Selective Labeling of Intracellular Parasite Proteins by Using Ricin (Protein Synthesis/Eimeria Tenella/Rhoo Cells) ANNE M

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2388-2392, March 1995 Biochemistry

Selective labeling of intracellular parasite proteins by using ricin (protein synthesis/Eimeria tenella/rhoO cells) ANNE M. GURNETT*, PAULA M. DULSKI*, SANDRA J. DARKIN RAT[RAY*, MARK J. CARRINGTONt, AND DENNIS M. SCHMATZ* *Department of Parasite Biochemistry and Molecular Biology, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065; and tDepartment of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, England Communicated by Edward M. Scolnick, Merck Research Laboratories, Rahway, NJ, August 26, 1994

ABSTRACT Studies focused on the synthesis by intracel- Antibodies have also been used to monitor the synthesis of lular parasites of developmentally regulated proteins have specific parasite proteins (5) in a variety of species by using been limited due to the lack of a simple method for selectively immunofluorescence, ELISA, and radioimmunoassay tech- labeling proteins produced by the parasite. A method has now niques (6, 7). Although useful, this approach is limited by the been developed in which ricin is employed to selectively inhibit number and specificity of available antisera. host-cell protein synthesis. Ricin is a heterodimer composed The limitations of these techniques led to a search for a of two subunits, a lectin and a glycosidase, and it binds to method to selectively label eukaryotic intracellular parasites. terminal galactose residues on the cell surface via the lectin. The toxic lectin from Ricinus communis, ricin, is a heterodimer Following endocytosis of the intact molecule, a disulfide bond that inhibits eukaryotic protein synthesis (8). It is composed of linking the two subunits is cleaved, and only the glycosidase a glycosidase A chain and a lectin B chain, which are linked by subunit enters the cytoplasm, where it inhibits cytoplasmic a disulfide bridge. The lectin binds to terminal galactose protein synthesis by catalyzing the cleavage of the 28S rRNA. residues found on the outside of most eukaryotic cells. Once Due to the loss of the receptor-binding lectin subunit, ricin bound, ricin is transported into the cell via receptor-mediated cannot permeate host-cell mitochondria or intracellular par- endocytosis. The glycosidase dissociates from the lectin, pos- asites, and, therefore, protein synthesis within these compart- sibly in the endoplasmic reticulum (9), and is released into the ments continues uninterrupted. This system has been used to cytosol. Without the lectin, the A chain cannot pass through a selectively label parasite proteins from Eimeria tenella and second membrane, resulting in its exclusion from mitochon- Toxoplasma gondii by using the avian cell line DU-24. In these dria and intracellular parasites. The glycosidase catalytically cells, mitochondrial protein synthesis was inhibited by using inactivates ribosomes by cleaving the N-glycosidic bond of a chloramphenicol. The use of the avian rhoO cell line DUS-3 specific adenine residue in the 28S ribosomal RNA (10). As provided an additional advantage, because these cells lack little as 1 ng of the toxin per ml can result in cell death (11). mitochondrial DNA. Therefore, those proteins radiolabeled The direct effect of the toxin is limited to protein synthesis, and with [35S]methionine/cysteine in ricin-treated, parasite- although other processes are inhibited indirectly, there is no infected rhoO cells are exclusively those of the intracellular effect on energy metabolism or oxidative phosphorylation in parasite. This technique should be applicable for studying the short term (11). protein synthesis by other intracellular parasites. Host-cell mitochondrial protein synthesis is not inhibited by ricin. It can be inhibited by chloramphenicol, but this drug may To understand host-parasite relationships and to identify both also prevent protein synthesis in parasite mitochondria and developmentally regulated and constitutively synthesized pro- could affect other parasite functions. To circumvent this teins in the eukaryotic intracellular parasite, techniques that problem, the avian cell line DUS-3 was used (12). DUS-3 cells, facilitate the study ofprotein synthesis and distinguish between also called rhoO cells, do not have a mitochondrial genome and parasite and host products are required. Proteins synthesized were selected because there would be no host mitochondrial by intracellular prokaryotes in eukaryotic hosts can be iden- protein synthesis in infected cells. The intracellular eukaryotic tified by labeling cells in the presence of emetine or cyclohex- parasites Eimeria tenella and T. gondii develop in this cell line imide (1, 2). These drugs specifically bind to 80S ribosomes and were used as model systems for this study. The