Molecular and Biochemical Parasitology, 45 ( 1991) 249-260
249
Elsevier MOLBIO 01487
Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies Marie A n n e Leriche and Jean Franqois D u b r e m e t z
Unitd 42 INSERM, Villeneuve d'Ascq, France (Received 8 August 1990; accepted 22 October 1990)
A subcellular fractionation procedure has been established to isolate the rhoptries of Toxoplasma gondii tachyzoites on a selfgenerating Percoll gradient. The rhoptry fraction also contains dense granules. Monoclonal antibodies have been raised against the fraction and used to identify major proteins in the organelles by immunoelectron microscopy and Western blotting. Six major rhoptry proteins (60.5 kDa, Pi 5.8; 55, 57, 59, 60 kDa, all of Pi over 8; 42 kDa, Pi 4.8) and 3 dense granule proteins (30 kDa; 28 kDa, Pi 7.5; 27 kDa, Pi 4.5) together with 5 other proteins of 57, 90, 120, 168, 220 kDa that have been located in the rhoptry area by indirect immunofluorescence have been identified. Key words: Toxoplasma gondii; Antigen; Monoclonal antibody; Rhoptry; Dense granule: Subcellular organelle; Subcellular fractionation
Introduction Host cell invasion by Sporozoa involves specialized organelles located in the apical part of the invasive stage. Three types of organelles, named rhoptries, micronemes and dense granules, have been distinguished by morphological criteria. Due to the importance of the invasion event in the life cycle of these parasites, the function of these organelles is being actively investigated in a number of genera, including Plasmodium, Toxoplasma, Eimeria and Sarcocystis. The primary goal of these studies is to identify the contents of the organelles as a prerequisite to functional studies. Most of the results reported so far have been obtained using monoclonal antibodies or an-
Correspondence (present) address: J.F. Dubremetz, U42 IN-
tibodies against recombinant proteins; these approaches do not provide complete information on the organelles' contents: the only way to fully characterize organelles is their isolation by subcellular fractionation. We have previously set up fractionation procedures for the isolation of micronemes and dense granules from Sarcocystis [1,2] and rhoptries and micronemes from Eimeria [3], which have provided some information on the contents and fate of these organelles. These procedures have now been modified and adapted to a model more suitable for experimental studies in vitro, i.e., Toxoplasma gondii. The present paper reports the isolation of a rhoptry-dense granule fraction from the tachyzoite stage of this organism and on the characterization of the major proteins contained in these organelles with monoclonal antibodies raised against the fraction.
SERM, 369, rue J. Guesde, 59650 Villeneuve d'Ascq, France.
Abbreviations: DTT, dithiothreitol; IFA, immunofluorescence assay; mAb, monoclonal antibody; NEPHGE, nonequilibrium pH gradient electrophoresis; NP-40, Nonidet P40; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2Delectrophoresis, 2-dimensional electrophoresis.
Materials and Methods Parasites. Tachyzoites of the RH strain of Toxoplasma gondii [4] were used throughout the study.
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
250 They were maintained routinely by intraperitoneal passage in Swiss mice.
oclonal antibodies was done by injection of hybridoma cells into pristan-primed BALB/c mice and collection of ascitic fluids.
Parasite culture. Mass production of tachyzoites was performed in vitro. Tachyzoites were grown in monolayers of Vero cells in 1-1 roller bottles in Dulbecco's MEM supplemented with 2% fetal calf serum. Parasites were harvested by scraping the monolayer at peak growth and purified by filtration on glass fibers [5].
Subcellularfractionation.
10 m purified tachyzoites were washed twice with homogenization buffer (TES: 250 mM sucrose/l mM EDTA/5 mM triethanolamine-HCl, pH 7.5) by centrifugation at 1250 z g for 10 min. They were resuspended in 20 ml TES and disrupted by French press homogenization at a pressure of 28 kg cm -2. Unbroken cells (about 5%), were sedimented at 2250 x g for 10 min. The resulting supernatant was referred to as the homogenate. All further centrifugations were performed on a Beckman L8 ultracentrifuge. The homogenate was centrifuged at 20 000 x gmax for 20 min in a SW28 rotor. The pellet was resuspended in TES and the suspension was adjusted to 30% Percoll (Pharmacia) at 3 x 108 cell equivalents ml -~, distributed in 10-ml polycarbonate tubes and centrifuged at 50 000 x gma× for 25 min in a 50Ti rotor (fixed angle rotor, 26°). The selfgenerated gradient was then collected from the bottom to the top of the tube into 14 fractions of 0.7 ml, numbered consecutively from 1 to 14. The fractions were then diluted with TES and centrifuged at 100000 x gmax for 90 min in the 50 Ti rotor. The organelle layer that sedimented on top of the Percoll pellet in each tube was collected in a minimal amount of TES and processed for further analysis.
Monoclonal antibody production.
Monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice immunized with the rhoptry fraction of T. gondii (as defined in Results). The fusion was performed according to Galfre et al. [6]. Screening of hybridomas was done by immunofluorescence assay (IFA) and by Western blotting of tachyzoite lysates (see below). Positive hybridomas were cloned by limit dilution. Mass production of mon-
lmmunofluorescence
assay. For hybridoma screening, purified RH tachyzoites were washed 3 times with phosphate-buffered saline (PBS: 150 mM NaC1/50 mM phosphate buffer, pH 7.4) and dried on standard IFA slides that were stored at -20° C. IFA was performed at 37°C after a 10 min fixation in cold acetone. The slides were incubated with monoclonal antibodies (1:100 dilutions of mouse ascitic fluid), washed and incubated in fluorescein conjugated rabbit anti mouse IgG antibodies.
Electron microscopy. For standard electron microscopy the material was fixed for 1 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, washed in the same buffer and post-fixed for 1 h in 1% osmium tetroxide in the same buffer. It was then dehydrated in ethanol and embedded in Epon. Embedded material was sectioned with a diamond knife and contrasted with uranyl acetate and lead hydroxide. The sections were observed with an Hitachi H600 electron microscope. Immunoelectron microscopy was performed on Lowicryl K4M embedded material as previously described [7,8].
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-PAGE was performed according to Laemmli [9] using 12, 10 or 8% separating gels. Mr markers (SDS HMW kit, Sigma; Low molecular weight markers, Pharmacia) were used for calibration. In some cases, dithiothreitol (DTT) was omitted from the SDS-PAGE sample buffer. Gels were stained with Coomassie Blue R or silver-stained [10].
