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Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach
International Journal for Parasitology 36 (2006) 1399–1407 www.elsevier.com/locate/ijpara
Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach Patrick de Venevelles a, Jean Franc¸ois Chich a, Wolfgang Faigle b, Be´renge`re Lombard b, Damarys Loew b, Pierre Pe´ry a, Marie Labbe´ a,* a
Unite´ de Virologie et Immunologie Mole´culaires UR892, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France b Laboratoire de Spectrome´trie de masse – Prote´omique, Institut Curie, 75005 Paris, France Received 18 May 2006; received in revised form 19 June 2006; accepted 23 June 2006
Abstract Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3–10 and 4–7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7 0 , the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions. 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Eimeria tenella; Sporozoites; Refractile bodies; Two-dimensional electrophoresis; Proteomics; Mass spectrometry
1. Introduction Eimeria tenella is a protozoan parasite belonging to the Apicomplexa phylum. Eimeria sp. are agents of chicken coccidiosis that costs almost USD 3 billion dollars each year worldwide, including disease impact and prevention costs. The appearance of chemoresistance against anticoccidial products, the cost and excessive use of these drugs in the avian industry, have led to research of new antigenic targets for the elaboration of vaccines. Refractile bodies (RB) are specific structures of the Eimeriidae family such as Eimeria or Caryospora genus parasites. RB are spherical electron-dense structures consti-
*
Corresponding author. Tel.: +33 6 62 47 96 08. E-mail address: [email protected] (M. Labbe´).
tuted of a fluid matrix (Vivier and Provost, 1977). These structures are considered to be devoid of a membrane. Their function remains unknown, despite previous studies. RB have been detected in the precocious asexual stages from the sporozoite until the first generation of schizont (Fayer and Hammond, 1969; Roberts and Hammond, 1970). The presence of RB was not found after first generation merozoites (Hammond et al., 1970). Two RB are present in the cytoplasm of sporozoite Eimeria genus parasites. The posterior RB occupies almost half of the posterior part of the sporozoite and the smaller anterior one is localised between the nucleus and the apical complex. Depending on the species of Eimeria, after host cell invasion by the sporozoite, the anterior RB is either divided into several small RB that disappear 10 h p.i. (Roberts et al., 1970; Speer and Hammond, 1970) or the anterior RB moves and merges with the posterior RB (Fayer and
0020-7519/$30.00 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.06.018
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Hammond, 1969). The unique remaining RB in the trophozoite is divided into several small RB in the immature schizont (Danforth and Augustine, 1989). Each small RB is found in the cytoplasm of the first generation merozoites budding from the schizont. Only a few proteins from sporozoites have been identified as RB proteins. An mAb raised against Eimeria acervulina was described to recognise RB (Danforth and Augustine, 1983). The in vitro development of first and second-generation schizonts is greatly inhibited by this 1209 mAb (Danforth and Augustine, 1989). It was shown (Kopko et al., 2000) that this antibody recognises SO7 0 (Liberator et al., 1989), the most abundant protein in sporozoites (de Venevelles et al., 2004) whose function is still unknown. An aspartyl protease called eimepsin has also been identified in sporozoite RB and the apical complex and seems to play a role in the invasion step (Jean et al., 2000). Another protein, exhibiting similarity with transhydrogenase and consensus sequence signature for hexose transport, is localised in RB and seems to participate in maltose metabolism in Eimeria sp. (Vermeulen et al., 1993). We have previously defined global protein expression of different E. tenella stages by a proteomic approach (de Venevelles et al., 2004). The two-dimensional electrophoresis (2-DE) gels of the global proteome of sporozoites have been established and may be used as reference maps for the definition of the sub-proteome of sporozoite organelles. In this study, we purified RB from sporozoites where they are most apparent. Then, by electrophoresis, we resolved the purified RB proteins and compared these sub-proteomic maps with the entire sporozoite proteome maps in order to determine which proteins are located in RB. Defining the sub-proteome of these RB gives new data to better understand the role of these structures during the parasite life cycle. 2. Material and methods 2.1. Parasite strain and sporozoite purification Sporozoites were purified from E. tenella sporulated oocysts obtained from the cæca of 2-week-old White Leghorn chickens infected with the PAPt38PA12 strain as described previously (de Venevelles et al., 2004). Second-generation merozoites were purified from the cæca of chickens 5 days p.i. following a protocol adapted from Stotish and Wang (1975). Cæca content was washed in Ringer medium containing Penicillin (2,000 UI/mL) and streptomycin (20 lg/mL). Cæca were then treated with 1 mg/mL of hyaluronidase in Roswell Park Memorial Institute medium containing 1 mg/mL BSA for 40 min at 37 C. After centrifugation (2,000g, 5 min), merozoites were purified on a 20-mL syringe filled with 2 cm of slightly pushed cotton wool. Before 2-DE, the sporozoite and merozoites were washed twice by centrifugation (1,000g, 10 min) in cell
wash buffer (0.25 M saccharose, Tris 20 mM, pH 7.4, containing a protease inhibitor cocktail (Complete Mini, EDTA-free, Roche Diagnostics, France)) in order to eliminate salts. 2.2. Refractile body purification RB were purified from freshly excysted sporozoites. Fifty million sporozoites were incubated with 1 mL of acetone for 40 min at 4 C, then centrifuged (4,000g, 8 min, 4 C). The pellet was resuspended in 500 lL PBS, sonicated during 90 s (2 lm amplitude) and filled to 1 mL with PBS. The RB suspension was deposed on a discontinuous Optiprep gradient (Axis-Shield, Oslo, Norway), realised with four layers of 20%, 25%, 30% and 35% of Optiprep. After centrifugation (1,200g, 55 min, 4 C), the bottom layer was removed with a needle and syringe, then RB were concentrated on a vivaspin 1,000 kDa (Vivascience AG, Germany) by centrifugation (500g, 5 min, 4 C). The RB were collected then washed two times with PBS (2,000g, 5 min, 4 C). For analysis of RB, samples were deposited on a glass slide by cytocentrifugation at 600g for 2 min (cytospin 2, Shandon). RB were visualised by May-Gru¨nwald Giemsa (MGG) staining or labelled with Nile Red (Sigma) at 0.0001% in PBS 1·. RB were also analysed by immunofluorescence (see Section 2.5) with purified antibodies (1 mg/ mL) directed against Eimepsin N3C8B12 (Jean et al., 2000) diluted 1:200. For 2-DE, RB were washed in wash buffer as described for sporozoites. 