sporozoites found in the eukaryotic host but not to 70S ribosomes found of E. tenella, an economically important parasite of chickens, in bacteria and mitochondria. However, in the case of intra- develop to first generation schizonts in the rhoO cells. Similarly, cellular protozoan parasites, this technique is not applicable, tachyzoites of T. gondii, a human pathogen, develop and since host and parasite are both eukaryotes. To address this release infectious progeny. problem, Pfefferkorn and Pfefferkorn (3) inhibited host-cell The work presented here demonstrates that ricin can selec- protein synthesis prior to infection and analyzed the effects on tively inhibit host-cell cytoplasmic protein synthesis, while growth of Toxoplasma gondii tachyzoites. This was achieved by having no effect on parasite protein synthesis. Furthermore, using temperature-sensitive, host-cell mutants or by pretreat- the use of rhoO cells as a host for these parasites eliminates any ment with irreversible protein-synthesis inhibitors. Although signal from host mitochondrial protein synthesis. The method inhibition of host-cell protein synthesis was achieved, incor- should be applicable to many intracellular parasite systems. poration of labeled amino acids into parasite proteins was low, and the host cells were viable only for a limited time following MATERIALS AND METHODS treatment. Another approach is to label infected cells and subsequently isolate the parasite free of host material (4). E. tenella Parasites. Chickens were infected orally with 7.5 x However, this can result in a loss of some parasite proteins and 104 E. tenella LS18 sporulated oocysts. The unsporulated contamination of the remainder with host cell products. oocysts were harvested from the ceca 7 days postinfection, purified according to the method of Schmatz et al. (13), and incubation at with continual The publication costs of this article were defrayed in part by page charge then sporulated by 29°C agitation payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: FBS, fetal bovine serum; TCA, trichloroacetic acid. 2388 Downloaded by guest on October 1, 2021 Biochemistry: Gurnett et al. Proc. Natl. Acad. Sci. USA 92 (1995) 2389 for 36 h. Sporozoites were prepared from sporulated oocysts cast in 2-mm i.d. glass tubes and used on the day of preparation. as described by Dulski and Turner (14). The gels were prerun at 200 V for 15 min, 300 V for 30 min, Host Cells and Parasite Growth. DU-24 (wild type) and and 400 V for 30 min. Samples were loaded onto the prerun DUS-3 (rhoO) chicken cell lines were a gift from R. Morais gels and covered with 20 ,ld of overlay buffer {8 M urea/1% (Department of Biochemistry, University of Montreal, Que- (vol/vol) Ampholines [a 4:1 mixture (vol/vol) of pH 5-7 and bec). Both cell lines were maintained at 37°C in Ham's F-12 pH 3.5-10 Ampholines]}, and the tubes were filled with 20mM medium supplemented with 10% heat-inactivated fetal bovine NaOH. Gels were run at 400 V for 15 h and 800 V for 2 h, serum (FBS), penicillin at 100 units/ml, streptomycin at 100 extruded into buffer 0 [10% (vol/vol) glycerol/2.3% SDS/ ,ug/ml, 10 mM Hepes at pH 7.4, and uridine at 50 ,tg/ml (12). 0.0625 M Tris-HCl, pH 6.8/5% (vol/vol) 2-mercaptoethanol], To initiate the cultures for labeling studies, 1.5 ml of a and incubated at room temperature for 30 min prior to suspension of 5 x 104 cells per ml was added to each well of electrophoresis in the second dimension. First-dimension gels a 12-well plate (12-well cluster dish, Costar). As E. tenella does were held in place on top of the second-dimension gels with 1% not develop at 37°C, the cells were maintained at 41°C for 24 agarose in buffer 0. Second-dimension SDS/polyacrylamide h prior to infection and throughout the remainder of the study. gels were run as described above. Freshly prepared E. tenella sporozoites were added to the host TCA Precipitation. Cells were labeled and washed as above, cells (1.5 x 105 sporozoites per well) and after 3 h the infected and 50 ,lI of 1 M NaOH was added to each well. After 10 min cells were washed to remove free sporozoites. Fresh Ham's at room temperature 1.5 ml of 20% TCA was added to each F-12 medium supplemented as before except that 2% heat- well, and the contents of each well were transferred to new inactivated FBS was added. The infected cultures were incu- tubes. Each well was washed with an additional 1.5 ml of 20% bated at 41°C for a maximum of 48 h with daily medium TCA and the two washes were pooled. Protein was allowed to changes. precipitate for 30 min on ice. Precipitated material was The RH strain of T. gondii was maintained in vitro on human collected on glass fiber filters (Whatman GF-C), which were foreskin fibroblast monolayers as previously described (15). then washed three times with 5 ml of 10% TCA and twice with Prior to infection of the avian rhoO cell