Two dimensional electrophoresis. The procedure was derived from O'Farrell [11] and Duncan and Hershey [12]. Samples were dissolved in 0.3% SDS/3% Nonidet 1:'40 (NP 40)/2% ampholines (Servalyt, 3-10)/9.5 M urea. The first dimension was run in cylindrical isoelectrofocusing gels containing 3.5% acrylamide/2% NP-40/2% ampholines (Servalyt 3-10)/9.8 M urea at 800 V for 16 h. One gel was used for measuring the pH gradient by cutting the gel in 2-mm pieces, soak-
251
ing the pieces in distilled water and measuring the resulting pH. The second dimension was then run in SDS-PAGE as described above. Nonequilibrium 2D-electrophoresis (NEPHGE) [13] was performed as above, but the sample was applied on the acidic instead of basic side and isoelectrofocusing was run at 400 V for 6 h.
Western blotting. Proteins were transferred to nitrocellulose (200 mA, 1 h; see ref. 14) after SDSPAGE. The nitrocellulose strips were saturated for 30 min in 5% non-fat dry milk in 15 mM TrisHC1 pH 8/150 mM NaCI/0.05% Tween 20 buffer (TNT). They were then incubated in mAb (mouse ascitic fluid) diluted 1:250 or rabbit serum diluted I:1000 in TNT for 1 h. After washing, the strips were incubated with peroxidase-conjugated or alkaline phospbatase conjugated anti-mouse IgG (Nordic) or anti-rabbit IgG (Tago) diluted 1:1000
in T N T and stained with diaminobenzidine-H202 or BCIP-NBT. Results When analyzed by electron microscopy, the 20000 × gmax pellet of French press homogenate was found to contain pellicles, mitochondria, rhoptries and dense granules. Further separation was achieved in the self-generated Percoll gradient that was analyzed both by electron microscopy and by SDS-PAGE and Western blotting using a previously described rhoptry antigen as marker [7]. Both procedures showed a great enrichment in rhoptries in fraction 1, decreasing in fractions 2 and 3 (Figs. 1 and 2). Pooled fractions 1 and 2, which corresponded to the first 1.4 ml of a 10 ml gradient, will be referred to below as the rhoptry fraction. By comparison with density markers
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Fig. 1. Electron microscopic analysis of the rhoptry fraction defined in the results section. (a) General view. Bar = 1 lLm.(b) Detail showing longitudinal and transverse rhoptry sections. Bar = 1 /zm (arrows point at dense granule contaminants).
252 1
2
3
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b a Fig. 2. SDS-PAGE (a) and Western blotting (b) of the fractions separated on the Percoll gradient as described in the Materials and Methods section. Fractions were numbered from the bottom to the top of the tube (fraction numbers are given at the top of panel b). In (a), pairs of consecutive fractions were pooled: the rhoptry fraction corresponds to lane 1 (12% acrylamide, reducing conditions, silver stained). In (b) the blot was probed by mAb T3 4A7 [7]; the reactivity in fractions 9, 10, 11 is due to organelles trapped in zoite ghosts (8% acrylamide, reducing conditions). The apparent Mr (× 10-3) of standard proteins are given. beads (Pharmacia), the density of these organelles in Percoll was approximately 1.13 g]cm - 3 . Electron microscopy showed that fractions 4, 5 and 6 contained mostly mitochondria, whereas the upper fractions contained pellicle fragments and ghosts some of which had trapped organelles, which explains the detection of rhoptry antigens on Western blots of the upper fractions. Recentrifugation of fractions at 100 000 × gmax for 90 min allowed for removal of Percoll that sedimented under the biological material. Complete removal could only be achieved using a Sephacryl
S-1000 column according to Hjorth et al. [15]. However, this procedure was time consuming and led to an extensive loss of material; it was therefore abandoned. Rhoptries isolated by the procedure described above appeared swollen compared to the organelles in situ: the peduncle was short and the posterior part of the organelle tended to be spherical in shape (Fig. 1) with a diameter of 0.3-0.4 #m, whereas it is more elongated with a width of about 0.2 # m in situ [7]. The morphology was better preserved when removing Percoll by
253 Sephacryl S-1000, but no difference was found between the two procedures after electrophoretic analysis, which suggested that the morphological alteration did not alter the organelle contents significantly. The rhoptry fraction also contained homogeneous dense organelles resembling dense granules. Further studies showed that both organelles were found in the fraction and that their contents could be distinguished by mAbs.
Electrophoretic analysis of the rhoptry fraction. SDS-PAGE analysis of the fraction demonstrated a complex protein profile, dominated by a major group of polypeptides in the 50-60-kDa range
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Fig. 3. SDS-PAGE analysis of the rhoptry fraction in 12% (A) and 8% (B) acrylamide gels, in non reducing (a and b) and reducing (c) conditions, stained with Coomassie Blue (a) or silver-stained (b and c). Mr markers were run in reducing conditions (arrows point to the 50-60 kDa range).
(Fig. 3A and B). Other proteins, present in lower but significant amounts were found at 220, 170, 125, 120, 105, 45, 42, 30, 28 and 27 kDa. When analyzing unreduced samples, some differences were found in the high-Mr range, but the major bands did not change significantly (Fig. 3A; b,c).
Monoclonal antibody characterization of the major components of the fraction. Monoclonal antibodies raised against the fraction were screened by immunofluorescence on tachyzoites. Three major IFA patterns were obtained: one was unipolar, revealing a compact group of structures located on one side of the nucleus and occupying approximately ]/6 to 1/3 of the organism; a typical example of this pattern was illustrated by mAb T3 4A7. When used in immunoelectronmicroscopy, this antibody reacted exclusively with rhoptries, as shown by Sadak et al. [7]. All of the monoclonal antibodies showing that IFA pattern recognized the rhoptries in IEM, A second pattern was similar to the previous one but slightly more anterior, though not apical; none of the monoclonal antibodies giving this pattern reacted in IEM, and they were therefore described as 'anterior'. The third group of responses was a bipolar picture made up of dots distributed on both sides of the nucleus; typical of this pattern was mAb T52B4 that reacted exclusively with dense granules by IEM [9]; the other mAbs of this group also reacted with dense granules on IEM. mAbs giving rise to each of these patterns were analyzed on Western blots to identify the corresponding antigens (Fig. 4). In contrast to the blotting patterns of mAbs of the second and third type, most of the rhoptryspecific mAbs reacted with proteins in the 55-60kDa range that were rather difficult to distinguish from one another, although the slight differences observed in migration suggested that they were indeed different. 2D gel electrophoresis was used for their definitive individual characterization. Using this procedure allowed us to show that some of the major polypeptides of the 55-60-kDa range migrated to a very basic Pi and N E P H G E was needed in that case for identification; for these molecules, the actual Pi could not be determined but they were characterized by their relative migration compared by reacting successively the different mAbs on the same nitrocellulose sheet. The
254
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Fig. 4. Western blot characterization of mAbs raised against the rhoptry fraction after SDS-PAGE in 6% (a~l), 8% (e-j) and 12% (k-o) acrylamide in reducing conditions. (a) T5 2A7; (b) T5 4G1; (c) T5 4HI; (d) T5 1BI1; (e) T5 2A3; (f) T5 3E2; (g) T3 4A7; (h) T2 2H3; (i) T5 3D1; (j) T5 1All; (k) T5 3H12; (1) T3 1E5; (m) T6 2HI1; (n) T4 1F5; (o) T5 2B4.