2.3. Immunoblot analysis Proteins from various fractions were separated by SDS– PAGE and were transferred to a Protran nitrocellulose membrane (Schleicher and Schull, Germany) for 45 min at 50 V on a mini-blot apparatus (Biorad). The membranes were rinsed with Tris Buffer Saline (10 mM Tris pH 7.4, 150 mM NaCl) containing 0.5% v/v Tween 20 (TBS-T) and incubated with blocking buffer (2% w/v skim milk in TBS-T) for 1 h. The nitrocellulose membranes were incubated with sera or purified mAb (1 mg/mL) diluted 1:500 in 1% w/v milk blocking buffer for 2 h at 37 C. After three washes in TBS-T for 5 min, the membranes were incubated with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (Paris, France) diluted 1:2,000 in 1% w/v milk blocking buffer for 1 h at 37 C. The membranes were washed three times with TBS-T for 5 min and one time with PBS for 20 min, then treated with Supersignal West Pico Chemiluminescent substrate solution (Pierce, USA) for 1 min. Nitrocellulose membranes were exposed to X-ray film for 30–60 s. 2.4. Electron microscopy The samples were fixed in a phosphate buffer 0.1 M, 4% paraformaldhehyde (PF) and 2% glutaraldehyde for 2 h at
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room temperature. After centrifugation (2,500g, 5 min), the pellets were resuspended in phosphate buffer 0.1 M, 4% PF for 24 h at 4 C, then in phosphate buffer 0.1 M, 0.1% PF for 24 h at 4 C. Fixed material was washed with phosphate buffer for 1 h then dehydrated in successive baths of ethanol 30% (30 min, 0 C), 50% (60 min, 20 C), 70% (60 min, 20 C), 90% (60 min, 20 C) and 100% (60 min, 20 C). The inclusion buffer LR White (London, England) is composed of 98.5% polyhydroxy-bisphenoldimethacrylate resin, 0.6% methacrylic acid and 0.9% benzoyl peroxide. The samples were included in an LR White–Ethanol 100% mix (1:1 v/v) for 60 min (20 C), LR White–Ethanol 100% mix (2:1 v/v) for 60 min (20 C) and three successive baths of pure LR White (60 min, 12 h, then 60 min at 20 C). Eighty nanometer sections were achieved with an ultramicrotome (Reichert Ultracut E, LEICA) and deposited on nickel grids. 2.5. Immunofluorescence analysis The sporozoites were incubated with PBS at room temperature or with Complete Medium 199 containing 1% FCS (CM) at 41 C for 1 h. Sporozoites were deposited on glass slides then dried. The sporozoites were fixed with PF 2% for 15 min at room temperature. After permeabilisation with Triton X-100 1% for 15 min, the sporozoites were washed three times with PBS then saturated with a solution of PBS–BSA 2% for 20 min. The parasites were incubated with mAb 1209 diluted to 1:500 for 1 h at 37 C then washed three times with PBS. After incubation with a rabbit anti-mouse secondary Ab conjugated to Alexa (Molecular probes) diluted to 1:500 for 1 h, the slides were washed three times with PBS. DABCO (Sigma, 0.02% in 10 mM Tris, pH 7.5; 90% glycerol) was added before microscopic observation. 2.6. Secretion analysis Freshly excysted sporozoites (2 · 106) were resuspended in 100 lL of PBS or CM and incubated at 4 C or 41 C, respectively, for 2 h. When indicated, 10 lM of staurosporine (Sigma) dissolved in dimethylsulfoxide (DMSO) or an appropriate volume of carrier DMSO was added. Sporozoites were then pelleted by centrifugation (1,200g, 3 min). For analysis by immunoblot of the supernatant containing excretory–secretory antigens (ESA), equivalents of proteins from 2 · 105 sporozoites were separated by SDS–PAGE and probed with mAb 1209, a generous gift of Dr. P. Augustine, diluted 1:500, or rabbit sera directed against EtMIC2 diluted 1:1,000, a generous gift of Dr. F. Tomley (IAH, Compton, UK). Augustine (1999) showed that mAb 1209 was directed against a multi-isoform protein. Silver-stained spots superimposed to 1209 immunodetected spots were identified by mass spectrometry as SO7 0 isoforms (de Venevelles et al., 2004 and unpublished results). Blots were revealed by chemiluminescence as described above. Chemiluminescent signals from immuno-
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blots were quantified using a charge-coupled device camera imaging system and GeneTool analysis software (GeneGnome, Syngene). For normalisation of data, quantifications were expressed as the percentage of total protein detected in arbitrary units (a.u.). 2.7. 2-DE Parasite proteins were solubilised by incubating the washed pellet for at least 1 h at room temperature in iso-electro focussing (IEF) rehydration buffer. Solubilised proteins were quantified using PlusOne 2-D Quant Kit (GE Healthcare Life Sciences) and were used immediately for 2-DE or stored at 80 C. 2-DE were realised on 4–7 or 3–10 linear gradients as described before (de Venevelles et al., 2004). Briefly, 300 lg of protein were resolved by iso-electro focussing and by SDS–PAGE when gels were coloured by silver nitrate staining. For Coomassie blue staining, 1 mg of proteins was deposited on gels. Cyber gels were constructed with the 4–7 linear gradients and the 7–10 part of the 3–10 linear gradients. Analysis of 2-D gels was performed using ImageMaster Software v4.01 (GE Healthcare Life Sciences). The density of each spot (i.e. area and intensity) was normalised to the volume of all detected spots on 2-DE of 300 lg solubilised proteins. 2.8. Protein identification by mass spectrometry Spots were excised from gels with a scalpel and stored at 20 C in deionised water with 1% acetic acid and prepared for mass spectrometry according to the method previously described (Hellman et al., 1995). For mass spectrometry sequencing, 20 lL of 60% acetonitrile and 5% formic acid were added to each tryptic peptide extract and the samples were sonicated for 10 min. The supernatant of each extract was collected, dried under a vacuum and adjusted to 800 fmol/lL. Tryptic-digested proteins were identified by comparing peptide mass fingerprinting (PMF) data from MALDITOF spectrometry (Voyager DE-Pro, Applied Biosystems, USA) and peptide fragmentation data (PFD) obtained from liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) (QSTAR Pulsar, Applied Biosystems, USA) with DNA or protein databases at NCBI using ‘‘Mascot in home’’ software (www.matrixscience.com). Peptide fragmentation data of unidentified proteins were compared by Mascot with Expressed Sequence Tag (EST) databases at NCBI and the Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/Eimeria/tenella/EST_clusters/) and with the E. tenella genome assembly (ftp://ftp.sanger.ac.uk/pub/pathogens/ Eimeria/tenella/genome/assemblies/). The identified EST, which were predicted to code for a spot, were compared with full gene non-redundant sequence databases (NCBI) to identify other similar proteins.