line, T. gondii 10 ml of ethanol. The filters were air dried, and the radioac- tachyzoites were purified from the host fibroblasts by tritura- tivity was quantitated by scintillation counting. tion of the infected monolayer through a 27-gauge needle Autoradiography. Cells were handled as described above (three times) followed by filtration of the suspension through with the following changes in protocol. Avian cells were seeded polycarbonate membrane filters with 3.0-,um pores (16). T. into Lab-tek 4-chamber slides (Nunc) at 5 x 104 rhoO cells per gondii tachyzoites were harvested by centrifugation and resus- well or 2.5 x 104 wild-type cells per well in 0.5 ml of F-12 pended in Ham's F-12 medium containing 1% heat-inactivated medium containing 10% FBS. Infections were performed as FBS. To infect cultures for labeling studies, freshly purified T. described using 1 X 105 parasites per well. At 48 h after gondii tachyzoites were added to confluent monolayers of rhoO infection, cells were pretreated for 1.5 h with methionine- and cells (in 6-well plates) at a 3:1 ratio of parasites to host cells. cysteine-free medium containing ricin and then pulse-labeled The plates were incubated at 37°C for 3 h, after which infected for 30 min with [35S]methionine/cysteine at 3.75 ,uCi/ml. cells were washed to remove free tachyzoites. Fresh F-12 Radioactivity in the cultures was chased by adding 1 ml of F-12 medium containing 1% FBS was added, and the cultures were medium to the cultures for 15 min. The cells were then washed incubated for 48 h at 37°C with daily medium changes. twice with phosphate-buffered saline (PBS; 2.7 mM KCI/1.2 Metabolic Labeling. At various times after infection the mM KH2PO4/138 mM NaCl/8.1 mM Na2HPO4), and fixed in culture medium was removed and replaced with 1.5 ml of 1% glutaraldehyde in PBS for 15 min at room temperature. methionine- and cysteine-free minimal essential medium The fixed cell monolayers were rinsed in water, stained with (GIBCO) containing 1 mM pyruvate, 50 ,ug uridine per ml, 2% hematoxylin and eosin, and photographed by using light dialyzed FBS, and with or without 1.7 jig of ricin per ml. After microscopy. The same slides were coated with liquid emulsion 1.5 h, 100 ,ug of emetine per ml and 200 ,ug of chloramphenicol (Kodak NTB-2 nuclear track emulsion) that had been pre- per ml were added as indicated. After an additional 5 min, warmed to 45°C. Slides were air dried, exposed for 1 day at 4°C, [35S]methionine/cysteine Express labeling mixture (a1200 processed at 10°C in Kodak D-19 developer for 2.5 min, rinsed Ci/mmol; 1 Ci = 37 GBq; DuPont/NEN) was added to the in water, fixed in Kodak rapid fixer for 5 min, rinsed in water culture medium to a final concentration of 7.5 ,uCi/ml. Cells for 2 min, and then air dried. Slides were reexamined, and were labeled for 2-3 h, washed twice with 1.0 ml of F-12 previously photographed areas were located and rephoto- medium without supplements, and harvested into 150 [lI of SDS sample buffer (17) for one-dimensional PAGE or 100 ,ul Eimeria tenella + + +- - of isoelectric sample buffer {9.5 M ricin - -+-++- -t-- focusing urea/2% (vol/vol) emetine - + + + + Nonidet P-40, 2% (vol/vol) Ampholine [a 4:1 mixture (vol/ chloramphenicol - - + - + vol) of pH 5-7 and pH 3.5-10 Ampholines]/5% (vol/vol) Size 2-mercaptoethanol} (18) for two-dimensional PAGE. Alter- Standards natively, cells were washed in preparation for trichloroacetic Mrx 10-3 acid (TCA) precipitation. For the time course studies, cells &i. were infected and incubated at 41°C for specified times prior - 96 to labeling. Conditions for metabolic labeling and drug treat- - 68 - 45 ment of T. gondii-infected cultures were identical, except that A* Am 0 ".. [35S]methionine/cysteine was added to a concentration of 45 - 30 -4i. ,tCi/ml, by adding 0.25 ml of labeling mixture per well. -oi.- i..6 - 18 Gel Electrophoresis. SDS/7-20% polyacrylamide gradient - 14 gels were prepared according to the method of Laemmli (17) and either stained with Coomassie brilliant blue R-250 in 20% acetic acid (vol/vol) or prepared for fluorog- methanol/7% FIG. 1. Fluorograph of newly synthesized proteins separated on an raphy. Gels were fixed in methanol/acetic acid for 16 h, treated SDS/7-20% polyacrylamide gel. Avian wild-type cells either unin- with Enlighten (NEN) for 30 min, dried, and exposed to film fected or infected with E. tenella were labeled 48 h postinfection with (Fuji RX medical x-ray film) at -70°C. [35S]methionine/cysteine in the presence (+) or absence (-) of Isoelectric Focusing/Two-Dimensional Polyacrylamide various inhibitors as described in Materials and Methods. The positions Gels. Isoelectric focusing gels (pH range 3.5-10; ref. 18) were of molecular weight markers are shown at the right. Downloaded by guest on October 1, 2021 2390 Biochemistry: Gumett et al. Proc. Natl Acad Sci USA 92 (1995) A A B (- Mr x 10-3