data obtained by such an analysis are illustrated in Figs. 5 and 6 and summarized in Table I. Discussion
The present report is the first on the isolation of T. gondii subcellular organelles. Both electron microscopy and Western blotting of the rhoptry fraction showed that it was highly enriched in this organelle. Dense granules were the only recognizable contaminants, as shown by raising mAbs against the fraction, since electron microscopy could not clearly differentiate these organelles on transverse sections. Coomassie Blue staining of SDS-PAGE of the fraction showed that proteins in the 50~0-kDa range are present in high amount and strongly suggested the prominence of rhop-
tries in the fraction since these proteins are recognized by rhoptry-specific mAb. It is not clear whether the fraction we have obtained actually reflects the relative amount of both organelles in tachyzoites or whether dense granules are lost somewhere during the purification. Dense granules might be more sensitive to homogenization or only partly sedimented during the 20000 x gmax centrifugation. All attempts to separate the two types of organelles quantitatively or to purify dense granules have failed so far. The major proteins contained in rhoptries were identified as the intensely Coomassie Blue staining spots observed on 2D gels: they comprise the 60.5-kDa, Pi 5.8 ROP 1 and 4 proteins of 60 (ROP4), 59.5 (ROP 5), 59 (ROP 3) and 55 (ROP 2) kDa of Pi >8 that are separated by NEPHGE.
255
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30-
B Fig. 5. 2D-electrophoresis of the rhoptry fraction after silver (A) or Coomassie Blue (B) staining. Some proteins have been identified with mAbs on Western blots of gels identical to the ones presented here and are indicated: (a) T5 2A3; (b,c,d) T3 4A7; (e) T5 3E2; (f T5 3H12; (g) T5 2B4. Isoelectric focusing, 800 V, 16 h. T is the control one-dimensional analysis of the fraction; 60 and 30 are the Mr of major proteins of the fraction used as markers.
ROP 1 is the product of the ROP A gene, and corresponds to the rhoptry protein recognized by mAb Tg49 [16], described by Schwartzman as inhibiting a penetration-enhancing factor of T. gondii (J, Boothroyd, personal communication). Indeed, T5 2A3, T5 2C3 and Tg49 reacted with the same molecule on 2D-gels; this identity was
confirmed at the gene level by hybridization between a probe specific for the ROP A gene supplied by J. Boothroyd and a T. gondii genomic construct that expressed a recombinant protein antigenically similar to ROP 1 (Puijalon and Dubremetz, unpublished). ROP 2, 3 and 4 correspond to the family of rhoptry antigens described
256
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60-
30-
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C
B
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257 TABLE I Antibody
IFA
IEM
Mr
T5 T5 T5 T5
Antigen
[Ref.]
Anterior Anterior Anterior Anterior
Neg Neg Neg Neg
220 168 120 90
T5 2A3 and T5 2C3
Rhoptry
Rhoptries
60.5
PEF ROP 1
[16]
T3 4A7
Rhoptry
Rhoptries
55+59+60
>8
ROP 2,3,4
17]
T2 2H3
Rhoptry
Rhoptries
60
>8
ROP 4
[71
T4 2F8 and T5 2D1
Rhoptry
Rhoptries
55+59
>8
ROP 2,3
171
T5 3E2
Rhoptry
Rhoptries
59.5
>8
ROP 5
T5 1A11 and T4 3HI
Rhoptry
Neg
57
nd
T3 IE5 and T5 3H12
Rhoptry
Rhoptries
42
4.7
ROP 6
[18]
T6 2HI1 T5 1F5 T5 2B4
Bipolar Bipolar Bipolar
Dense granules Dense granules Dense granules
30 28 27
nd 7.8 4.8
GRA 3 GRA 2 GRA 1
[21] [8,20]
2A7 4GI 4H 1 1B 11
Pi 7.2-7.6 7.5 7.4 4.7 5.8
Antibody, monoclonal antibody identification; IFA, immunofluorescence pattern; IEM, immunoelectron microscopic localization; Mr, apparent Mr in SDS-PAGE, reducing conditions; Pi, isoelectric point measured on 2D-electrophoresis; nd, not done. b y S a d a k et al. [7]. R O P 5 has not been d e s c r i b e d before. K i m a t a and T a n a b e [17] have d e s c r i b e d a r h o p t r y antigen o f 66 k D a that c o u l d c o r r e s p o n d to one o f the p r o t e i n s d e s c r i b e d here (the d i s c r e p a n c y c o u l d o r i g i n a t e f r o m the fact that b o v i n e s e r u m a l b u m i n w h i c h is u s e d as a 6 7 - k D a Mr s t a n d a r d c o m i g r a t e s with the 6 0 - k D a r h o p t r y m o l e c u l e s in n o n - r e d u c i n g conditions). R O P 7 c o r r e s p o n d s to the 4 2 - k D a r h o p t r y p r o t e i n d e s c r i b e d b e f o r e [18]. Since w e h a v e a l r e a d y s h o w n that R O P 2, 3 and 4 share a n t i g e n i c d e t e r m i n a n t s and therefore belong to a f a m i l y , the q u e s t i o n is raised w h e t h e r the o t h e r p r o t e i n s that w e h a v e identified, or the m i n o r ones w h i c h are also f o u n d on N E P H G E in
the s a m e Mr range c o u l d also be related to the f a m i l y . W e n e e d to p u r i f y the proteins, to raise m o n o s p e c i f i c p o l y c l o n a l a n t i b o d i e s , and to c l o n e and s e q u e n c e the c o r r e s p o n d i n g genes to s o l v e this question. Interesting is the fact that h i g h l y b a s i c m o l e c u l e s are f o u n d in rhoptries since the interaction o f basic p r o t e i n s with m e m b r a n e s is a m o n g the h y p o t h e s e s p r o p o s e d for the m e c h a n i s m o f host cell i n v a s i o n b y S p o r o z o a . T h e higher-Mr m o l e c u l e s identified and l o c a t e d b y I F A anterior to the rhoptries c o u l d c o r r e s p o n d e i t h e r to r h o p t r y p e d u n c l e antigens as d e s c r i b e d in Plasmodiumfalciparum [ 19], or to m i c r o n e m e antigens a l t h o u g h these o r g a n e l l e s were not f o u n d
+___
Fig. 6. NEPHGE of the rhoptry fraction (A) and Western blots of gels identical to A (B to E) probed with mAbs. B was probed with T5 3E2; C was probed with T3 4A7; D was probed with T4 2F8 (c,d), developed and then probed with T5 2C3 (a, and several degradation products of the PEF protein which is highly sensitive to degradation; see also ref. 16); E was probed with T5 3E2 (e), developed, probed with T3 4A7 (b,c,d), developed, and probed with T5 2A3 (a). Electrophoretic conditions for first dimension, 400 V, 6 h. Panels B, C, D and E were originally of the same size as A but were reduced for convenience.
