15 minutes at 3 7 ° C , then sepa
rated by SDS-PAGE and stained for FPIX or in parallel with C o o m a s s i e .
RESULTS
E F F E C T O F D R U G TREATMENT ON T H E BINDING O F F P I X T O PROTEINS
R
ather high drug c o n c e n t r a t i o n s relative to t h o s e k n o w n to inhibit parasite growth in culture w e r e used in this investigation. Such high c o n c e n t r a tions w e r e c h o s e n b e c a u s e it has b e e n s h o w n b e f o r e that shorter times o f e x p o s u r e to the drugs required higher c o n c e n t r a t i o n s for inhibition o f parasite growth (Krugliak & Ginsburg, 1 9 9 1 ) . Treatment o f infected cells with C Q or AQ resulted in the association o f FPIX with several parasite proteins. T h e s e proteins w e r e visualized after SDS-PAGE o f disrupted free parasites and staining both with Coomassie and for FPIX (Fig. 1 ) . T h e e q u a l densities o f the C o o m a s s i e b a n d s in the various lanes (Fig. 1A) indicate that equal a m o u n t s o f proteins w e r e l o a d e d in e a c h lane and that the differences in intensities o f the FPIX-stained bands (Fig. 1B)
P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0
Mémoire 4 1
F A M I N O. & G I N S B U R G H.
are a g e n u i n e reflection o f selective FPIX binding. T h e identity o f the C o o m a s s i e b a n d s a m o n g the different treatments, suggest that drug treatment per se d o e s induce or d o w n regulates parasite proteins. C o m p a rison o f the Coomassie-stained gels with their FPIX- stained counterparts clearly indicates that FPIX binding demonstrates varying specificity (the relative intensities o f C o o m a s s i e b a n d s and FPIX b a n d s are distinctly dif
ferent). T h e F P I X - a s s o c i a t e d proteins c a n n o t b e high m o l e c u l a r weight products o f h e m o g l o b i n digestion since, u n d e r the conditions o f the SDS-PAGE, h e m o globin tetramers are dissociated into m o n o m e r s ( a s c e r tained using lysates o f uninfected erythrocytes; data not s h o w n ) . T h e FPIX-binding proteins are all o f greater m o l e c u l a r w e i g h t s than the h e m o g l o b i n m o n o m e r ( 1 6 . 7 k D a ) that are always present in the gels o f C Q - treated parasites. This represents the remnants o f undi
gested h e m o g l o b i n in the free parasite (Famin & Gins- burg, 2 0 0 2 ) . FPIX binding appears to b e insignificant in drug-free parasites, rises to high levels after treat
ment o f intact infected cells with either C Q o r AQ, but is generally absent, or present at very l o w levels, after treatment with M Q . Treatment with Q result in l o w e r levels than with C Q treatment but higher than with MQ treatment. T h e levels o f protein-associated FPIX vary quite n o t i c e a b l y a m o n g e x p e r i m e n t s due to s o m e variations in culture conditions and parasite stage, but the relative effect o f the drugs is always o b s e r v e d . T o confirm the p r e s e n c e o f FPIX o n these proteins, the
extracts w e r e treated with low c o n c e n t r a t i o n s o f H202, k n o w n to degrade FPIX. This treatment considerably r e d u c e d the intensity o f F P I X on all protein b a n d s and almost destroyed it c o m p l e t e l y o n the high m o l e c u l a r weight s p e c i e s (Fig. 2 ) . T h e effects o f verapamil, an e n h a n c e r o f C Q action in resistant strains, including the FCR3 strain used in this investigation, and o f H202, w a s also tested. T h e effect o f C Q o n the level o f FPIX bin
ding was e n h a n c e d by verapamil a b o v e that s e e n with CQ (Fig. 3 ) . Since F P I X is the intracellular ligand for C Q (Bray et al, 1 9 9 9 ) , this i n c r e a s e m a y explain the ability o f verapamil to i n c r e a s e the net uptake o f C Q into drug-resistant strains. T h e p r e s e n c e o f H202 redu
c e d the effect o f CQ. It is not clear why verapamil itself c a u s e d a slight i n c r e a s e in FPIX binding, but for that matter the m o d e o f antimalarial action o f this drug a l o n e , is not k n o w n .
T O T A L P R O T E I N - B O U N D F P I X IN C Q - T R E A T E D I N F E C T E D CELLS
T h e q u a n t i f i c a t i o n o f F P I X b o u n d to p r o t e i n s , as revealed by specific staining, w a s a c c o m p l i s h e d using BSA as a prototypic protein. T h e intensity o f the FPIX signal w a s correlated with the a m o u n t o f loaded FPIX (data not s h o w n ) . Integration o f staining intensities o f all protein b a n d s a s s o c i a t e d with F P I X (after S D S - PAGE o f parasite cytosol following C Q treatment) in conjunction with staining intensities o f k n o w n amounts o f FPIX-BSA loaded o n the s a m e gel, a l l o w e d the cal-
42 P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0
Fig. I. - A s s o c i a t i o n of F P I X with p a r a s i t e p r o t e i n s .
I n f e c t e d c e l l s w e r e t r e a t e d for four h o u r s with 1 0 u M C Q , A Q , Q o r M Q . P a r a s i t e s w e r e freed a n d d i s r u p t e d a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e s u p e r n a t a n t f o l l o w i n g c e n t r i f u g a t i o n w a s run o n S D S - P A G E a n d s t a i n e d for p r o t e i n s w i t h C o o m a s s i e B l u e ( 1 A ) a n d for F P I X (1B).
Mémoire
d i l a t i o n o f total intact FPIX a m o u n t . This calculation yields a concentration o f ~ 165 µM ( b a s e d o n the num
b e r o f cells used for the preparation o f free parasites, the n u m b e r o f infected cells/1 and the relative v o l u m e
M W ( KDA ) 2 5 0 -
9 8 -
Fig. 2. - A s s e s s m e n t o f F P I X a s p r o t e i n ligand.