- 96 - 68 I I - 45 PAGE PAGE

- 30

.o 18 - 14 l1 Lc L 1_I l_ C (-) D 1 2 u 3 4 CO B

Mrx 10-3 PAGE I PAGE -96 - 68 .45 30 18 FIG. 3. Two-dimensional gel fluorographs of newly synthesized 14 proteins derived from E. tenella-infected or uninfected rhoO cells that were labeled 48 h postinfection in the presence or absence of ricin. Equivalent amounts of TCA-precipitable radioactive material (25,000 cpm) were loaded on each gel. (A) Ricin-treated, infected rhoO cells. 1 2 " 3 4 co (B) Control uninfected rhoO cells. (C) Mixture of samples shown inA FIG. 2. (A) Fluorograph of newly synthesized proteins of E. and B. (D) Same as C but with labels to designate proteins as either tenella-infected or uninfected rhoO cells. Proteins were separated on an host-specific (H) or parasite-specific (P). IEF, isoelectric focusing SDS/7-20% polyacrylamide gel. Track 1, E. tenella-infected, ricin- dimension; PAGE, SDS/polyacrylamide gel electrophoresis dimen- treated cells, 120-h exposure; track 2, uninfected, ricin-treated cells, sion. 120-h exposure; track 3, E. tenella-infected control cells, 3-h exposure; track 4, uninfected control cells, 3-h exposure. Control cells were not with ricin resulted in the detection of other proteins, presum- treated with ricin. (B) Same gel as in A stained with Coomassie blue. ably derived from the parasite. Ricin-treated, E. tenella- std, molecular mass standards. infected wild-type cells were then exposed to chloramphenicol, resulting in the inhibition of host-cell mitochondrial protein graphed to allow a direct comparison of silver grains with synthesis. The synthesis of parasite proteins was unaffected by stained cells. chloramphenicol but inhibited by emetine (Fig. 1). When uninfected rhoO cells were treated with ricin, no RESULTS labeled proteins were detected (Fig. 2A), as these cells do not The level of ricin used in these experiments was optimized by possess a mitochondrial genome. However, when E. tenella- titration (data not shown). In the avian wild-type (DU-24) and infected rhoO cells were treated with ricin, labeled proteins rhoO (DUS-3) cell lines, 1.7 jig of ricin per ml was the minimal were detected, suggesting that this synthesis originated in the concentration required to achieve total inhibition of cytoplas- parasite. The parasite-derived protein synthesis was sensitive mic protein synthesis after a 90-min exposure. to emetine and resistant to chloramphenicol (data not shown). When wild-type, uninfected cells were preincubated with Infected and uninfected cultures labeled in the absence of ricin either ricin or emetine, an identical subset ofnewly synthesized were indistinguishable, even on a short exposure (Fig. 2). The proteins was labeled (Fig. 1). This synthesis was inhibited by Coomassie blue-stained gel (Fig. 2B) demonstrates that chloramphenicol, which is characteristic of protein synthesis roughly equivalent amounts of protein were loaded into each within mitochondria. When E. tenella-infected wild-type cells lane. were treated with emetine, the only protein synthesis detect- To confirm that labeled parasite proteins were qualitatively able was mitochondrial; however, treatment of the same cells different from the labeled host-cell proteins, two-dimensional