258 in the fraction since they were not s e d i m e n t e d at 20 000 × gma× × 20 min. T h e s e proteins need to be i n v e s t i g a t e d further. T h r e e d e n s e g r a n u l e p r o t e i n s were also identified. C o m i g r a t i o n and identical Pi s h o w e d that the 2 7 - k D a p r o t e i n c o r r e s p o n d e d to the protein c l o n e d by C e s b r o n - D e l a u w et al. [20]. T h e 2 8 - k D a p r o tein is p r o b a b l y identical to the antigen d e s c r i b e d b y S i b l e y and S h a r m a [21] and p a r t l y s e q u e n c e d b y Prince et al. [22]. T h e 3 0 - k D a protein d o e s not c o m i g r a t e with the 3 2 - k D a p r o t e i n d e s c r i b e d by S i b l e y and K r a h e n b u h l ( m A b 1G5; ref. 23), as s h o w n b y c o m p a r i n g m A b T62H1 1 and m A b 1G5 on the s a m e W e s t e r n blot (D. S i b l e y , personal c o m m u n i c a t i o n ) . T h e isolation o f o r g a n e l l e s is an i m p o r t a n t step t o w a r d s the e l u c i d a t i o n o f their function. In the p r e s e n t case, it has also s h o w n h o w c o m p l e x the contents o f these o r g a n e l l e s were and therefore h o w difficult it will be to a s s i g n a function to the i n d i v i d u a l c o m p o n e n t s identified. A s i m i l a r situation occurs in P. falciparum w h e r e an i n c r e a s i n g v a r i e t y o f r h o p t r y p r o t e i n s are d e s c r i b e d . P r e l i m i nary i n v e s t i g a t i o n s have also s h o w n a novel lipid c o m p o s i t i o n o f the fraction ( F o u s s a r d , L e r i c h e and D u b r e m e t z , s u b m i t t e d for p u b l i c a t i o n ) , that m a y also be i m p o r t a n t in the i n v a s i o n process. A t t e m p t s at i n v e s t i g a t i n g f u n c t i o n a l activities o f the fraction ( P E F activity, i n h i b i t i o n o f i n v a s i o n by specific a n t i b o d i e s , e n z y m e activities) have been u n s u c c e s s f u l so far. M o r e w o r k is needed; the p r e s e n t report d e s i g n a t e s m a j o r targets for future research and suggests that m i n o r c o m p o n e n t s are also p r e s e n t and n e e d further investigation.
Acknowledgements T h e authors are i n d e b t e d to C. A n s e l , I. Briche, A. L o y e n s , M. M o r t u a i r e and N. V a n p o u i l l e for e x p e r t assistance. T h a n k s are due to J. B o o t h r o y d , M.F. C e s b r o n - D e l a u w , D. S i b l e y and J. S c h w a r t z m a n for e x c h a n g i n g p r o b e s and c o m m u n i c a t i n g u n p u b l i s h e d results. T h e s t i m u l a t i n g interest o f O. P u i j a l o n is g r a t e f u l l y a c k n o w l e d g e d . M . A . L e r i c h e was the r e c i p i e n t o f a d o c t o r a l f e l l o w s h i p f r o m M i n i s t e r e de la R e c h e r c h e et de la T e c h n o l o g i e . This w o r k was s u p p o r t e d by I N S E R M and C N R S .
References 1 Dubremetz, J.F. and Dissous, C. (1980) Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia). Mol. Biochem. Parasitol. 1, 279-289. 2 Entzeroth, R., Dubremetz, J.F., Hodick, D. and Ferreira, E. (1986) Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa). Eur. J. Cell Biol. 41, 182-188. 3 Dubremetz, J.F., Ferreira, E. and Dissous, C. (1989) Isolation and partial characterization of rhoptries and micronemes from Eimeria nieschulzi zoites (Sporozoa, Coccidia). Parasitol. Res. 75, 449M-54. 4 Sabin, A.B. (1941) Toxoplasmic encephalitis in children. J. Am. Med. Assoc. 116, 801-807. 5 Grimmwood, B.G., Hechemy, K. and Stevens, R.W. (1979) Toxoplasma gondii: Purification of trophozoites propagated in cell culture. Exp. Parasitol. 48, 282-286. 6 Galfre, G., Howe, S.C., Milstein, C., Butcher, G.W. and Howard, J.C. (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550-552. 7 Sadak, A., Taghy, Z., Fortier, B. and Dubremetz, J.F. (1988) Characterization of a family of rhoptry proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 29, 203-212. 8 Leriche, M.A, and Dubremetz, J.F. (1990) Exocytosis of dense granules after host-cell invasion by Toxoplasma gondii. Parasitol. Res. 76, 559-562. 9 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680~85. 10 Morrissey, J.H. (1981) Silver staining ['or proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117, 307-310. 11 O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4002-4021. 12Duncan, R. and Hershey, J.W.B. (1984) Evaluation of isoelectric focusing running conditions during twodimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis: Variation of gel patterns with changing conditions and optimized isoelectric focusing conditions. Anal. Biochem. 138, 144-155. 13 O'Farrell, P.Z., Goodman, H.M. and O'Farrell, P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142. 14 Towbin, H., Staehelin, T. and Gordon, J. (1978) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. 15 Hjorth, R. and Pertoft, H. (1982) Removal of Percoll from microsomal vesicles by gel filtration on Sephacryl S-1000 superfine. Biochim. Biophys. Acta 688, 1-4. 16 Schwartzmann, J.D. and Krug, E.C. (1989) Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries. Exp. Parasitol. 68, 74-82. 17 Kimata, I. and Tanabe, K. (1987) Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell. J. Cell Sci. 88, 231 239. 18 Dubremetz, J.F., Sadak, A,, Taghy, Z. and Fortier, B. (1987) Characterization of a 42-kDa rhoptry antigen of Toxoplasma gondii. In: Host-parasite Cellular and Molecu-
259 lar Interactions in Protozoal Infections, (Chang, K.-P. and Snary, D., eds.), pp. 365-369, Springer Verlag, Berlin. 19 Roger, N., Dubremetz, J.F., Delplace, P., Fortier, B., Tronchin, G. and Vernes, A. (1988) Characterization of a 225-kilodalton rhoptry protein of Plasmodiumfalciparum. Mol. Biochem. Parasitol. 27, 135-142. 20 Cesbron-Delauw, M.F., Guy, B., Torpier, G., Pierce, R.J., Lenzen, G., Cesbron, J.Y., Charif, H., Lepage, P., Darcy, F., Lecocq, J.P. and Capron, A. (1989) Molecular characterization of a 23 K major antigen secreted by Toxoplasma gondii. Proc. Natl. Acad. Sci. USA 86, 7537-7541.