I n f e c t e d c e l l s w e r e t r e a t e d with 1 0 µM C Q o r Q for four h o u r s . F r e e p a r a s i t e s w e r e o b t a i n e d b y s a p o n i n lysis a n d d i s r u p t e d . Parallel s a m p l e s w e r e left a s c o n t r o l s o r t r e a t e d w i t h 1 0 uM H202 for s e v e r a l m i n u t e s at r o o m t e m p e r a t u r e , e l e c t r o p h o r e s e d b y S D S - P A G E a n d s t a i n e d for F P I X . B a n d at ~ 16 k D A is h e m o g l o b i n m o n o m e r s .
of the parasite in the infected cell). This is a tiny frac
tion o f the total ~ 4 mM FPIX that is g e n e r a t e d up to the t r o p h o z o i t e stage during h e m o g l o b i n digestion a n d that is not p o l y m e r i z e d into h e m o z o i n . F P I X b o u n d to parasite proteins is about half the amount found to b e associated with the m e m b r a n e fraction of similarly treated cells ( Z h a n g et al., 1999).
I D E N T I T Y O F F P I X - B I N D I N G PARASITE P R O T E I N S Since the parasite lysate contains many proteins (Fig. 1 A ) and their staining for FPIX resulted in broad diffuse bands (Fig. 1 B ) , it was impossible to isolate the FPIX-binding proteins from the gels in pure form. An alternative way to identify FPIX-binding proteins in parasite cytosol was undertaken. Free parasites were dismpted by freezing and thawing and after centrifugation to remove cell debris and hemozoin, the lysate was equilibrated with FPIX-agarose beads. T h e unbound proteins w e r e removed and bound proteins were eluted with sample buffer, electrophoresed
Fig. 3. - Effect o f v e r a p a m i l a n d H2O2 o n C Q - i n d u c e d a s s o c i a t i o n o f F P I X w i t h p a r a s i t e p r o t e i n s .
I n f e c t e d c e l l s w e r e i n c u b a t e d for four h o u r s at c o n d i t i o n s d e s c r i b e d in t h e l e g e n d t o F i g u r e 1 w i t h o u t o r with i n c r e a s i n g c o n c e n t r a t i o n s o f C Q ± 10 u M v e r a p a m i l , o r w i t h C Q + 8 0 µM H2O2. P a r a s i t e s w e r e f r e e d b y s a p o n i n lysis, d i s n i p t e d a n d t h e s u p e r n a t a n t w a s e l e c t r o p h o r e s e d o n S D S - P A G E . G e l s w e r e s t a i n e d for F P I X .
Fig. 4 . - I d e n t i f i c a t i o n o f p a r a s i t e p r o t e i n s that b i n d t o h e m i n - a g a - r o s e b e a d s .
T r o p h o z o i t e s w e r e freed from h o s t c e l l s , triturated a n d t h e s u p e r n a t a n t w a s e x p o s e d t o h e m i n a g a r o s e b e a d s a s d e s c r i b e d in M a t e rial a n d m e t h o d s . B o u n d p r o t e i n s w e r e e l e c t r o p h o r e s e d a n d s t a i n e d for i d e n t i f i c a t i o n . I n d i v i d u a l b a n d s w e r e cut o u t from t h e g e l s a n d t h e i r p r o t e i n s w e r e s e q u e n c e d . I d e n t i f i c a t i o n o f b a n d s : l - H s p 9 0 ; 2 - H s p 7 0 ; 3 - e n o l a s e a n d e l o n g a t i o n f a c t o r 1-α; 4 - p h o s h o g l y - c e r a t e k i n a s e ; 5 - g l y c e r a l d e h y d e 3 - p h o s p h a t e d e h y d r o g e n a s e ; 6 - L- lactate d e h y d r o g e n a s e a n d g a m e t o c y t o g e n e s i s o n s e t - s p e c i f i c protein.
B a n d s l a b e l e d w i t h * a r e a l s o seen in t h e l y s a t e s of C Q - t r e a t e d c e l l s ( F i g . 1 B ) .
P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0
Mémoire
43and stained with Coomassie (Fig. 4 ) . T h e parasite lysate proteins that were eluted from the beads were probably bound non-specifically since their elution did not depend on ionic strength, i.e., the same proteins could b e eluted with 0.1 M and 1.0 M KC1. No binding o f lysate proteins could b e observed with blank agarose beads. S o m e o f the proteins that were eluted from the hemin agarose beads migrated on the gel identically to those found to bind FPIX in CQ-treated infected cells, notably those having ~ 50, - 3 6 and ~ 3 9 kDa molecular weight. While identical migration does not confirm identity o f proteins, uniqueness may b e suggested. Major bands were excised from the gel and sequenced. Several parasite proteins were thus detected: heat s h o c k proteins 7 0 and 9 0 , enolase, phosphoglycerate kinase, glyceraldehyde 3 - phosphate dehydrogenase, L-lactate dehydrogenase and g a m t o c y t o g e n e s i s o n s e t - s p e c i f i c protein. T h e g e n e s coding for all these proteins are present in the g e n o m e o f P. falciparum.
A C T I V I T Y O F F P I X - I N H I B I T A B L E E N Z Y M E S IN PARASITE C Y T O S O L B E F O R E A N D A F T E R D R U G T R E A T M E N T
W e investigated the activity o f e n z y m e s reported to b e inhibited by FPIX in normal erythrocytes ( Z e r e z et al., 1 9 8 7 ) . T h e format w a s to test e n z y m e activity in the parasite, using the erythrocyte as a positive control, and then test it again after treatment with CQ.
T o verify the effect o f FPIX on 6 - p h o s p h o g l u c o n a t e dehydrogenase, it was first tested on a commercially available e n z y m e . At 0.01 U, 5 µM FPIX caused an inhi
bition o f 50 % in e n z y m e activity at maximal substrate concentration. Results s h o w n in Figure 5A depict the
Fig. 5. - I n h i b i t i o n o f 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e activity b y F F I X .