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,,£7 ^ FIG. 4. Ricin-treated, [35S]- methionine/cysteine-labeled, E. tenella-infected rhoO cells were fixed, stained, and photographed -10 prior to being coated with liquid *t photographic emulsion and processed for autoradiography. (A) * 4 0 Hematoxylin- and eosin-stained field of E. tenella-infected rhoO ...... :..:'As cells before being coated with ~b .#:-i;f A.j k. emulsion. (B) Same field as in A after autoradiography. (Bar = 10 ' * fZ . Am.) Downloaded by guest on October 1, 2021 ~~~~~~~~~.1= Biochemistry: Gurnett et al. Proc. Natl Acad Sci. USA 92 (1995) 2391

+ + Ricin labeled in the presence of emetine, while chloramphenicol has + Emetine no apparent effect, indicating that protein synthesis in sporo-

.- Chloramphenicol zoites is eukaryotic in nature (Fig. 5). Mitochondrial protein synthesis was not detected in emetine-treated sporozoites. E. tenella-infected rhoO cells pretreated with ricin were labeled at various times throughout the 48 h of schizont development. Constitutively synthesized and developmentally regulated parasite proteins were identified by comparing labeled sporozoite Mr x 10-3 proteins with those of the developing schizont (Fig. 6). In Fig. - 96 6F, examples of differentially regulated parasite proteins have MIv# been indicated, and these are described in the Conclusions. 68 Results with T. gondii-infected, ricin-treated rhoO cells were .. 45 qualitatively similar to those obtained for E. tenella-infected cells as shown in Fig. 7. This demonstrates that the method can be extended to other intracellular parasites. The T. gondii - 30 proteins labeled in the presence of ricin could be distinguished from both E. tenella proteins and host-cell proteins (data not *. 18 shown). - 14 CONCLUSIONS Sporozoites A comparison of protein synthesis in avian wild-type cells with FIG. 5. Free sporozoites were labeled with [35S]methionine/ protein synthesis in their rhoO counterparts when both cell cysteine in the presence of ricin, emetine, or chloramphenicol, and 2.2 types are labeled in the presence of the inhibitors emetine or x 106 sporozoites were loaded onto each lane of an SDS/poly- ricin clearly shows that protein synthesis occurs exclusively in acrylamide gel. Molecular weight markers are shown on the right. the wild-type cells (Figs. 1 and 2). The synthesis of proteins in the wild-type cells in the presence of the inhibitors is consistent polyacrylamide gel electrophoresis of the samples was per- with previous studies on proteins made within the mitochon- formed (Fig. 3). Protein synthesis by control, uninfected rhoO dria (19-21). Further evidence to support this is provided by host cells and ricin-treated, infected rhoO cells was analyzed experiments in which chloramphenicol, a known inhibitor of separately (Fig. 3 A and B) and together (Fig. 3 C and D). bacterial and mitochondrial protein synthesis, abolishes the Comparison of the panels showed that few, if any, parasite remaining incorporation into proteins (Fig. 1). proteins comigrated with those synthesized in the uninfected Metabolic labeling of parasite-infected, ricin-treated cells host cells. Fig. 3D is identical to Fig. 3C, except that proteins resulted in the selective labeling of the intracellular parasites, have been labeled as host-specific (H) or parasite-specific (P) as demonstrated by comparing proteins synthesized by in- to identify the origin of each protein in the mixture. fected cells with those synthesized by uninfected cells (Figs. 1, Parasite-specific protein labeling was confirmed by direct 2, and 7). Both avian wild-type and rhoO cells are capable of autoradiography (Fig. 4). Infected rhoO cells were fixed, supporting the intracellular development of certain stages of stained, and photographed. The cells were then coated with E. tenella and T. gondii; however, the rhoO cells offer a distinct photographic emulsion, exposed, and developed, and the same advantage over the wild-type cells, since there is no host areas were located and rephotographed. By using this method, mitochondrial protein synthesis. the areas of intense labeling (Fig. 4B) were shown to corre- Visual confirmation of the selective labeling of intracellular spond to mature stained schizonts (Fig. 4A). The appearance parasites was facilitated by the use of direct autoradiography of the schizonts exposed to the photographic emulsion was not of labeled rhoO cells infected with E. tenella. As shown in Fig. enhanced by the prestaining procedure (data not shown). 4, when a field of infected cells (Fig. 4A) was examined in a Extracellular sporozoites incorporated label in the presence culture treated with ricin, the silver grains were located over of ricin, suggesting that ricin is not effective against the the developing parasites, with minimal background labeling extracellular parasite (Fig. 5). Extracellular sporozoites are not over the host cells (Fig. 4B).