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3. Results 3.1. RB purification Different methods were assessed to purify the RB. The RB purification protocol has to propose a low level of contamination, a sufficient output in order to realise several 2DE gels and a protein state compatible with the subsequent electrophoresis and mass spectrometry analysis. Sporozoites were lysed with acetone and sonication. This treatment allowed the releasing of RB from sporozoites while microscopic observations showed that RB seem to conserve their integrity. RB were then purified with a discontinuous gradient (35% Optiprep) and fraction 4 corresponding to the more purified fraction was collected. The purification yield was 25%. The purified RB fraction was analysed by microscopy after MGG colouration and no other organelle was detectable (Fig. 1A). All purified RB were recognised by an anti-eimepsin mAb (Fig. 1B) and by Nile red suggesting the presence of lipids or highly hydrophobic proteins in RB (Fig. 1C). The purified RB obtained by this method were predominantly posterior RB, with only a few small anterior RB being present in fraction 4 of the discontinuous gradient. The content of fraction 4 was analysed by electron microscopy (Fig. 1D). No surrounding membrane was detectable in posterior RB. Few RB showed the presence of a piece of sporozoite membrane still attached to the RB. In some cases, electron microscopy analysis shows the presence of holes inside the RB; this could result from the acetone treatment and could suggest the presence of lipids in the refractile matrix of these structures. No other
A
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C
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parasite organelles, such as mitochondria, were observed in this purified fraction. In order to control the purity of our RB purification, immunoblots were performed on each fraction obtained from each step of the RB purification protocol (Fig. 2). As expected, Ab directed against RB protein Eimepsin and RB protein SO7 0 show the presence of both proteins in fraction 4 of the discontinuous gradient. Two other Ab directed against non-RB proteins were used. The first is directed against a recombinant protein corresponding to the repetitive domain Rep found in EtMIC3 (Labbe´ et al., 2005) and the second is directed against the ribosomal protein S3a (Ouarzane et al., 1998). Immunoblots have shown that these proteins were no more detectable after treatment of sporozoites with acetone. Thus acetone treatment of sporozoites followed by centrifugation enriched RB. 3.2. Definition of the RB sub-proteome Proteins from the RB purified fraction were separated by 2-DE and silver stained. They were then compared with the reference maps of sporozoites to determine spots with increased or decreased amounts (Fig. 3). Six RB gels were realised in the pH range 3–10 and 4–7 and an average of 80 spots were detected by ImageMaster software. Few proteins were detected in the basic pH range 7–10 but almost all proteins were located in the pH range 5–7. In order to define proteins present mainly in RB, we compared the normalised density of each spot of the RB gels with its equivalent spot from sporozoite gels using
B
15 µm
D
1 µm
Fig. 1. Microscopic analysis of purified refractile bodies (RB). Purified RB (35% Optiprep) were observed independently after: (A) May-Gru¨nwald Giemsa colouration, (B) immunolabelling by anti-eimespin mAb, (C) Nile red colouration. (D) Purified posterior RB were observed by electron microscopy.
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Anti – 6S2 _______________________
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Anti-Rep _______________________ Spz Act S
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Anti SO7 ______________________ Sp
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Anti-S3a _______________________ Spz Act S
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Fig. 2. Enrichment or loss of Eimerian proteins during refractile body (RB) purification. Following the purification of RB, immunoblots were performed with aliquots from different steps of purification. (Spz) Sporozoites, (Act) Sporozoites following acetone incubation, (S) Sporozoites following acetone incubation and sonication, (F1, F2, F3 and F4). Different fractions of discontinuous Optiprep gradient. Blots were probed with: (A) polyclonal Ab anti-eimepsin, (B) mAb 1209-C2 anti-SO7 0 , (C) anti-Rep serum and (D) anti-S3a serum. Black arrows show the position of specific bands labelled by sera or Ab.
Fig. 3. 2-DE of sporozoite and RB proteins. Sporozoite proteins (300 lg) and RB proteins were separated by 2-DE using immobilised linear pH gradient strips. Gels were silver stained and analysed by ImageMasterTM 4.01 software. (A) Identification of sporozoite proteins was achieved by tandem mass spectrometry analysis of abundant spots. (B) Comparison of gels with ImageMasterTM allowed identification of proteins on RB gel. Normalised volumes of RB spots were compared to sporozoite spots. The increase of a normalised volume of RB spots is indicated by squares and decreased by circles. The protein names described on RB gels refer to Table 1.
ImageMaster software (Table 1). If the two spots have the same normalised density, the ratio (spot density of RB/spot density of sporozoites) is equal to 1.00. Both sporozoite and purified RB gels were achieved using 300 lg of solubilised proteins. This quantity corresponds to 20 · 106 sporozoites or to 50 · 106 purified RB. Since the large majority of purified RB were the larger ones, it can be assessed that one sporozoite gives only one purified RB. Consequently, RB gels contained 2.75 times more RB than sporozoite gels. Some spots that were not resolved in spo-
rozoite gels because of their low abundance (de Venevelles et al., 2004) were clearly detected in RB gels. They were analysed by MS/MS and allowed the identification of new proteins, especially new isoforms of eimepsin. The RB proteins and relative abundance are shown in Table 1. RB spots which were enriched more than 2.75 times as compared with sporozoite spots were considered as proteins located exclusively in RB. Finally, 14 proteins or isoforms were enriched with a factor equal or superior to 2.75 times.
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Table 1 Abundance of proteins detected in refractile bodies (RB) compared with Eimeria tenella sporozoite proteins identified by mass spectrometry
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Among these proteins, eimepsin and its different isoforms were the most abundant. Several isoforms of the sporozoite protein SO7 0 were also enriched on RB gels. Other enzymes involved in cell metabolism such as serine protease, reductase, hydrolase, subtilase and ubiquitin were also abundant in RB. In order to determine if RB proteins are expressed during the second generation of merozoites, we compared spot density corresponding to these proteins between the sporozoite and second-generation merozoite gels (300 lg of proteins were equivalent to 55 · 106 merozoites). The ratio of the normalised density between the sporozoite and merozoite is shown in Table 1. Among the RB proteins, 11 RB spots expressed in sporozoites were also present in second-generation merozoites as dehalogenase-like, hydrolase, subtilase and unknown proteins. On RB gels,
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truncated isoforms of eimepsin were detected from 30 to 40 kDa (Fig. 3). These spots were detected on second-generation merozoite gels but not on sporozoite gels. 3.3. Trafficking and secretion of the RB SO7 0 protein The S07 0 protein was defined as an RB protein. Freshly excysted sporozoites were incubated in PBS or activated at 41 C in CM. The localisation of SO7 0 was analysed by immunofluorescence with the mAb 1209-C2 (Fig. 4). As expected, SO7 0 was located in anterior and posterior RB in sporozoites incubated in PBS (Fig. 4A). In contrast, SO7 0 appeared at the apical complex of sporozoites after incubation with CM at 41 C (Fig. 4B). At the same time, immunolabelling of the anterior RB by mAb 1209 decreased in most of the sporozoites.