21 Sibley, L.D. and Sharma, S.D. (1987) Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice. Infect. Immun. 55, 2137-2141. 22 Prince, J.B., Araujo, F.J., Remington, J.S., Burg, J.L., Boothroyd, J.C. and Sharrna, S.D. (1989) Cloning of cDNAs encoding a 28-kilodalton antigen of Toxoplasma gondii. Mol. Biochem. Parasitol. 34, 3-13. 23 Sibley, L.D. and Krahenbuhl, J.L. (1988) Modification of host cell phagosomes by Toxoplasma gondii involves redistribution of surface proteins and secretion of a 32-kDa protein. Eur. J. Cell Biol. 47, 81-87.
249
Elsevier MOLBIO 01487
Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies Marie A n n e Leriche and Jean Franqois D u b r e m e t z
Unitd 42 INSERM, Villeneuve d'Ascq, France (Received 8 August 1990; accepted 22 October 1990)
A subcellular fractionation procedure has been established to isolate the rhoptries of Toxoplasma gondii tachyzoites on a selfgenerating Percoll gradient. The rhoptry fraction also contains dense granules. Monoclonal antibodies have been raised against the fraction and used to identify major proteins in the organelles by immunoelectron microscopy and Western blotting. Six major rhoptry proteins (60.5 kDa, Pi 5.8; 55, 57, 59, 60 kDa, all of Pi over 8; 42 kDa, Pi 4.8) and 3 dense granule proteins (30 kDa; 28 kDa, Pi 7.5; 27 kDa, Pi 4.5) together with 5 other proteins of 57, 90, 120, 168, 220 kDa that have been located in the rhoptry area by indirect immunofluorescence have been identified. Key words: Toxoplasma gondii; Antigen; Monoclonal antibody; Rhoptry; Dense granule: Subcellular organelle; Subcellular fractionation
Introduction Host cell invasion by Sporozoa involves specialized organelles located in the apical part of the invasive stage. Three types of organelles, named rhoptries, micronemes and dense granules, have been distinguished by morphological criteria. Due to the importance of the invasion event in the life cycle of these parasites, the function of these organelles is being actively investigated in a number of genera, including Plasmodium, Toxoplasma, Eimeria and Sarcocystis. The primary goal of these studies is to identify the contents of the organelles as a prerequisite to functional studies. Most of the results reported so far have been obtained using monoclonal antibodies or an-
Correspondence (present) address: J.F. Dubremetz, U42 IN-
tibodies against recombinant proteins; these approaches do not provide complete information on the organelles' contents: the only way to fully characterize organelles is their isolation by subcellular fractionation. We have previously set up fractionation procedures for the isolation of micronemes and dense granules from Sarcocystis [1,2] and rhoptries and micronemes from Eimeria [3], which have provided some information on the contents and fate of these organelles. These procedures have now been modified and adapted to a model more suitable for experimental studies in vitro, i.e., Toxoplasma gondii. The present paper reports the isolation of a rhoptry-dense granule fraction from the tachyzoite stage of this organism and on the characterization of the major proteins contained in these organelles with monoclonal antibodies raised against the fraction.
SERM, 369, rue J. Guesde, 59650 Villeneuve d'Ascq, France.
Abbreviations: DTT, dithiothreitol; IFA, immunofluorescence assay; mAb, monoclonal antibody; NEPHGE, nonequilibrium pH gradient electrophoresis; NP-40, Nonidet P40; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2Delectrophoresis, 2-dimensional electrophoresis.
Materials and Methods Parasites. Tachyzoites of the RH strain of Toxoplasma gondii [4] were used throughout the study.
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
250 They were maintained routinely by intraperitoneal passage in Swiss mice.
oclonal antibodies was done by injection of hybridoma cells into pristan-primed BALB/c mice and collection of ascitic fluids.
Parasite culture. Mass production of tachyzoites was performed in vitro. Tachyzoites were grown in monolayers of Vero cells in 1-1 roller bottles in Dulbecco's MEM supplemented with 2% fetal calf serum. Parasites were harvested by scraping the monolayer at peak growth and purified by filtration on glass fibers [5].
Subcellularfractionation.
10 m purified tachyzoites were washed twice with homogenization buffer (TES: 250 mM sucrose/l mM EDTA/5 mM triethanolamine-HCl, pH 7.5) by centrifugation at 1250 z g for 10 min. They were resuspended in 20 ml TES and disrupted by French press homogenization at a pressure of 28 kg cm -2. Unbroken cells (about 5%), were sedimented at 2250 x g for 10 min. The resulting supernatant was referred to as the homogenate. All further centrifugations were performed on a Beckman L8 ultracentrifuge. The homogenate was centrifuged at 20 000 x gmax for 20 min in a SW28 rotor. The pellet was resuspended in TES and the suspension was adjusted to 30% Percoll (Pharmacia) at 3 x 108 cell equivalents ml -~, distributed in 10-ml polycarbonate tubes and centrifuged at 50 000 x gma× for 25 min in a 50Ti rotor (fixed angle rotor, 26°). The selfgenerated gradient was then collected from the bottom to the top of the tube into 14 fractions of 0.7 ml, numbered consecutively from 1 to 14. The fractions were then diluted with TES and centrifuged at 100000 x gmax for 90 min in the 50 Ti rotor. The organelle layer that sedimented on top of the Percoll pellet in each tube was collected in a minimal amount of TES and processed for further analysis.
Monoclonal antibody production.
Monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice immunized with the rhoptry fraction of T. gondii (as defined in Results). The fusion was performed according to Galfre et al. [6]. Screening of hybridomas was done by immunofluorescence assay (IFA) and by Western blotting of tachyzoite lysates (see below). Positive hybridomas were cloned by limit dilution. Mass production of mon-
lmmunofluorescence
assay. For hybridoma screening, purified RH tachyzoites were washed 3 times with phosphate-buffered saline (PBS: 150 mM NaC1/50 mM phosphate buffer, pH 7.4) and dried on standard IFA slides that were stored at -20° C. IFA was performed at 37°C after a 10 min fixation in cold acetone. The slides were incubated with monoclonal antibodies (1:100 dilutions of mouse ascitic fluid), washed and incubated in fluorescein conjugated rabbit anti mouse IgG antibodies.
Electron microscopy. For standard electron microscopy the material was fixed for 1 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, washed in the same buffer and post-fixed for 1 h in 1% osmium tetroxide in the same buffer. It was then dehydrated in ethanol and embedded in Epon. Embedded material was sectioned with a diamond knife and contrasted with uranyl acetate and lead hydroxide. The sections were observed with an Hitachi H600 electron microscope. Immunoelectron microscopy was performed on Lowicryl K4M embedded material as previously described [7,8].
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-PAGE was performed according to Laemmli [9] using 12, 10 or 8% separating gels. Mr markers (SDS HMW kit, Sigma; Low molecular weight markers, Pharmacia) were used for calibration. In some cases, dithiothreitol (DTT) was omitted from the SDS-PAGE sample buffer. Gels were stained with Coomassie Blue R or silver-stained [10].
Two dimensional electrophoresis. The procedure was derived from O'Farrell [11] and Duncan and Hershey [12]. Samples were dissolved in 0.3% SDS/3% Nonidet 1:'40 (NP 40)/2% ampholines (Servalyt, 3-10)/9.5 M urea. The first dimension was run in cylindrical isoelectrofocusing gels containing 3.5% acrylamide/2% NP-40/2% ampholines (Servalyt 3-10)/9.8 M urea at 800 V for 16 h. One gel was used for measuring the pH gradient by cutting the gel in 2-mm pieces, soak-
251
ing the pieces in distilled water and measuring the resulting pH. The second dimension was then run in SDS-PAGE as described above. Nonequilibrium 2D-electrophoresis (NEPHGE) [13] was performed as above, but the sample was applied on the acidic instead of basic side and isoelectrofocusing was run at 400 V for 6 h.
Western blotting. Proteins were transferred to nitrocellulose (200 mA, 1 h; see ref. 14) after SDSPAGE. The nitrocellulose strips were saturated for 30 min in 5% non-fat dry milk in 15 mM TrisHC1 pH 8/150 mM NaCI/0.05% Tween 20 buffer (TNT). They were then incubated in mAb (mouse ascitic fluid) diluted 1:250 or rabbit serum diluted I:1000 in TNT for 1 h. After washing, the strips were incubated with peroxidase-conjugated or alkaline phospbatase conjugated anti-mouse IgG (Nordic) or anti-rabbit IgG (Tago) diluted 1:1000
in T N T and stained with diaminobenzidine-H202 or BCIP-NBT. Results When analyzed by electron microscopy, the 20000 × gmax pellet of French press homogenate was found to contain pellicles, mitochondria, rhoptries and dense granules. Further separation was achieved in the self-generated Percoll gradient that was analyzed both by electron microscopy and by SDS-PAGE and Western blotting using a previously described rhoptry antigen as marker [7]. Both procedures showed a great enrichment in rhoptries in fraction 1, decreasing in fractions 2 and 3 (Figs. 1 and 2). Pooled fractions 1 and 2, which corresponded to the first 1.4 ml of a 10 ml gradient, will be referred to below as the rhoptry fraction. By comparison with density markers
47" ........ ~,
:v )
.
.
.
.
.
Fig. 1. Electron microscopic analysis of the rhoptry fraction defined in the results section. (a) General view. Bar = 1 lLm.(b) Detail showing longitudinal and transverse rhoptry sections. Bar = 1 /zm (arrows point at dense granule contaminants).
252 1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
10 11 12 13
q 67-
67-
30-
20-
b a Fig. 2. SDS-PAGE (a) and Western blotting (b) of the fractions separated on the Percoll gradient as described in the Materials and Methods section. Fractions were numbered from the bottom to the top of the tube (fraction numbers are given at the top of panel b). In (a), pairs of consecutive fractions were pooled: the rhoptry fraction corresponds to lane 1 (12% acrylamide, reducing conditions, silver stained). In (b) the blot was probed by mAb T3 4A7 [7]; the reactivity in fractions 9, 10, 11 is due to organelles trapped in zoite ghosts (8% acrylamide, reducing conditions). The apparent Mr (× 10-3) of standard proteins are given. beads (Pharmacia), the density of these organelles in Percoll was approximately 1.13 g]cm - 3 . Electron microscopy showed that fractions 4, 5 and 6 contained mostly mitochondria, whereas the upper fractions contained pellicle fragments and ghosts some of which had trapped organelles, which explains the detection of rhoptry antigens on Western blots of the upper fractions. Recentrifugation of fractions at 100 000 × gmax for 90 min allowed for removal of Percoll that sedimented under the biological material. Complete removal could only be achieved using a Sephacryl
S-1000 column according to Hjorth et al. [15]. However, this procedure was time consuming and led to an extensive loss of material; it was therefore abandoned. Rhoptries isolated by the procedure described above appeared swollen compared to the organelles in situ: the peduncle was short and the posterior part of the organelle tended to be spherical in shape (Fig. 1) with a diameter of 0.3-0.4 #m, whereas it is more elongated with a width of about 0.2 # m in situ [7]. The morphology was better preserved when removing Percoll by
253 Sephacryl S-1000, but no difference was found between the two procedures after electrophoretic analysis, which suggested that the morphological alteration did not alter the organelle contents significantly. The rhoptry fraction also contained homogeneous dense organelles resembling dense granules. Further studies showed that both organelles were found in the fraction and that their contents could be distinguished by mAbs.
Electrophoretic analysis of the rhoptry fraction. SDS-PAGE analysis of the fraction demonstrated a complex protein profile, dominated by a major group of polypeptides in the 50-60-kDa range
a
b
c
a b c iiiiiiii
67.
2 0 5 - iiii:iiiiiiii;:
45.
11630
.... 94-
~,i~iiiiiiil,il, 6720-
!~i~!'i ~i,i ~i !i~i~
14_ 45 -
A
B
Fig. 3. SDS-PAGE analysis of the rhoptry fraction in 12% (A) and 8% (B) acrylamide gels, in non reducing (a and b) and reducing (c) conditions, stained with Coomassie Blue (a) or silver-stained (b and c). Mr markers were run in reducing conditions (arrows point to the 50-60 kDa range).