(-) (+) A B C Mrxl10-3 -

200m| 97.4 . 69' FIG. 6. Two-dimensional gel 46. fluorographs of newly synthe- :~~~~~~~~~~~~~~~~~~~~~...... sized 30~ parasite proteins. (A) Free 21.5 : sporozoites were labeled with

14.3 [35S]methionine/cysteine as in sporozoites - 12 hrs Fig. 5. [35S]Methionine/cysteine- D E PAGE labeled, ricin-treated, E. tenella- infected rhoO cells were labeled for 3 h at 12 h .~ ~~-...... ~... ~ ~~~~~~~~~~~ postinfection (B), 24 h postinfection (C), 36 h postinfection (D), and 48 h postinfection (E). (F) Same fluorograph shown in E, except that differentially regulated proteins are numbered from 1 to 7. Mo- lecular weight markers are shown at the left ofA. Downloaded by guest on October 1, 2021 2392 Biochemistry: Gurnett et al. Proc. Natl Acad Sci. USA 92 (1995)

Ricin those labeled in the free-living sporozoite. This indicates that + + + + + + Parasite the majority of proteins labeled in ricin-treated, infected host + + + + Emetine cells are synthesized by the parasite and not by the host cell in + response to parasitic infection. As ricin inhibits host-cell protein synthesis, it was expected that ricin would also inhibit protein synthesis in the free-living parasite; however, protein synthesis in E. tenella sporozoites was unaffected by ricin treatment (Fig. 5). The trilamellar membrane, which surrounds the sporozoite, may exclude ricin 10-3 from the cytoplasm. Alternatively, the terminal galactose - 96 receptor or the receptor-mediated endocytosis portion of the ricin-entry process may be absent or quiescent in this stage. Another possibility is that parasite protein synthesis is resistant to ricin, as has been shown for the protozoan Tetrahymena (23). If protein synthesis in Eimeria or, as postulated by Wilde et al. (23), in the protozoa in general is resistant to inhibition by ricin, these differential drug sensitivities may be useful in the - 30 design of future antiprotozoals. As with the intracellular parasite, mitochondrial protein synthesis was not detected in the sporozoite in the presence of emetine. If there is synthesis 18 in this compartment, it is below the limits of detection by our -14 methods. In conclusion, ricin selectively inhibits host-cell cytoplasmic 1 2 3 4 5 6 protein synthesis and facilitates selective labeling of proteins FIG. 7. Fluorograph of newly synthesized proteins separated on an synthesized by intracellular parasites. It has been used to SDS/7-20% polyacrylamide gel. Proteins were isolated from [35S]me- selectively label not only E. tenella and T. gondii as shown here, thionine/cysteine-labeled, ricin-treated, rhoO cells infected with T. but also Theileria parva in infected lymphocytes (M.J.C., gondii as described in Materials and Methods. Lanes 1 and 2, ricin- treated, uninfected rhoO cells; lanes 3 and 4, ricin-treated, infected unpublished data). The method can be used to identify stage- rhoO cells; and lanes 5 and 6, emetine- and ricin-treated, infected rhoO specific and constitutive proteins and should be generally cells. The positions of molecular weight markers are as shown. applicable to many intracellular parasites. Host-cell mitochondrial protein synthesis can be inhibited by chloramphenicol. By using the ricin/rhoO technique, it was found that intra- When appropriate, the use of host cells lacking a mitochon- cellular parasite protein synthesis in E. tenella (Fig. 1) and T. drial genome provides an additional advantage by eliminating gondii (Fig. 7) was inhibited by the eukaryotic protein synthesis the need for inhibitors of mitochondrial protein synthesis. inhibitor emetine but was unaffected by chloramphenicol, a prokaryotic protein synthesis inhibitor. This confirms the 1. Abshire, K. Z. & Neihardt, F. C. (1993) J. Bacteriol. 175, 3734-3743. eukaryotic nature of protein synthesis in E. tenella, as previ- 2. Brundage, R. A., Smith, G. A., Camilli, A., Theriot, J. A. & Portnoy, D. A. (1993) Proc. Natl. Acad. Sci. USA 90, 11890-11894. ously shown (22). Theoretically, parasite mitochondrial pro- 3. Pfefferkorn, E. R. & Pfefferkorn, L. C. (1981) Exp. Parasitol. 