B
aRB Apex pRB pRB
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Fig. 4. Trafficking of SO7 0 refractile body (RB) protein prior to sporozoite invasion. The mAb 1209 was used to detect SO7 0 protein in sporozoites. (A) Immunolabelling of the RB protein SO7 0 on freshly purified sporozoites. (B) Immunolabelling on sporozoites activated by incubation with FCS 5% at 41 C. (aRB) anterior Refractile Body. (pRB) posterior Refractile Body.
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Fig. 5. Secretion of SO7 protein. (A) Sporozoites were incubated either in PBS at 4 C or at 41 C in medium containing FCS (CM). Excretory/secretory antigens were collected by centrifugation and analysed by immunoblot probed with mAb 1209 or with sera raised against EtMic2 protein. Secreted proteins were quantified using GeneTool analysis software. The results are an average of three independent experiments. (B) Sporozoites were incubated at 41 C in CM in the presence of staurosporine (STS). Inhibition of secretion of SO7 0 and EtMic2 by staurosporine was observed when immunoblots were compared with solvent controls.
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To determine whether the relocalised protein SO7 0 was secreted in the culture medium, sporozoites were incubated in CM and the presence of SO7 0 was looked for in ESA (Fig. 5A). As for the EtMIC2 protein used as microneme secretion control, the SO7 0 protein was secreted only when incubated in CM at 41 C. This secretion was inhibited by the kinase inhibitor STS known to prevent microneme release (Fig. 5B). 4. Discussion RB is a structure unique to the Eimeriidae family whose composition is still unknown. In an attempt to define the function of this structure, its sub-proteome was compared with the sporozoite proteome that we previously established (de Venevelles et al., 2004). The definition of the RB sub-proteome by a proteomic approach needed to purify RB with a protocol compatible with 2-DE and mass spectrometry analysis. This protocol required the fixation of RB during purification. Acetone appeared to be the most valuable chemical product to maintain RB integrity and is compatible with subsequent steps of precipitation and solubilisation (Jacobs et al., 2001; Jiang et al., 2004). Nevertheless, it remains difficult to determine if all purified RB proteins have been solubilised compared with sporozoite protein solubilisation. The different controls performed during RB purification show that enrichment was sufficient to carry on the subproteomic approach. Analysis of enriched fractions with Ab directed against eimerian proteins strongly suggested that RB were separated from cytoplasm or small organelles. After separation of RB proteins by 2-DE, 16 proteins were enriched on RB gels corresponding with three known E. tenella proteins and three newly identified proteins. Other proteins were without significant homology with proteins present in databases. Analysis could be improved in future with complete genome annotation. Numerous proteins were also found in gels with an enrichment factor less than 2.75 times. These proteins are probably not exclusively located in RB. Among eimepsin and SO7 0 , this work confirmed the RB localisation of these already known proteins. Nevertheless, transhydrogenase, which has been shown to be localised in RB by immunolabelling (Vermeulen et al., 1993), was impoverished here by a factor of 1.56 times on RB gels compared with sporozoite gels. The RB proteome analysis suggests different functions for these structures. Indeed, eimepsin and SO7 0 , the two most abundant RB proteins, have been described to take part in invasion since mAb directed against these proteins can inhibit this step (Augustine, 1999, 2001; Jean et al., 2000). The exact role of these proteins remains to be determined. Eimepsin is an aspartyl protease like the Plasmodium falciparum plasmepsins that are localised in the food vacuole and participate in heme degradation (Liu et al., 2005). A serine protease was also detected in RB. This pro-
tein was homologous to subtilase 2, a P. falciparum protein localised in the secretory organelles of merozoites just before invasion (Withers-Martinez et al., 2004), which is also essential for invasion of the host cell (Uzureau et al., 2004). The presence of all these proteins in RB seems to confirm the proposed function of this structure as a reservoir for proteins involved in invasion. Other RB proteins identified here are implicated in the maintenance of cellular homeostasis. A lactate dehydrogenase involved in energetic metabolism was detected. A group of proteins involved in redox mechanisms was also found with a protein homologous to a haloacid dehalogenase type hydrolase, an enzyme implicated in detoxification in bacteria (Munro et al., 2000), a carbonyl reductase and one isoform of the 2-cys peroxiredoxin. An ubiquitin family protein was also detected. This protein is present in intracellular stages of P. falciparum and its corresponding transcripts are increased in trophozoites and schizonts and in response to heat shock (Horrocks and Newbold, 2000). The comparison of RB with other parasitic organelles remains difficult, because no lipidic membrane seems to delimitate RB. Fat inclusion bodies have been described in Toxoplasma gondii and P. falciparum. These structures, devoid of membrane, are proposed to be an energetic and membrane biogenesis reservoir for the parasite (Vielemeyer et al., 2004). Moreover, these lipid inclusion bodies play a role in the G3P pathway and participate in the diacyl and triacylglycerol synthesis in apicomplexan parasites (Vielemeyer et al., 2004). The labelling of RB with ‘‘Nile red’’ suggests that RB are lipid rich or contain highly hydrophobic proteins. Several peptides of RB proteins presented homologies with an acylCoA synthase by MS/MS analysis. These results suggest that RB could be related to lipid bodies but studies of their biochemical composition by other approaches are required. The particular fate of RB during the asexual stage of Eimeria sp. Life-cycle remains unknown. Eimepsin is localised in RB and in cytoplasmic granules and just before invasion is relocalised at the apex of sporozoites (Jean et al., 2000). In this study, we show that SO7 0 is also localised at the apex after sporozoite activation and secreted in the culture medium. Eimerian EST database analysis revealed that only a few EST corresponding to SO7 0 are detected in sporozoites, suggesting that SO7 0 is not actively translated before invasion and consequently SO7 0 at the apex could result from the traffic of the protein from RB. All these observations strongly suggest that a part of the RB content is redirected to the sporozoite apex during the invasion processes. We compared all the sequences of the proteins detected in RB in order to underline a signal for trafficking to the RB and secretory pathway. No common sequence was detected between these proteins but all RB proteins contain a signal peptide. Analysis by MS/MS of SO7 0 shows that isoforms containing a peptide signal are enriched in RB.