E n z y m e activity in l y s a t e s in a b s e n c e a n d in p r e s e n c e of i n c r e a s i n g F P I X c o n c e n t r a t i o n s w a s d e t e r m i n e d a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A. E n z y m e activity of e r y t h r o c y t e l y s a t e w a s c h a r a c t e r i z e d b y d e r i v i n g K m ( u M ) a n d V m a x (x10-3 a b s o r b a n c e u n i t s / m i n ) v a l u e s , r e s p e c t i v e l y : C o n t r o l - • (filled c i r c l e s ) - 2 8 . 3 ± 7 . 1 ; 12+9. [FPIX] = 0 . 5 µ.M - O ( e m p t y c i r c l e s ) - 2 3 . 4 ± 7 . 8 ; 1 0 . 2 ± 0 . 9 . [FPIX] = 1 µM - • ( f i l l e d t r i a n g l e s ) - 27.6 ± 1.9; 7 . 4 ± 0 1 . [ F P I X ] = 2 µM - A ( e m p t y t r i a n g l e s ) - 4 9 . 0 ± 1 1 . 1 : 8.6 ± 0.6. B . E n z y m e acti
vity in p a r a s i t e l y s a t e o b t a i n e d from c o n t r o l a n d C Q ( 1 0 µM)-treated i n f e c t e d c e l l s . C o n t r o l - • (filled c i r c l e s ) - 8 . 8 ± 5 . 4 : 4 . 9 ± 0 . 9 . C Q - t r e a t e d - O ( e m p t y c i r c l e s ) - 8 . 2 ± 3 . 0 ; 3.1 ± 0 . 3 . C. Effect o f d r u g t r e a t m e n t o n 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e in t h e c o m p a r t m e n t s of i n f e c t e d c e l l s . F i l l e d b a r s - u n i n f e c t e d c e l l s ; E m p t y b a r s - h o s t c e l l c o m p a r t m e n t ; G r e y b a r s - p a r a s i t e c o m p a r t m e n t . S h o w n a r e r e p r e s e n t a t i v e r e s u l t s of s e v e r a l s i m i l a r e x p e r i m e n t s . I n f e c t e d e r y t h r o c y t e s w e r e i n c u b a t e d for f o u r h o u r s in a b s e n c e o r in p r e s e n c e of C Q a n d Q ( 1 0 pM e a c h ) . C e l l s w e r e s e p a r a t e d b y P e r c o l l g r a d i e n t a n d e n z y m e activity w a s d e t e r m i n e d in u n i n f e c t e d c e l l s , in t h e s u p e r n a t a n t of s a p o n i n l y s e d i n f e c t e d c e l l s a n d in t h e l y s a t e of free p a r a s i t e s . A c t i v i t i e s relative t o u n t r e a t e d c o n t r o l s a r e s h o w n . N o t i c e that o n l y t h e activity of p a r a s i t e e n z y m e is a f f e c t e d . 6 - P G A - 6 - p h o s p h o g l u c o n i c a c i d .
effect o f FPIX on the activity o f 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e , the s e c o n d e n z y m e o f the h e x o s e m o n o p h o s p h a t e shunt (HMS) in erythrocyte cytosol.
Symbols depict the experimental results and lines are the best fit to the Michaelis-Menten equation. Increasing [FPIX] first results in non-competitive inhibition - V m a x is reduced - and then the mechanism o f inhibition s e e m s to b e o f a c o m p e t i t i v e nature, i.e., Km is increased. Although this observation is rather unusual, higher FPIX concentrations were not tested because they are outside the range o f relevance. T h e overall activity
44 Memoire P a r a s i t e , 2 0 0 3 , 10. 3 9 - 5 0
F E R R I P R O T O P O R P H I N FX B I N D I N G T O M A L A R I A P A R A S I T E P R O T E I N S
is reduced by ≤ 30 %. No inhibition o f e n z y m e activity could b e s e e n in the p r e s e n c e o f CQ alone (data not s h o w n ) . W h e n trophozoite-infected RBC w e r e treated with CQ and the e n z y m e activity measured in the para
site lysate, a 4 0 % inhibition o f non-competitive nature was observed (Fig. 5 B ) . Results shown in Figure 5C were obtained from the following procedure: parasite cultures at the trophozoite stage w e r e treated with CQ o r Q.
Infected cells w e r e separated from uninfected by Per- coll gradient centrifugation. Enzyme activity was then tested in uninfected cells and in the host and parasite compartments o f infected cells. CQ exposure did not affect the enzyme in uninfected cells or in the host com
partment o f infected cells. Enzyme activity in the para
site compartment was reduced by 4 0 % ( c o m p a r e with Figure 5A) as would b e e x p e c t e d from the putative bin
ding o f FPIX to the enzyme. Q had n o effect, as would b e e x p e c t e d from the lack o f FPIX accumulation under the influence o f this drug ( s e e Fig. 1 B ) .
As reported by Zerez et al. ( 1 9 8 7 ) . w e also found that FPIX inhibits the activity o f adenylate kinase in unin
fected erythrocytes. Inhibition was o f the uncompeti
tive type. Km (uM) increased from 0.19 ± 0.02 in control to 0.47 ± 0.23 in the p r e s e n c e o f 4 uM FPIX, and V m a x (in x 1 0- 2 a b s o r b a n c e units/min) declined from 4 . 4 5 ± 0.2 to 0.4 ± 0 . 0 1 , respectively. W e could not detect any activity o f this e n z y m e in the parasite cytosol, although such activity has b e e n inferred indirectly (Kanaani &
Ginsburg, 1 9 8 9 ) and the g e n e coding for this e n z y m e can b e predicted from the s e q u e n c i n g project o f the P. falciparum g e n o m e : several o p e n reading frames that predictably c o d e for this e n z y m e have b e e n found o n several c h r o m o s o m e : P F D 0 7 5 5 c o n c h r o m o s o m e 4 (annotated by Sanger), P F 1 0 _ 0 0 8 6 on c h r o m o s o m e 10 (annotated T I G R ) , and P l a s m o D B using various algo
rithms for g e n e prediction could find it on c h r o m o s o m e 8 - c h r 8 . g e n _ 2 3 3 (predicted by Genefinder), chr8.glm_
163 (predicted by GlimmerM) and chr8.phat_150 (pre
dicted by FullPhat).
W h e r e a s FPIX most potently inhibited the activity o f the erythrocyte 5'-pyrimidine nucleotidase (Fig. 6 ) , n o inhi
bition o f this e n z y m e in either the host cell or the para
site compartment could be seen after treatment with CQ or with Q. FPIX was also tested on commercially avai
lable enzyme (0.1 m g / m l ) and at 5 pM inhibited enzyme activity by 8 0 %. T h e s e results suggest that this enzyme is the most sensitive to FPIX o f those tested.