(Fig. 3A and B). Other proteins, present in lower but significant amounts were found at 220, 170, 125, 120, 105, 45, 42, 30, 28 and 27 kDa. When analyzing unreduced samples, some differences were found in the high-Mr range, but the major bands did not change significantly (Fig. 3A; b,c).
Monoclonal antibody characterization of the major components of the fraction. Monoclonal antibodies raised against the fraction were screened by immunofluorescence on tachyzoites. Three major IFA patterns were obtained: one was unipolar, revealing a compact group of structures located on one side of the nucleus and occupying approximately ]/6 to 1/3 of the organism; a typical example of this pattern was illustrated by mAb T3 4A7. When used in immunoelectronmicroscopy, this antibody reacted exclusively with rhoptries, as shown by Sadak et al. [7]. All of the monoclonal antibodies showing that IFA pattern recognized the rhoptries in IEM, A second pattern was similar to the previous one but slightly more anterior, though not apical; none of the monoclonal antibodies giving this pattern reacted in IEM, and they were therefore described as 'anterior'. The third group of responses was a bipolar picture made up of dots distributed on both sides of the nucleus; typical of this pattern was mAb T52B4 that reacted exclusively with dense granules by IEM [9]; the other mAbs of this group also reacted with dense granules on IEM. mAbs giving rise to each of these patterns were analyzed on Western blots to identify the corresponding antigens (Fig. 4). In contrast to the blotting patterns of mAbs of the second and third type, most of the rhoptryspecific mAbs reacted with proteins in the 55-60kDa range that were rather difficult to distinguish from one another, although the slight differences observed in migration suggested that they were indeed different. 2D gel electrophoresis was used for their definitive individual characterization. Using this procedure allowed us to show that some of the major polypeptides of the 55-60-kDa range migrated to a very basic Pi and N E P H G E was needed in that case for identification; for these molecules, the actual Pi could not be determined but they were characterized by their relative migration compared by reacting successively the different mAbs on the same nitrocellulose sheet. The
254
a
b
c
d
e f g h i
j
k Imno
205205-i +
| 116-
116-
4594-
11+
9467-
'[-|
30-
"+
2045-
Fig. 4. Western blot characterization of mAbs raised against the rhoptry fraction after SDS-PAGE in 6% (a~l), 8% (e-j) and 12% (k-o) acrylamide in reducing conditions. (a) T5 2A7; (b) T5 4G1; (c) T5 4HI; (d) T5 1BI1; (e) T5 2A3; (f) T5 3E2; (g) T3 4A7; (h) T2 2H3; (i) T5 3D1; (j) T5 1All; (k) T5 3H12; (1) T3 1E5; (m) T6 2HI1; (n) T4 1F5; (o) T5 2B4.
data obtained by such an analysis are illustrated in Figs. 5 and 6 and summarized in Table I. Discussion
The present report is the first on the isolation of T. gondii subcellular organelles. Both electron microscopy and Western blotting of the rhoptry fraction showed that it was highly enriched in this organelle. Dense granules were the only recognizable contaminants, as shown by raising mAbs against the fraction, since electron microscopy could not clearly differentiate these organelles on transverse sections. Coomassie Blue staining of SDS-PAGE of the fraction showed that proteins in the 50~0-kDa range are present in high amount and strongly suggested the prominence of rhop-
tries in the fraction since these proteins are recognized by rhoptry-specific mAb. It is not clear whether the fraction we have obtained actually reflects the relative amount of both organelles in tachyzoites or whether dense granules are lost somewhere during the purification. Dense granules might be more sensitive to homogenization or only partly sedimented during the 20000 x gmax centrifugation. All attempts to separate the two types of organelles quantitatively or to purify dense granules have failed so far. The major proteins contained in rhoptries were identified as the intensely Coomassie Blue staining spots observed on 2D gels: they comprise the 60.5-kDa, Pi 5.8 ROP 1 and 4 proteins of 60 (ROP4), 59.5 (ROP 5), 59 (ROP 3) and 55 (ROP 2) kDa of Pi >8 that are separated by NEPHGE.
255
÷
,-
r
Q ! Q,
0
m
s
60-
W o
Q I
Q Q
t
a i
60-
30-
B Fig. 5. 2D-electrophoresis of the rhoptry fraction after silver (A) or Coomassie Blue (B) staining. Some proteins have been identified with mAbs on Western blots of gels identical to the ones presented here and are indicated: (a) T5 2A3; (b,c,d) T3 4A7; (e) T5 3E2; (f T5 3H12; (g) T5 2B4. Isoelectric focusing, 800 V, 16 h. T is the control one-dimensional analysis of the fraction; 60 and 30 are the Mr of major proteins of the fraction used as markers.
ROP 1 is the product of the ROP A gene, and corresponds to the rhoptry protein recognized by mAb Tg49 [16], described by Schwartzman as inhibiting a penetration-enhancing factor of T. gondii (J, Boothroyd, personal communication). Indeed, T5 2A3, T5 2C3 and Tg49 reacted with the same molecule on 2D-gels; this identity was
confirmed at the gene level by hybridization between a probe specific for the ROP A gene supplied by J. Boothroyd and a T. gondii genomic construct that expressed a recombinant protein antigenically similar to ROP 1 (Puijalon and Dubremetz, unpublished). ROP 2, 3 and 4 correspond to the family of rhoptry antigens described
256
÷
60-
30-
A
C
B
c\
• a e
d/"
D
E
257 TABLE I Antibody
IFA
IEM
Mr
T5 T5 T5 T5
Antigen
[Ref.]