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In conclusion, this sub-proteomic analysis shows that RB is a complex structure, composed of numerous proteins, often with several isoforms and maybe lipids. RB is a dynamic structure constituted of proteins that may be addressed to other organelles where they interact with cellular metabolism and participate in invasion of the host cell by the parasite. Acknowledgements We thank Christine Longin and Sophie Chat for TEM preparation (Unite´ GPL – Jouy-en-Josas). We acknowledge Wendy Brand-Williams, Language Editor, for her help in English language correctness. Patrick de Venevelles is the recipient of a grant from the University of Versailles Saint-Quentin-en-Yvelines. References Augustine, P.C., 1999. Reduced invasion of cultured cells pretreated with a monoclonal antibody elicited against refractile body antigens of avian coccidial sporozoites. J. Eukaryot. Microbiol. 46, 254–258. Augustine, P.C., 2001. Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites. J. Eukaryot. Microbiol. 48, 177–181. Danforth, H.D., Augustine, P.C., 1983. Specificity and crossreactivity of immune serum and hybridoma antibodies to various species of avian coccidia. Poult. Sci. 62, 2145–2151. Danforth, H.D., Augustine, P.C., 1989. Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development. Exp. Parasitol. 68, 1–7. de Venevelles, P., Chich, J.F., Faigle, W., Loew, D., Labbe´, M., GirardMisguich, F., Pe´ry, P., 2004. Towards a reference map of Eimeria tenella sporozoite proteins by two-dimensional electrophoresis and mass spectrometry. Int. J. Parasitol. 34, 1321–1331. Fayer, R., Hammond, D.M., 1969. Morphological changes in Eimeria bovis sporozoites during their first day in cultured mammalian cells. J. Parasitol. 55, 398–401. Hammond, D.M., Speer, C.A., Roberts, W., 1970. Occurrence of refractile bodies in merozoites of Eimeria species. J. Parasitol. 56, 189–191. Hellman, U., Wernstedt, C., Gonez, J., Heldin, C.H., 1995. Improvement of an ‘‘In-Gel’’ digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451–455. Horrocks, P., Newbold, C.I., 2000. Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-shock control. Mol. Biochem. Parasitol. 105, 115–125. Jacobs, D.I., van Rijssen, M.S., van der Heijden, R., Verpoorte, R., 2001. Sequential solubilization of proteins precipitated with trichloroacetic
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acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis. Proteomics 1, 1345– 1350. Jean, L., Grosclaude, J., Labbe´, M., Tomley, F., Pe´ry, P., 2000. Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process. Int. J. Parasitol. 30, 1099–1107. Jiang, L., He, L., Fountoulakis, M., 2004. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J. Chromatogr. A 1023, 317–320. Kopko, S.H., Martin, D.S., Barta, J.R., 2000. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies. Poult. Sci. 79, 336–342. Labbe´, M., de Venevelles, P., Girard-Misguich, F., Bourdieu, C., Guillaume, A., Pe´ry, P., 2005. Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection. Mol. Biochem. Parasitol. 140, 43–53. Liberator, P.A., Hsu, J., Turner, M.J., 1989. Tandem trinucleotide repeats throughout the nucleotide sequence of a cDNA encoding an Eimeria tenella sporozoite antigen. Nucleic Acids Res. 17, 7104. Liu, J., Gluzman, I.Y., Drew, M.E., Goldberg, D.E., 2005. The role of Plasmodium falciparum food vacuole plasmepsins. J. Biol. Chem. 280, 1432–1437. Munro, A.W., Taylor, P., Walkinshaw, M.D., 2000. Structures of redox enzymes. Curr. Opin. Biotechnol. 11, 369–376. Ouarzane, M., Labbe´, M., Pe´ry, P., 1998. Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein. Gene 225, 125–130. Roberts, W.L., Hammond, D.M., 1970. Ultrastructural and cytologic studies of the sporozoites of four Eimeria species. J. Protozool. 17, 76–86. Roberts, W.L., Hammond, D.M., Anderson, L.C., Speer, C.A., 1970. Ultrastructural study of schizogony in Eimeria callospermophili. J. Protozool. 17, 584–592. Speer, C.A., Hammond, D.M., 1970. Nuclear divisions and refractilebody changes in sporozoites and schizonts of Eimeria callospermophili in cultured cells. J. Parasitol. 56, 461–467. Stotish, R.L., Wang, C.C., 1975. Preparation and purification of merozoites of Eimeria tenella. J. Parasitol. 61, 700–703. Uzureau, P., Barale, J.C., Janse, C.J., Waters, A.P., Breton, C.B., 2004. Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events. Cell Microbiol. 6, 65–78. Vermeulen, A.N., Kok, J.J., van den Boogaart, P., Dijkema, R., Claessens, J.A., 1993. Eimeria refractile body proteins contain two potentially functional characteristics: transhydrogenase and carbohydrate transport. FEMS Microbiol. Lett. 110, 223–229. Vielemeyer, O., McIntosh, M.T., Joiner, K.A., Coppens, I., 2004. Neutral lipid synthesis and storage in the intraerythrocytic stages of Plasmodium falciparum. Mol. Biochem. Parasitol. 135, 197–209. Vivier, E., Provost, J., 1977. Observations compare´es sur des inclusions ordonne´es mine´rales et organiques. Biol. Cellulaire 30, 159–164. Withers-Martinez, C., Jean, L., Blackman, M.J., 2004. Subtilisin-like proteases of the malaria parasite. Mol. Microbiol. 53, 55–63.
Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach Patrick de Venevelles a, Jean Franc¸ois Chich a, Wolfgang Faigle b, Be´renge`re Lombard b, Damarys Loew b, Pierre Pe´ry a, Marie Labbe´ a,* a
Unite´ de Virologie et Immunologie Mole´culaires UR892, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France b Laboratoire de Spectrome´trie de masse – Prote´omique, Institut Curie, 75005 Paris, France Received 18 May 2006; received in revised form 19 June 2006; accepted 23 June 2006
Abstract Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3–10 and 4–7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7 0 , the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions. 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Eimeria tenella; Sporozoites; Refractile bodies; Two-dimensional electrophoresis; Proteomics; Mass spectrometry
1. Introduction Eimeria tenella is a protozoan parasite belonging to the Apicomplexa phylum. Eimeria sp. are agents of chicken coccidiosis that costs almost USD 3 billion dollars each year worldwide, including disease impact and prevention costs. The appearance of chemoresistance against anticoccidial products, the cost and excessive use of these drugs in the avian industry, have led to research of new antigenic targets for the elaboration of vaccines. Refractile bodies (RB) are specific structures of the Eimeriidae family such as Eimeria or Caryospora genus parasites. RB are spherical electron-dense structures consti-
*
Corresponding author. Tel.: +33 6 62 47 96 08. E-mail address: [email protected] (M. Labbe´).
tuted of a fluid matrix (Vivier and Provost, 1977). These structures are considered to be devoid of a membrane. Their function remains unknown, despite previous studies. RB have been detected in the precocious asexual stages from the sporozoite until the first generation of schizont (Fayer and Hammond, 1969; Roberts and Hammond, 1970). The presence of RB was not found after first generation merozoites (Hammond et al., 1970). Two RB are present in the cytoplasm of sporozoite Eimeria genus parasites. The posterior RB occupies almost half of the posterior part of the sporozoite and the smaller anterior one is localised between the nucleus and the apical complex. Depending on the species of Eimeria, after host cell invasion by the sporozoite, the anterior RB is either divided into several small RB that disappear 10 h p.i. (Roberts et al., 1970; Speer and Hammond, 1970) or the anterior RB moves and merges with the posterior RB (Fayer and
0020-7519/$30.00 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.06.018
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Hammond, 1969). The unique remaining RB in the trophozoite is divided into several small RB in the immature schizont (Danforth and Augustine, 1989). Each small RB is found in the cytoplasm of the first generation merozoites budding from the schizont. Only a few proteins from sporozoites have been identified as RB proteins. An mAb raised against Eimeria acervulina was described to recognise RB (Danforth and Augustine, 1983). The in vitro development of first and second-generation schizonts is greatly inhibited by this 1209 mAb (Danforth and Augustine, 1989). It was shown (Kopko et al., 2000) that this antibody recognises SO7 0 (Liberator et al., 1989), the most abundant protein in sporozoites (de Venevelles et al., 2004) whose function is still unknown. An aspartyl protease called eimepsin has also been identified in sporozoite RB and the apical complex and seems to play a role in the invasion step (Jean et al., 2000). Another protein, exhibiting similarity with transhydrogenase and consensus sequence signature for hexose transport, is localised in RB and seems to participate in maltose metabolism in Eimeria sp. (Vermeulen et al., 1993). We have previously defined global protein expression of different E. tenella stages by a proteomic approach (de Venevelles et al., 2004). The two-dimensional electrophoresis (2-DE) gels of the global proteome of sporozoites have been established and may be used as reference maps for the definition of the sub-proteome of sporozoite organelles. In this study, we purified RB from sporozoites where they are most apparent. Then, by electrophoresis, we resolved the purified RB proteins and compared these sub-proteomic maps with the entire sporozoite proteome maps in order to determine which proteins are located in RB. Defining the sub-proteome of these RB gives new data to better understand the role of these structures during the parasite life cycle. 2. Material and methods 2.1. Parasite strain and sporozoite purification Sporozoites were purified from E. tenella sporulated oocysts obtained from the cæca of 2-week-old White Leghorn chickens infected with the PAPt38PA12 strain as described previously (de Venevelles et al., 2004). Second-generation merozoites were purified from the cæca of chickens 5 days p.i. following a protocol adapted from Stotish and Wang (1975). Cæca content was washed in Ringer medium containing Penicillin (2,000 UI/mL) and streptomycin (20 lg/mL). Cæca were then treated with 1 mg/mL of hyaluronidase in Roswell Park Memorial Institute medium containing 1 mg/mL BSA for 40 min at 37 C. After centrifugation (2,000g, 5 min), merozoites were purified on a 20-mL syringe filled with 2 cm of slightly pushed cotton wool. Before 2-DE, the sporozoite and merozoites were washed twice by centrifugation (1,000g, 10 min) in cell
wash buffer (0.25 M saccharose, Tris 20 mM, pH 7.4, containing a protease inhibitor cocktail (Complete Mini, EDTA-free, Roche Diagnostics, France)) in order to eliminate salts. 2.2. Refractile body purification RB were purified from freshly excysted sporozoites. Fifty million sporozoites were incubated with 1 mL of acetone for 40 min at 4 C, then centrifuged (4,000g, 8 min, 4 C). The pellet was resuspended in 500 lL PBS, sonicated during 90 s (2 lm amplitude) and filled to 1 mL with PBS. The RB suspension was deposed on a discontinuous Optiprep gradient (Axis-Shield, Oslo, Norway), realised with four layers of 20%, 25%, 30% and 35% of Optiprep. After centrifugation (1,200g, 55 min, 4 C), the bottom layer was removed with a needle and syringe, then RB were concentrated on a vivaspin 1,000 kDa (Vivascience AG, Germany) by centrifugation (500g, 5 min, 4 C). The RB were collected then washed two times with PBS (2,000g, 5 min, 4 C). For analysis of RB, samples were deposited on a glass slide by cytocentrifugation at 600g for 2 min (cytospin 2, Shandon). RB were visualised by May-Gru¨nwald Giemsa (MGG) staining or labelled with Nile Red (Sigma) at 0.0001% in PBS 1·. RB were also analysed by immunofluorescence (see Section 2.5) with purified antibodies (1 mg/ mL) directed against Eimepsin N3C8B12 (Jean et al., 2000) diluted 1:200. For 2-DE, RB were washed in wash buffer as described for sporozoites. 