Aldolase is a k e y e n z y m e in the glycolytic pathway, the s o l e s o u r c e o f m e t a b o l i c energy for both host cell and parasite (Sherman, 1 9 7 9 ) . FPIX inhibited a c o m mercially available e n z y m e ( 0 . 0 1 1 unit/ml) by 5 0 % at 5 uM. T h e activity o f this e n z y m e , in normal erythro
cytes and in parasite cytosol, was inhibited by FPIX with a Ki o f 5-7 pM. However, the activity o f the para
site e n z y m e after CQ treatment was inhibited by only 15 %, w h e r e a s Q had n o effect at all (data not s h o w n ) .
Fig. 6. - Activity o f 5 ' - p y r i m i d i n e n u c l e o t i d a s e in n o r m a l e r y t h r o c y t e s a n d in t h e c o m p a r t m e n t s o f c o n t r o l a n d C Q - t r e a t e d i n f e c t e d c e l l s .
A. E n z y m e activity w a s d e t e r m i n e d in lysates of n o r m a l e r y t h r o c y t e s u s i n g 5 0 uM c y t i d y l m o n o p h o s p h a t e (al V m a x ) , in a b s e n c e o r pre
s e n c e of t w o c o n c e n t r a t i o n s o f F P I X . B . E n z y m e activity w a s simi
larly m e a s u r e d in the lysates o f n o r m a l e r y t h r o c y t e s ( R B C ) a n d in t h e host cell a n d p a r a s i t e c o m p a r t m e n t s o f i n f e c t e d c e l l s , after four h o t i r s of i n c u b a t i o n e i t h e r in a b s e n c e of d r u g o r in t h e p r e s e n c e of 1 0 u M o f C Q (filled b a r s ) o r 10 uM Q ( e m p t y b a r s ) . % o f activity w a s c a l c u l a t e d w i t h r e s p e c t t o e n z y m e activity in u n t r e a t e d c e l l s . S h o w n a r e r e p r e s e n t a t i v e results o f s e v e r a l similar e x p e r i m e n t s .
B I N D I N G O F F P I X T O PURE PROTEINS
Several commercially available enzymes, chosen as exam
ples for n o particular reason, w e r e tested for their abi
lity to bind FPIX. After a brief e x p o s u r e to 5 0 uM FPIX, e n z y m e s w e r e run o n SDS-PAGE (without D T T ) and stained for FPIX and with C o o m a s s i e . All e n z y m e s b o u n d F P I X , but c o m p a r i s o n o f t h e d e n s i t i e s o f Coomassie-stained bands to their FPIX-stained coun
terparts, indicates a variable degree o f binding (Fig. 7 ) . Thus, alcohol d e h y d r o g e n a s e , triose p h o s p h a t e iso- merase and α-glycerophosphate dehydrogenase bound FPIX m o r e avidly than BSA. c y t o c h r o m e c oxidase or G S H r e d u c t a s e . S o m e impurities r e s o l v e d o n the
Parasite. 2 0 0 3 . 10. 3 9 - 5 0
Mémoire 45
FAMIN O. & GINSBURG H.
Fig. 7. - B i n d i n g of FPIX t o v a r i o u s p r o t e i n s .
V a r i o u s proteins w e r e i n c u b a t e d with 5 0 µM F P I X , e l e c t r o p h o r e s e d o n S D S - P A G E a n d stained for F P I X ( u p p e r p a n e l ) a n d with C o o m a s s i e ( l o w e r p a n e l ) . L a n e designation: 1 - B S A ; 2 - c y t o c h r o m e c o x i d a s e ; 3 - h e x o k i n a s e ; 4 - alcohol d e h y d r o g e n a s e ; 5 - G S H reductase; 6 - triose p h o s p h a t e i s o m e r a s e ; 7 - a-glycerophosphate dehytlrogena.se; 8 - mix
ture o f all p r o t e i n s i n c u b a t e d with 5 0 uM FPIX; 9 - mixture o f all pro
teins with 3 5 0 uM F P I X .
C o o m a s s i e stained gel did not bind FPIX at all. W h e n all proteins w e r e m i x e d together, proteins with l o w e r affinity for FPIX such as BSA (lane f ) , c y t o c h r o m e C o x i d a s e (lane 2 ) , and h e x o k i n a s e (lane 3 ) , c o u l d not b e s e e n at l o w FPIX c o n c e n t r a t i o n (lane 8 ) . But they are detectable w h e n FPIX concentration w a s increased (lane 9 ) . T h e s e results clearly indicate that FPIX binds to s o m e proteins with higher affinity than to others, a n d that t h e affinity o f b i n d i n g at s u b - s a t u r a t i n g c o n c e n t r a t i o n s determines the partition o f FPIX bet
w e e n the different p r o t e i n s that are present. This c o n c l u s i o n pertains to the differential binding o f FPIX to parasite proteins following the treatment o f intact cells with CQ or AQ.