52, tein synthesis could occur in the presence of emetine, but there 129-136. was no detectable protein synthesis in the intracellular parasite 4. Sugimoto, C., Conrad, P. A., Ito, S., Brown, W. C. & Grab, D. J. under these conditions. It is not clear whether labeled parasite (1988) Acta Trop. 45, 203-216. mitochondrial proteins are absent, below the limits of detec- 5. Nantulya, V. M. (1991) Experientia 47, 142-145. 6. tion, or excluded from SDS/polyacrylamide gels. It is also Olson, J. (1990) Antimicrob. Agents Chemother. 34, 1435-1439. 7. Merli, A., Canessa, A. & Melioli, G. (1985) J. Clin. Microbiol. 21, possible that parasite mitochondrial protein synthesis is sen- 88-91. sitive to emetine. 8. Olsnes, S. & Pihl, A. (1982) in MolecularAction of Toxins and Viruses, To confirm that protein synthesis seen in ricin-treated, eds. Cohen, P. & van Heyningen, S. (Elsevier, Amsterdam), pp. infected rhoO cells was parasite derived, it was important to 51-105. demonstrate that these newly synthesized proteins were dis- 9. Pelham, H. R. B., Roberts, L. M. & Lord, J. M. (1992) Trends Cell tinct from those in the Biol. 2, 183-185. synthesized uninfected host cell. A 10. Endo, Y. & Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130. comparison of proteins synthesized by infected and uninfected 11. Lin, J.-Y., Liu, K., Chen, C.-C. & Tung, T.-C. (1971) Cancer Res. 31, cells and separated on one-dimensional SDS/polyacrylamide 921-924. gels (Fig. 2) shows distinct patterns of synthesis in the host and 12. Morais, R., Desjardins, P., Turmel, C. & Zinkewich-Peotti, K. (1988) the parasite. Further analysis using two-dimensional polyacryl- In Vitro Cell. Dev. Biol. 24, 649-658. amide gels (Fig. 3) shows that the origin of each protein can 13. Schmatz, D. M., Crane, M. S. J. & Murray, P. K. (1984) J. Protozool. 31, 181-183. be defined as host-specific or parasite-specific by comparing 14. Dulski, P. M. & Turner, M. J. (1988) Avian Dis. 32, 235-239. samples run individually with those run together. 15. Pfefferkorn, E. R. & Pfefferkorn, L. C. (1976) Exp. Parasitol. 39, During the development of intracellular parasites, proteins 365-376. are synthesized and turned over at different rates. Some 16. Sibley, L. D., Krahenbuhl, J. L., Adams, G. M. W. & Weidner, E. proteins are synthesized at a constant level in sporozoites and (1986) J. Cell Biol. 103, 867-874. throughout schizogony. Examples of such constitutively syn- 17. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 18. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021. thesized proteins include proteins 6 and 7 indicated in Fig. 6F. 19. Attardi, G. & Ching, E. (1979) Methods Enzymol. 56, 66-79. Other proteins appear to be unique to the developing schizont 20. Desjardins, P. & Morais, R. (1990) J. Mol. Biol. 212, 599-634. (Fig. 6F, proteins 1-4) or are synthesized at specific times 21. Chomyn, A. & Lai, S.-A. T. (1990) in Structure, Function and Bio- during schizogony (Fig. 6F, protein 5). This type of analysis can genesis of Energy Transfer Systems, eds. Quagiariello, E., Papa, S., be performed on intracellular parasites only if a selective Palmieri, F. & Saccone, C. (Elsevier, Amsterdam), pp. 179-185. 22. inhibitor of host-cell protein synthesis is present and serves to Profous-Juchelka, H. (1993) in Proceedings of Sixth International Coccidiosis Conference, eds. Barta, J. & Fernando, A. (Moffitt Print underscore the utility of the ricin technique. The two- Craft Ltd., Guelph, ON), p. 143. dimensional polyacrylamide gels also demonstrate that many 23. Wilde, C. G., Boguslawski, S. & Houston, L. L. (1979) Biochem. of the labeled intracellular parasite proteins comigrate with Biophys. Res. Commun. 91, 1082-1088. Downloaded by guest on October 1, 2021