2.3. Immunoblot analysis Proteins from various fractions were separated by SDS– PAGE and were transferred to a Protran nitrocellulose membrane (Schleicher and Schull, Germany) for 45 min at 50 V on a mini-blot apparatus (Biorad). The membranes were rinsed with Tris Buffer Saline (10 mM Tris pH 7.4, 150 mM NaCl) containing 0.5% v/v Tween 20 (TBS-T) and incubated with blocking buffer (2% w/v skim milk in TBS-T) for 1 h. The nitrocellulose membranes were incubated with sera or purified mAb (1 mg/mL) diluted 1:500 in 1% w/v milk blocking buffer for 2 h at 37 C. After three washes in TBS-T for 5 min, the membranes were incubated with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (Paris, France) diluted 1:2,000 in 1% w/v milk blocking buffer for 1 h at 37 C. The membranes were washed three times with TBS-T for 5 min and one time with PBS for 20 min, then treated with Supersignal West Pico Chemiluminescent substrate solution (Pierce, USA) for 1 min. Nitrocellulose membranes were exposed to X-ray film for 30–60 s. 2.4. Electron microscopy The samples were fixed in a phosphate buffer 0.1 M, 4% paraformaldhehyde (PF) and 2% glutaraldehyde for 2 h at
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room temperature. After centrifugation (2,500g, 5 min), the pellets were resuspended in phosphate buffer 0.1 M, 4% PF for 24 h at 4 C, then in phosphate buffer 0.1 M, 0.1% PF for 24 h at 4 C. Fixed material was washed with phosphate buffer for 1 h then dehydrated in successive baths of ethanol 30% (30 min, 0 C), 50% (60 min, 20 C), 70% (60 min, 20 C), 90% (60 min, 20 C) and 100% (60 min, 20 C). The inclusion buffer LR White (London, England) is composed of 98.5% polyhydroxy-bisphenoldimethacrylate resin, 0.6% methacrylic acid and 0.9% benzoyl peroxide. The samples were included in an LR White–Ethanol 100% mix (1:1 v/v) for 60 min (20 C), LR White–Ethanol 100% mix (2:1 v/v) for 60 min (20 C) and three successive baths of pure LR White (60 min, 12 h, then 60 min at 20 C). Eighty nanometer sections were achieved with an ultramicrotome (Reichert Ultracut E, LEICA) and deposited on nickel grids. 2.5. Immunofluorescence analysis The sporozoites were incubated with PBS at room temperature or with Complete Medium 199 containing 1% FCS (CM) at 41 C for 1 h. Sporozoites were deposited on glass slides then dried. The sporozoites were fixed with PF 2% for 15 min at room temperature. After permeabilisation with Triton X-100 1% for 15 min, the sporozoites were washed three times with PBS then saturated with a solution of PBS–BSA 2% for 20 min. The parasites were incubated with mAb 1209 diluted to 1:500 for 1 h at 37 C then washed three times with PBS. After incubation with a rabbit anti-mouse secondary Ab conjugated to Alexa (Molecular probes) diluted to 1:500 for 1 h, the slides were washed three times with PBS. DABCO (Sigma, 0.02% in 10 mM Tris, pH 7.5; 90% glycerol) was added before microscopic observation. 2.6. Secretion analysis Freshly excysted sporozoites (2 · 106) were resuspended in 100 lL of PBS or CM and incubated at 4 C or 41 C, respectively, for 2 h. When indicated, 10 lM of staurosporine (Sigma) dissolved in dimethylsulfoxide (DMSO) or an appropriate volume of carrier DMSO was added. Sporozoites were then pelleted by centrifugation (1,200g, 3 min). For analysis by immunoblot of the supernatant containing excretory–secretory antigens (ESA), equivalents of proteins from 2 · 105 sporozoites were separated by SDS–PAGE and probed with mAb 1209, a generous gift of Dr. P. Augustine, diluted 1:500, or rabbit sera directed against EtMIC2 diluted 1:1,000, a generous gift of Dr. F. Tomley (IAH, Compton, UK). Augustine (1999) showed that mAb 1209 was directed against a multi-isoform protein. Silver-stained spots superimposed to 1209 immunodetected spots were identified by mass spectrometry as SO7 0 isoforms (de Venevelles et al., 2004 and unpublished results). Blots were revealed by chemiluminescence as described above. Chemiluminescent signals from immuno-
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blots were quantified using a charge-coupled device camera imaging system and GeneTool analysis software (GeneGnome, Syngene). For normalisation of data, quantifications were expressed as the percentage of total protein detected in arbitrary units (a.u.). 2.7. 2-DE Parasite proteins were solubilised by incubating the washed pellet for at least 1 h at room temperature in iso-electro focussing (IEF) rehydration buffer. Solubilised proteins were quantified using PlusOne 2-D Quant Kit (GE Healthcare Life Sciences) and were used immediately for 2-DE or stored at 80 C. 2-DE were realised on 4–7 or 3–10 linear gradients as described before (de Venevelles et al., 2004). Briefly, 300 lg of protein were resolved by iso-electro focussing and by SDS–PAGE when gels were coloured by silver nitrate staining. For Coomassie blue staining, 1 mg of proteins was deposited on gels. Cyber gels were constructed with the 4–7 linear gradients and the 7–10 part of the 3–10 linear gradients. Analysis of 2-D gels was performed using ImageMaster Software v4.01 (GE Healthcare Life Sciences). The density of each spot (i.e. area and intensity) was normalised to the volume of all detected spots on 2-DE of 300 lg solubilised proteins. 2.8. Protein identification by mass spectrometry Spots were excised from gels with a scalpel and stored at 20 C in deionised water with 1% acetic acid and prepared for mass spectrometry according to the method previously described (Hellman et al., 1995). For mass spectrometry sequencing, 20 lL of 60% acetonitrile and 5% formic acid were added to each tryptic peptide extract and the samples were sonicated for 10 min. The supernatant of each extract was collected, dried under a vacuum and adjusted to 800 fmol/lL. Tryptic-digested proteins were identified by comparing peptide mass fingerprinting (PMF) data from MALDITOF spectrometry (Voyager DE-Pro, Applied Biosystems, USA) and peptide fragmentation data (PFD) obtained from liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) (QSTAR Pulsar, Applied Biosystems, USA) with DNA or protein databases at NCBI using ‘‘Mascot in home’’ software (www.matrixscience.com). Peptide fragmentation data of unidentified proteins were compared by Mascot with Expressed Sequence Tag (EST) databases at NCBI and the Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/Eimeria/tenella/EST_clusters/) and with the E. tenella genome assembly (ftp://ftp.sanger.ac.uk/pub/pathogens/ Eimeria/tenella/genome/assemblies/). The identified EST, which were predicted to code for a spot, were compared with full gene non-redundant sequence databases (NCBI) to identify other similar proteins.