DISCUSSION
W
e previously demonstrated that treatment o f infected cells with CQ a n d A Q , but not Q or M Q , results in the accumulation o f FPIX in the m e m b r a n e fraction in correlation with parasite killing (Ginsburg et al., 1 9 9 8 ; Zhang et al, 1 9 9 9 ) . W es u b s e q u e n t l y s h o w e d that the accumulation c o u l d b e due to the inhibition o f G S H - d e p e n d e n t degradation o f FPIX by CQ and AQ (Famin et al, 1999). Since these drugs, and to a m u c h lesser extent Q a n d M Q , inhibit the degradation o f B S A - b o u n d FPIX b y G S H (Famin et al. 1 9 9 9 ) , w e s u s p e c t e d that u n d e g r a d e d FPIX could also affect protein ( e n z y m e ) - d e p e n d e n t p r o c e s s e s . T h e binding o f FPIX c a n have physiological signifi
c a n c e , as it is k n o w n to inhibit several erythrocytic e n z y m e s ( Z e r e z et al, 1 9 8 7 ) and to regulate protein synthesis through its binding to heme-regulated inhi
bitor o f eukaryotic initiation factor 2 subunit a ( e l F - 2 a ; Surolia & P a d m a n a b a n , 1 9 9 1 ) . Here, w e h a v e clearly s h o w n that treatment o f infected cells with C Q or AQ c a u s e s an increase o f FPIX association with various parasite proteins in a c o n c e n t r a t i o n - d e p e n d e n t manner. Contrary to our previous suggestion that most of the FPIX g e n e r a t e d b y h e m o g l o b i n digestion exits the food v a c u o l e to b e d e g r a d e d b y GSH in the para
site's cytosol (Ginsburg et al, 1998), it has b e e n recently s h o w n the vast majority o f FPIX is biomineralized into h e m o z o i n (Egan et al, 2 0 0 2 ) . Although this conforms to the lack o f FPIX association with parasite proteins in a b s e n c e o f drug. However, inhibition o f FPIX b i o - mineralization by CQ or AQ could allow the exit o f free FPIX from the food v a c u o l e a n d its association with the m e m b r a n e s o f the infected cells ( Z h a n g et al, 1 9 9 9 ) . This increase w a s found to parallel parasite killing w h e r e the drugs w e r e used at the s a m e c o n c e n trations ( F a m i n & Ginsburg, 2 0 0 2 ) . Q a n d M Q did not c a u s e such an increase although they killed the para
sites. T h e latter two drugs also failed to i n d u c e a c c u mulation o f u n d e g r a d e d FPIX in the m e m b r a n e frac
tion o f drug-treated infected cells (Ginsburg et al, 1998; Zhang et al, 1 9 9 9 ) p r o b a b l y due to their failure to inhibit the degradation o f m e m b r a n e - and BSA-asso- ciated FPIX b y GSH ( F a m i n et al, 1 9 9 9 ) . B o t h CQ and AQ w e r e effective in these p r o c e s s e s . T h e s e results u n d e r s c o r e again that the m e c h a n i s t i c details o f the antimalarial action for CQ a n d AQ differ from Q and MQ, although all these drugs contain the aminoqui- n o l i n e n u c l e u s (Merkli & Richie, 1 9 8 0 ; Lambros &
Notsch, 1984; K n o w l e s et al, 1 9 8 4 ; W e b s t e r et al, 1985a, b; Ward et a l . , 1 9 9 5 ) .
An alternative m e c h a n i s m for FPIX destruction has b e e n suggested (Loria et al, 1 9 9 9 ) , w h e r e b y H202 g e n e r a t e d during the oxidation o f o x y - h e m o g l o b i n to m e t h e m o g l o b i n in the food v a c u o l e destroys FPIX. At the s a m e time, FPIX reduces H202 due to its peroxi- dative capacity. B o t h p r o c e s s e s are inhibited b y C Q , but m u c h less s o by Q o r M Q . T h e w e a k n e s s e s o f this m e c h a n i s m have b e e n discussed at length e l s e w h e r e (Ginsburg & Krugliak, 1 9 9 9 ) , and it has b e e n recently s h o w n that parasites d e v e l o p well in CO-treated ery
throcytes (Monti et al, 2 0 0 2 ) w h e r e H202 is not e x p e c ted to b e p r o d u c e d by h e m o g l o b i n degradation. It has
46 P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0
Mémoire
FERRtPROTOPORPHIN I X B I N D I N G TO MALARIA PARASITE PROTEINS
b e e n s h o w n that H202 e n h a n c e s the antimalarial action o f CQ (Malhotra et al, 1 9 9 0 ) , but here w e have o b s e r ved that the c o m b i n e d treatment o f intact cells with CQ and H202 results in d e c r e a s e d binding o f FPIX to proteins. This could raise the question w h e t h e r drug- induced binding o f FPIX to parasite proteins is involved in CQ action.
In t h e p r e s e n t investigation w e h a v e s h o w n that various erythrocytic e n z y m e s w e r e inhibited in vitro to variable extents and by different m o d e s o f inhibition b y FPIX. T h e s a m e e n z y m e s , e x c e p t adenylate kinase, were also present in the parasite and similarly inhibited by F P I X . H o w e v e r , w h e n t h e a c t i v i t i e s o f t h e s e e n z y m e s w e r e measured after treatment o f infected cells with CQ, they w e r e only marginally affected, e x c e p t for 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e w h o s e activity in the parasite c o m p a r t m e n t was reduced by 4 0 %. It could b e suggested that drug treatment c o u l d down-regulate the expression o f this e n z y m e , but this s e e m s rather unlikely given the relatively long half-life o f h o u s e k e e p i n g e n z y m e s and the short time o f drug treatment. T h e inhibition o f 6-phosphogluconate dehy
d r o g e n a s e indicates that a k e y e n z y m e o f the antioxi
dant defense o f the parasite, this the s e c o n d e n z y m e in HMS, is affected by CQ exposure. This result concurs with our previous demonstration that CQ inhibits the HMS activity o f infected cells (Atamna et al, 1994). But such inhibition is also e x p e c t e d if much less GSH would b e oxidized w h e n the degradation o f FPIX is inhibited by CQ (Ginsburg et al, 1998; Famin et al, 1 9 9 9 ) . HMS activity provides NADPH that is used to reduce oxidized glutathione by glutathione reductase.
Since treatment o f intact infected cells with CQ never resulted in a decline in host cell e n z y m e activity, w e c o n c l u d e that free FPIX d o e s not reach the host cell c o m p a r t m e n t . W e verified this independently by e l e c trophoresis o f host cell cytosol and staining o f FPIX after drug treatment (data not s h o w n ) . Thus, binding to proteins or to m e m b r a n e s effectively retains all intact FPIX within the parasite.
T h e fact that s o m e parasite e n z y m e s can b e inhibited b y FPIX in vitro, but not w h e n tested after extraction from cells that have b e e n treated with CQ, can b e due either to the fact that not e n o u g h free FPIX is avai
lable for inhibition, or that association o f the e n z y m e with o t h e r cytosolic c o m p o n e n t s in the intact cell pro
tects it from the inhibitory effect o f FPIX. W e have estimated the total level o f non-specifically b o u n d FPIX in CQ treated cells to b e 156 pM. This is distri
buted a m o n g all FPIX binding proteins according to their relative affinity for the ligand, as has b e e n s h o w n h e r e for the differential binding o f FPIX to a random assortment o f proteins (Fig. 8 ) . That 6 - p h o s p h o g l u c o nate d e h y d r o g e n a s e was found to b e equally inhibited both in vitro and in extracts o f CQ-treated cells (Fig. 5 ) suggests that this e n z y m e has relatively high affinity
P a r a s i t e . 2 0 0 3 . 10. 3 9 - 5 0
for FPIX. At the other end o f the spectrum was 5-pyri- micline nucleotidase, which was the most sensitive to FPIX in parasite and erythrocyte extracts vitro, but was not affected by treatment with either CQ or Q (Fig. 6 ) . This e n z y m e may have a low affinity for FPIX that will b e competed-out by other proteins with higher affinity, or can b e protected from inhibition through associa
tion with other cytosolic proteins. Binding o f FPIX to these parasite enzymes is not likely to underlie the anti
malarial m o d e o f action o f CQ.