Anterior Anterior Anterior Anterior
Neg Neg Neg Neg
220 168 120 90
T5 2A3 and T5 2C3
Rhoptry
Rhoptries
60.5
PEF ROP 1
[16]
T3 4A7
Rhoptry
Rhoptries
55+59+60
>8
ROP 2,3,4
17]
T2 2H3
Rhoptry
Rhoptries
60
>8
ROP 4
[71
T4 2F8 and T5 2D1
Rhoptry
Rhoptries
55+59
>8
ROP 2,3
171
T5 3E2
Rhoptry
Rhoptries
59.5
>8
ROP 5
T5 1A11 and T4 3HI
Rhoptry
Neg
57
nd
T3 IE5 and T5 3H12
Rhoptry
Rhoptries
42
4.7
ROP 6
[18]
T6 2HI1 T5 1F5 T5 2B4
Bipolar Bipolar Bipolar
Dense granules Dense granules Dense granules
30 28 27
nd 7.8 4.8
GRA 3 GRA 2 GRA 1
[21] [8,20]
2A7 4GI 4H 1 1B 11
Pi 7.2-7.6 7.5 7.4 4.7 5.8
Antibody, monoclonal antibody identification; IFA, immunofluorescence pattern; IEM, immunoelectron microscopic localization; Mr, apparent Mr in SDS-PAGE, reducing conditions; Pi, isoelectric point measured on 2D-electrophoresis; nd, not done. b y S a d a k et al. [7]. R O P 5 has not been d e s c r i b e d before. K i m a t a and T a n a b e [17] have d e s c r i b e d a r h o p t r y antigen o f 66 k D a that c o u l d c o r r e s p o n d to one o f the p r o t e i n s d e s c r i b e d here (the d i s c r e p a n c y c o u l d o r i g i n a t e f r o m the fact that b o v i n e s e r u m a l b u m i n w h i c h is u s e d as a 6 7 - k D a Mr s t a n d a r d c o m i g r a t e s with the 6 0 - k D a r h o p t r y m o l e c u l e s in n o n - r e d u c i n g conditions). R O P 7 c o r r e s p o n d s to the 4 2 - k D a r h o p t r y p r o t e i n d e s c r i b e d b e f o r e [18]. Since w e h a v e a l r e a d y s h o w n that R O P 2, 3 and 4 share a n t i g e n i c d e t e r m i n a n t s and therefore belong to a f a m i l y , the q u e s t i o n is raised w h e t h e r the o t h e r p r o t e i n s that w e h a v e identified, or the m i n o r ones w h i c h are also f o u n d on N E P H G E in
the s a m e Mr range c o u l d also be related to the f a m i l y . W e n e e d to p u r i f y the proteins, to raise m o n o s p e c i f i c p o l y c l o n a l a n t i b o d i e s , and to c l o n e and s e q u e n c e the c o r r e s p o n d i n g genes to s o l v e this question. Interesting is the fact that h i g h l y b a s i c m o l e c u l e s are f o u n d in rhoptries since the interaction o f basic p r o t e i n s with m e m b r a n e s is a m o n g the h y p o t h e s e s p r o p o s e d for the m e c h a n i s m o f host cell i n v a s i o n b y S p o r o z o a . T h e higher-Mr m o l e c u l e s identified and l o c a t e d b y I F A anterior to the rhoptries c o u l d c o r r e s p o n d e i t h e r to r h o p t r y p e d u n c l e antigens as d e s c r i b e d in Plasmodiumfalciparum [ 19], or to m i c r o n e m e antigens a l t h o u g h these o r g a n e l l e s were not f o u n d
+___
Fig. 6. NEPHGE of the rhoptry fraction (A) and Western blots of gels identical to A (B to E) probed with mAbs. B was probed with T5 3E2; C was probed with T3 4A7; D was probed with T4 2F8 (c,d), developed and then probed with T5 2C3 (a, and several degradation products of the PEF protein which is highly sensitive to degradation; see also ref. 16); E was probed with T5 3E2 (e), developed, probed with T3 4A7 (b,c,d), developed, and probed with T5 2A3 (a). Electrophoretic conditions for first dimension, 400 V, 6 h. Panels B, C, D and E were originally of the same size as A but were reduced for convenience.
258 in the fraction since they were not s e d i m e n t e d at 20 000 × gma× × 20 min. T h e s e proteins need to be i n v e s t i g a t e d further. T h r e e d e n s e g r a n u l e p r o t e i n s were also identified. C o m i g r a t i o n and identical Pi s h o w e d that the 2 7 - k D a p r o t e i n c o r r e s p o n d e d to the protein c l o n e d by C e s b r o n - D e l a u w et al. [20]. T h e 2 8 - k D a p r o tein is p r o b a b l y identical to the antigen d e s c r i b e d b y S i b l e y and S h a r m a [21] and p a r t l y s e q u e n c e d b y Prince et al. [22]. T h e 3 0 - k D a protein d o e s not c o m i g r a t e with the 3 2 - k D a p r o t e i n d e s c r i b e d by S i b l e y and K r a h e n b u h l ( m A b 1G5; ref. 23), as s h o w n b y c o m p a r i n g m A b T62H1 1 and m A b 1G5 on the s a m e W e s t e r n blot (D. S i b l e y , personal c o m m u n i c a t i o n ) . T h e isolation o f o r g a n e l l e s is an i m p o r t a n t step t o w a r d s the e l u c i d a t i o n o f their function. In the p r e s e n t case, it has also s h o w n h o w c o m p l e x the contents o f these o r g a n e l l e s were and therefore h o w difficult it will be to a s s i g n a function to the i n d i v i d u a l c o m p o n e n t s identified. A s i m i l a r situation occurs in P. falciparum w h e r e an i n c r e a s i n g v a r i e t y o f r h o p t r y p r o t e i n s are d e s c r i b e d . P r e l i m i nary i n v e s t i g a t i o n s have also s h o w n a novel lipid c o m p o s i t i o n o f the fraction ( F o u s s a r d , L e r i c h e and D u b r e m e t z , s u b m i t t e d for p u b l i c a t i o n ) , that m a y also be i m p o r t a n t in the i n v a s i o n process. A t t e m p t s at i n v e s t i g a t i n g f u n c t i o n a l activities o f the fraction ( P E F activity, i n h i b i t i o n o f i n v a s i o n by specific a n t i b o d i e s , e n z y m e activities) have been u n s u c c e s s f u l so far. M o r e w o r k is needed; the p r e s e n t report d e s i g n a t e s m a j o r targets for future research and suggests that m i n o r c o m p o n e n t s are also p r e s e n t and n e e d further investigation.
Acknowledgements T h e authors are i n d e b t e d to C. A n s e l , I. Briche, A. L o y e n s , M. M o r t u a i r e and N. V a n p o u i l l e for e x p e r t assistance. T h a n k s are due to J. B o o t h r o y d , M.F. C e s b r o n - D e l a u w , D. S i b l e y and J. S c h w a r t z m a n for e x c h a n g i n g p r o b e s and c o m m u n i c a t i n g u n p u b l i s h e d results. T h e s t i m u l a t i n g interest o f O. P u i j a l o n is g r a t e f u l l y a c k n o w l e d g e d . M . A . L e r i c h e was the r e c i p i e n t o f a d o c t o r a l f e l l o w s h i p f r o m M i n i s t e r e de la R e c h e r c h e et de la T e c h n o l o g i e . This w o r k was s u p p o r t e d by I N S E R M and C N R S .
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