T h e s e c o n d approach for the identification o f parasite proteins that bind FPIX was achieved through the bin
ding o f parasite cytosolic proteins to hemin-agarose b e a d s , their separation by electrophoresis and subse
quent s e q u e n c i n g . Although the binding o f FPIX to these proteins may b e non-specific, they are singled- out from the multitude o f other proteins present in the parasite lysate. FPIX is an amphipathic m o l e c u l e that may indiscriminately affect protein conformation and h e n c e , its effect o n e n z y m e activity may not require specific binding to active or modulating sites. This approach has revealed four additional putative target enzymes: e n o l a s e , p h o s p h o g l y c e r a t e kinase, glyceral- dehyde 3-phosphate deydrogenase and L-lactate dehy
drogenase, all m e m b e r s o f the glycolytic pathway that is the sole generator o f ATP in both parasites and host cells (Sherman, 1979). Another glycolytic enzyme, aldo
lase, was identified by the first approach. G l u c o s e c o n s u m p t i o n was inhibited by CQ in dose- and stage- d e p e n d e n t m a n n e r ( Y a y o n et al., 1 9 8 3 ) , and since gly
colysis is the major consumer o f glucose, it is suggested that inhibition o f glycolytic e n z y m e s by FPIX could b e the c a u s e . O n the o t h e r hand, d e c r e a s e d g l u c o s e c o n s u m p t i o n could result from disruption o f ion and pH h o m e o s t a s i s (Lee et al, 1 9 8 8 ) due to permeabili- zation o f m e m b r a n e s by accumulation o f undegraded FPIX (Zhang et al, 1 9 9 9 ) . Parenthetically, a radioiodi- nated p h o t o r e a c t i v e a n a l o g u e o f c h l o r o q u i n e w a s s h o w n to bind specifically to L-lactate d e h y d r o g e n a s e o f P. falciparum (Menting et al, 1 9 9 7 ) . It could well b e that this binding is mediated by FPIX. However, it has b e e n recently s h o w n that CQ binds within the NADH binding p o c k e t o f the e n z y m e , acting as a competitive inhibitor for this critical glycolytic e n z y m e (Read et al. 1 9 9 9 ) . But the high Ki (1 m M ) indicates that such inhibition cannot mediate parasiticidal acti
vity.
H s p 7 0 and H s p 9 0 w e r e other parasite proteins that b o u n d to hemin-agarose b e a d s . Several Hsp's have b e e n identified in the Plasmodium falciparum g e n o m e and s o m e murine parasite s p e c i e s without assignment o f a definitive biological role. H s p 7 0 is e x p r e s s e d in sporozoite, erythrocytic and gametocyte stages (Kumar et al, 1993; K a p p e s et al, 1993) and has b e e n impli
cated in invasion o f merozoites into uninfected ery
throcytes (Tardieux et al. 1998). No allusion to func-
Mémoire 47
FAMIN O. & GINSBURG H.
tional interaction b e t w e e n H s p 7 0 and FPIX could b e found in the literature and it is doubtful that such asso
ciation would have an immediate c o n s e q u e n c e for parasite viability.
Hsp90 is o n e o f the most abundant chaperonins in the cytosol o f eukaryotic cells and is an obligate c o m p o nent o f fundamental cellular p r o c e s s e s such as hor
m o n e signaling and cell cycle control, steroid r e c e p tors, cell c y c l e kinases, and p53-mediated p r o c e s s e s ( s e e Scheibel & B u c h n e r , 1 9 9 8 for a review). A single g e n e (pfhsp86) e n c o d i n g
Hsp90
has b e e n found in the P. falciparum g e n o m e . It is constitutively e x p r e s s e d in both trophozoite and g a m e t o c y t e stage parasites ( B o n - nefoy et al, 1994). T h e pfhsp86 g e n e maps within a c h r o m o s o m e 7 s e g m e n t that is linked to CQ r e s p o n s e in a falciparum cross but its alleles could not predict CQ r e s p o n s e (Su & W e l l e m s , 1 9 9 4 ) . It may b e interesting to investigate if FPIX interferes with o n e or m o r e o f the various functions o f Hsp90 as a basis for C Q toxicity.
Elongation factor f a ( E F - α ) was also retrieved from parasite cytosol by its ability to bind FPIX. E F - l a is involved in translation o f mRNA, in cytoskeletal orga
nization, in activation o f the phosphatidylinositol-4 kinase in the regulation o f actin polymerization and in modulating the rate o f apoptosis (Duttaroy et al, 1998).
E F - l a is e n c o d e d by m o r e than o n e g e n e in the g e n o m e o f three malaria parasites, P. knowlesi, P. ber- ghei and P. falciparum ( V i k e n o o g et al., 1 9 9 8 ) . Inter
e s t i n g l y , E F - l a c o - s e g r e g a t e s with a C Q - r e s i s t a n t marker in P. chabaudi (Carlton et al, 1998). T h e mani
fold activities o f E F - l a could well b e affected by FPIX, but n o e v i d e n c e for that could b e found in the litera
ture.
G a m e t o c y t o g e n e s i s onset-specific protein ( P f g 2 7 / 2 5 ) is e x p r e s s e d early in the differentiation o f g a m e t o c y t e s (Alano et al, 1 9 9 1 ; Lobo et al, 1 9 9 4 ) . It was also retrieved b y binding to agarose-hemin beads. G a m e t o c y t o g e n e s i s is k n o w n to increase in r e s p o n s e to stress (Sinden et al., 1 9 9 6 ) . S o m e n o n - g a m e t o c y t o - cydal antimalarial drugs increase transmission o f para
sites to mosquitoes ( s e e Butcher, 1997 for review). Sub- curative c h e m o t h e r a p y with CQ increases g a m e t o c y t e production and infectivity to mosquitoes in the rodent malaria P. chabaucli and P. vinckei petteri in vivo ( B u c k ling et al., 1997; Gautret et al., 2 0 0 0 ) and g a m e t o c y togenesis in P. falciparum in vitro ( B u c k l i n g et al, 1 9 9 9 ) . Disruption o f infected cells following CQ treat
ment may release FPIX that could serve as a g a m e t o - c y t o g e n i c signal b y activating the factor. However, such binding has probably little or nothing to do with the inhibition o f parasite growth by C Q treatment.
In conclusion, CQ and A Q c a u s e the accumulation o f protein-bound FPIX in the parasite, and binding o f FPIX to several e n z y m e s involved in glycolysis may underlie the o b s e r v e d inhibition o f this m e t a b o l i c
pathway by CQ ( Y a y o n et al, 1 9 8 3 ) . Binding to o t h e r proteins is probably inconsequential for drug action.
W e have previously suggested (Ginsburg et al., 1 9 9 8 ) that the antimalarial action o f CQ (and A Q ) is mediated b y the accumulation o f FPIX in the m e m b r a n e fraction o f the infected cell, that leads to m e m b r a n e p e r m e a - bilization and the irreversible perturbation o f ion h o m e o s t a s i s and the eventual disruption o f cellular metabolism. T o elucidate w h i c h o f these alternative m o d e s o f inhibition is o f most c o n s e q u e n c e for para
site death requires further scrutiny.
ACKNOWLEDGEMENTS
This research (No. 1 8 7 / 9 8 ) was supported by a grant from the Israel S c i e n c e Foundation.
REFERENCES
A L A N O P., P R E M A W A N S A S., B R U C E M.C. & C A R T E R R. A stage spe
cific gene expressed at the onset of gametocytogenesis in Plasmodium falciparum. Molecular and Biochemical Para
sitology, 1 9 9 1 . 46, 8 1 - 8 8 .
A T A M N A H.. P A S C A R M O N A G . & G I N S B U R G H. Hexose-mono- phosphate shunt activity in intact Plasmodium falcipantm infected erythrocytes and in free parasites. Molecular and Biochemical Parasitology, 1 9 9 4 , 6 7 , 7 9 - 8 9 .
A T A M N A H. & G I N S B U R G H. Heme degradation in the presence of glutathione - A proposed mechanism to account for the high levels of non-heme iron found in the membranes of hemoglobinopathic red blood cells. Journal of Biological Chemistry, 1 9 9 5 . 2 7 0 , 2 4 8 7 6 - 2 4 8 8 3 .
B E U T L E R E. Red Cell Metabolism. A Manual of Biochemical
Methods. Grune & Stratton. New York, 1 9 8 4 .
B O N N E F O Y S., A T T A I . G . , L A N G S L E Y G . , T E K A I A F. & M E R C E R E A U - P U I J A L O N O. Molecular characterization of the heat shock protein 9 0 gene of the human malaria parasite Plasmo
dium falcipantm. Molecular and Biochemical Parasitology.
1 9 9 4 , 6 7 , 1 5 7 - 1 7 0 .
B R A Y P . G . , J A N N E H O., R A Y N E S K.J., M U N G T I I I N M., G I N S B U R G H.
& W A R D S.A. Cellular uptake of chloroquine is dependent
on binding to ferriprotoporphyrin IX and is independent of NHF. activity in Plasmodium falciparum. Journal of Cell Biology. 1 9 9 9 . 145. 3 6 3 - 3 7 6 .
B U C K L I N G A.G.J., T A Y L O R L.H., C A R L T O N J.M.R. & R E A D A.F.
Adaptive changes in Plasmodium transmission strategies following chloroquine chemotherapy. Proceedings of the Royal Society. London (Biology). 1 9 9 7 . 264. 5 5 3 - 5 5 9 . B U C K L I N G A., R A N F O R D - C A R T W R I G H T L.C.. M I L E S A. & R E A D A.F.
Chloroquine increases Plasmodium falcipantm gameto
cytogenesis in vitro. Parasitology, 1 9 9 9 , 118, 3 3 9 - 3 4 6 .
B U F C H E R G.A. Antimalarial drugs and the mosquito trans
mission of Plasmodium. International Journal of Parasi
tology, 1 9 9 7 , 27, 9 7 5 - 9 8 7 .
C A R L T O N J . , M A C K I N N O N M. & W A L L I K E R D . A chloroquine resis
tance locus in the rodent malaria parasite P l a s m o d i u m cha-
48- Mémoire P a r a s i t e , 2 0 0 3 , 10, 3 9 - 5 0
F E R R I P R O T O P O R P H I N IX F U N D I N G T O M A L A R I A P A R A S I T E P R O T E I N S
baudi. Molecular and Biochemical Parasitology, 1998, 93, 57-72.
D U T T A R O Y A., B O U R B E A U D., W A N G X.L. & W A N G F.. Apoptosis rate can be accelerated or decelerated by overexpression or reduction of the level of elongation factor-la. Experi
mental Cell Research, 1998, 238, 168-176.
E G A N T . J . . C O M B R I N C K J . M . , E G A N J . , H E A R N E G.R., M A R Q U E S H.M., N T E N T E N T S., SEWEL B . T . , S M I T H P . J . , T A Y L O R D., V A N S C H A L K W Y K D.A. & W A L D E N J . C . The fate of haem iron in the malaria parasite Plasmodium falciparum. Bioche
mical Journal. 2002. 365. 343-347.
F A M I N O., K R U G L I A K M. & G I N S B U R G H. Kinetics of inhibition of glutathione-mediated degradation of ferriprotopor- phyrin IX by antimalarial drugs. Biochemical Pharmaco
logy. 1999. 58, 59-68.
F A M I N O. & G I N S B U R G H. Differential effects of 4-aminoqui- noline-containing antimalarial drugs on hemoglobin diges
tion in Plasmodium falciparum-infected erythrocytes. Bio
chemical Pharmacology. 2002, 63. 393-398.
F R A N C I S R.T. Jr & B E C K E R R.R. Specific indication of homeo- proteins in polacrylamide gels using a double-staining pro
cess. Analytical Biochemistry. 1984, 136, 509-514.
F R A N C I S S . E . , S U L L I V A N D.J. Jr & GOLDBERG D . E . Hemoglobin metabolism in the malaria parasite Plasmodium falci
pantm. Annual Reviews in Microbiology. 1997, 51, 97-123.
G A U T R E T P., L A N D A U I., T A I L H A R D A T L., M I L T G E N F . , C O Q U E L I N F . , V O / . A T . , C H A B A U D A . G . & J A C Q U E M I N J . L . The effects of sub- curative doses of chloroquine on Plasmodium vinckei petteri gametocytes and on their infectivity to mosquitoes.
International Journal of Parasitology, 2000, 30, 1193-1198.
G I N S B U R G H.. F A M I N O., Z H A N G J.M. & K R U G L I A K M. Inhibition of glutathione-dependent degradation of heme by chlo
roquine and amodiaquine as a possible basis for their anti
malarial mode of action. Biochemical Pharmacology, 1998, 56, 1305-1313.
G I N S B U R G H. & K R U G L I A K M. Chloroquine - some open ques
tions on its antimalarial mode of action and resistance.
Drug Resistance Update, 1999, 2, 180-187.
G O L D B E R G D . E . & S L A T E R A.F. Plasmodium and the infected erythrocyte - the pathway of hemoglobin degradation in malaria parasite. Parasitology Today, 1992, 8, 280-283.
K A N A A N I J . & G I N S B U R G H. Metabolic interconnection between the human malarial parasite Plasmodium falciparum and its host erythrocyte: Regulation of A T P levels by means of an adenylate translocator and adenylate kinase. Journal of Biological Chemistry, 1989, 264, 3194-3199.
K A P P E S B . , S U E T T E R L I N B . W . , H O F E R - W A R B I N E K R., H U M A R R. &
F R A N K L I N R.M. T W O major phosphoproteins of Plasmo
dium falcipantm are heat shock proteins. Molecular and Biochemical Parasitology, 1993, 5 9 , 8 3 - 9 4 .
K N O W L E S G . , D A V I D S O N W . L . , JOLLEY D . & A L P E R S M.P. The rela
tionship between the in vitro response of Plasmodium fal
ciparum to chloroquine. quinine and mefloquine. Tran
sactions of the Royal Socity of Tropical Medicine and Hygiene. 1984. 78, 146-150.
K R U G L I A K M. & G I N S B U R G H. Studies on the antimalarial mode of action of quinoline-containing drugs: time-dependence and irreversibility of drug action, and interactions with
compounds that alter the function of the parasite's food vacuole. Life Sciences, 1991, 49, 1213-1219.
K U M A R N., N A G A S A W A H., S A C C I J.B. J R . S I N A B . J . , A I K A W A M..
A T K I N S O N C , U P A R A N U K R A W P., K U B I A K L . B . , A Z A D A . F . &
HOLINGDALE M.R. Expression of members of the heat-shock
protein-70 family in the exoerythrocytic stages of Plas
modium berghei and Plasmodium falciparum. Parasito
logy Research. 1993. 79. 109-113.
KEINER S., B R E U E R W . V . , G I N S B U R G II., A L L Y S.B. & C A B A N T -
C H I K Z.I. Characterization of permeation pathways in the
plasma membrane of human erythrocytes infected with early stages of Plasmodium falcipantm: association with parasite development. Journal of Cell Physiology, 1985,
125 521-527.
L A M B R O S C.J. & V A N D E R B E R G J.P. Synchronization of Plasmo
dium falciparum erythrocytic stages in culture, Journal of Parasitology. 1979. 65. 4 1 8 - 4 2 0 .
L A M B R O S C. & N O T S C I I I . D . Plasmodium falciparum: meflo
quine resistance produced in vitro. Bulletin of the World Health Organization. 1984, 62. 433-438.
L E E P.. Y E Z., V A N D Y K E K . & K I R K R . G . X-ray microanalysis of Plasmodium falcipantm and infected red blood cells:
effect of qinghaosu and chloroquine on potassium, sodium, and phosphorus composition. American Journal of Tro
pical Medicine and Hygiene. 1988, 39, 157-165.
Lot«) C.A., K O N I N G S R.N.H. & K U M A R N. Expression of early gametocyte-stage antigens Pfg27 and Pfs16 in synchronized gametocytes and non-gametocyte producing clones of Plasmodium falciparum. Molecular and Biochemical Para
sitology. 1994, 68, 151-154.
L O R I A P., M I L L E R S., F O L E Y M. & T I L L E Y L. Inhibition of the per- oxidative degradation of haem as the basis of action of chloroquine and other quinoline antimalarials. Bioche
mical Journal. 1999, 339, 363-370.
M A L H O T R A K . , S A L M O N D., LI. B. & V I L D E J . L . Potentiation of chloroquine activity against Plasmodium falciparum by the peroxidase-hydrogen peroxide system. Antimicrobial Agents and Chemotherapy. 1990. 34. 1981-1985.
M E N T I N G J . G . . T I L L E Y L., D E A D Y L . W . . Nc. K . . S I M P S O N R.J..
C O W M A N A . F . & F O L E Y M. The antimalarial drug, chloro
quine, interacts with lactate dehydrogenase from Plasmo
dium falciparum. Molecular and Biochemical Parasitology.
1997, 88. 215-224.
M E R K L I B. & RICHLE-: R.W. Studies on the resistance to single and combined antimalarials in the Plasmodium berghei mouse model. Acta Tropica. 1980, 37, 228-231.
M O N T I D., B A S I L I C O N., PARAPINT S., P A S I N I E., O L L I A R O P. & T A R A -
MELLI D. Does chloroquine really act through oxidative
stress ? FEHS Letters, 2002, 522, 3-5.
R E A D J . A . , W I L K I N S O N K . W . , T R A N T E R R., S E S S I O N S R.B. & B R A D Y
R.L. Chloroquine binds in the cofactor binding site of Plas
modium falciparum lactate dehydrogenase, fournal of Biological Chemistry. 1999, 274, 10213-10218.
S C H E I B E L T. & B U C H N E R J . The Hsp90 complex - a super-cha- perone machine as a novel drug target. Biochemical Phar
macology, 1998. 56. 675-682.
S H E R M A N I.W. Biochemistry of Plasmodium (Malarial Para
sites). Microbiological Reviews, 1979, 43, 453-493.
P a r a s i t e . 2 0 0 3 . 10. 3 9 - 5 0
Mémoire 49