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3. Results 3.1. RB purification Different methods were assessed to purify the RB. The RB purification protocol has to propose a low level of contamination, a sufficient output in order to realise several 2DE gels and a protein state compatible with the subsequent electrophoresis and mass spectrometry analysis. Sporozoites were lysed with acetone and sonication. This treatment allowed the releasing of RB from sporozoites while microscopic observations showed that RB seem to conserve their integrity. RB were then purified with a discontinuous gradient (35% Optiprep) and fraction 4 corresponding to the more purified fraction was collected. The purification yield was 25%. The purified RB fraction was analysed by microscopy after MGG colouration and no other organelle was detectable (Fig. 1A). All purified RB were recognised by an anti-eimepsin mAb (Fig. 1B) and by Nile red suggesting the presence of lipids or highly hydrophobic proteins in RB (Fig. 1C). The purified RB obtained by this method were predominantly posterior RB, with only a few small anterior RB being present in fraction 4 of the discontinuous gradient. The content of fraction 4 was analysed by electron microscopy (Fig. 1D). No surrounding membrane was detectable in posterior RB. Few RB showed the presence of a piece of sporozoite membrane still attached to the RB. In some cases, electron microscopy analysis shows the presence of holes inside the RB; this could result from the acetone treatment and could suggest the presence of lipids in the refractile matrix of these structures. No other
A
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C
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parasite organelles, such as mitochondria, were observed in this purified fraction. In order to control the purity of our RB purification, immunoblots were performed on each fraction obtained from each step of the RB purification protocol (Fig. 2). As expected, Ab directed against RB protein Eimepsin and RB protein SO7 0 show the presence of both proteins in fraction 4 of the discontinuous gradient. Two other Ab directed against non-RB proteins were used. The first is directed against a recombinant protein corresponding to the repetitive domain Rep found in EtMIC3 (Labbe´ et al., 2005) and the second is directed against the ribosomal protein S3a (Ouarzane et al., 1998). Immunoblots have shown that these proteins were no more detectable after treatment of sporozoites with acetone. Thus acetone treatment of sporozoites followed by centrifugation enriched RB. 3.2. Definition of the RB sub-proteome Proteins from the RB purified fraction were separated by 2-DE and silver stained. They were then compared with the reference maps of sporozoites to determine spots with increased or decreased amounts (Fig. 3). Six RB gels were realised in the pH range 3–10 and 4–7 and an average of 80 spots were detected by ImageMaster software. Few proteins were detected in the basic pH range 7–10 but almost all proteins were located in the pH range 5–7. In order to define proteins present mainly in RB, we compared the normalised density of each spot of the RB gels with its equivalent spot from sporozoite gels using
B
15 µm
D
1 µm
Fig. 1. Microscopic analysis of purified refractile bodies (RB). Purified RB (35% Optiprep) were observed independently after: (A) May-Gru¨nwald Giemsa colouration, (B) immunolabelling by anti-eimespin mAb, (C) Nile red colouration. (D) Purified posterior RB were observed by electron microscopy.
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A
Anti – 6S2 _______________________
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F3
F4
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53 41
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Anti-Rep _______________________ Spz Act S
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Anti SO7 ______________________ Sp
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Anti-S3a _______________________ Spz Act S
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F2 F3
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Fig. 2. Enrichment or loss of Eimerian proteins during refractile body (RB) purification. Following the purification of RB, immunoblots were performed with aliquots from different steps of purification. (Spz) Sporozoites, (Act) Sporozoites following acetone incubation, (S) Sporozoites following acetone incubation and sonication, (F1, F2, F3 and F4). Different fractions of discontinuous Optiprep gradient. Blots were probed with: (A) polyclonal Ab anti-eimepsin, (B) mAb 1209-C2 anti-SO7 0 , (C) anti-Rep serum and (D) anti-S3a serum. Black arrows show the position of specific bands labelled by sera or Ab.
Fig. 3. 2-DE of sporozoite and RB proteins. Sporozoite proteins (300 lg) and RB proteins were separated by 2-DE using immobilised linear pH gradient strips. Gels were silver stained and analysed by ImageMasterTM 4.01 software. (A) Identification of sporozoite proteins was achieved by tandem mass spectrometry analysis of abundant spots. (B) Comparison of gels with ImageMasterTM allowed identification of proteins on RB gel. Normalised volumes of RB spots were compared to sporozoite spots. The increase of a normalised volume of RB spots is indicated by squares and decreased by circles. The protein names described on RB gels refer to Table 1.
ImageMaster software (Table 1). If the two spots have the same normalised density, the ratio (spot density of RB/spot density of sporozoites) is equal to 1.00. Both sporozoite and purified RB gels were achieved using 300 lg of solubilised proteins. This quantity corresponds to 20 · 106 sporozoites or to 50 · 106 purified RB. Since the large majority of purified RB were the larger ones, it can be assessed that one sporozoite gives only one purified RB. Consequently, RB gels contained 2.75 times more RB than sporozoite gels. Some spots that were not resolved in spo-
rozoite gels because of their low abundance (de Venevelles et al., 2004) were clearly detected in RB gels. They were analysed by MS/MS and allowed the identification of new proteins, especially new isoforms of eimepsin. The RB proteins and relative abundance are shown in Table 1. RB spots which were enriched more than 2.75 times as compared with sporozoite spots were considered as proteins located exclusively in RB. Finally, 14 proteins or isoforms were enriched with a factor equal or superior to 2.75 times.
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Table 1 Abundance of proteins detected in refractile bodies (RB) compared with Eimeria tenella sporozoite proteins identified by mass spectrometry
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Among these proteins, eimepsin and its different isoforms were the most abundant. Several isoforms of the sporozoite protein SO7 0 were also enriched on RB gels. Other enzymes involved in cell metabolism such as serine protease, reductase, hydrolase, subtilase and ubiquitin were also abundant in RB. In order to determine if RB proteins are expressed during the second generation of merozoites, we compared spot density corresponding to these proteins between the sporozoite and second-generation merozoite gels (300 lg of proteins were equivalent to 55 · 106 merozoites). The ratio of the normalised density between the sporozoite and merozoite is shown in Table 1. Among the RB proteins, 11 RB spots expressed in sporozoites were also present in second-generation merozoites as dehalogenase-like, hydrolase, subtilase and unknown proteins. On RB gels,
A
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truncated isoforms of eimepsin were detected from 30 to 40 kDa (Fig. 3). These spots were detected on second-generation merozoite gels but not on sporozoite gels. 3.3. Trafficking and secretion of the RB SO7 0 protein The S07 0 protein was defined as an RB protein. Freshly excysted sporozoites were incubated in PBS or activated at 41 C in CM. The localisation of SO7 0 was analysed by immunofluorescence with the mAb 1209-C2 (Fig. 4). As expected, SO7 0 was located in anterior and posterior RB in sporozoites incubated in PBS (Fig. 4A). In contrast, SO7 0 appeared at the apical complex of sporozoites after incubation with CM at 41 C (Fig. 4B). At the same time, immunolabelling of the anterior RB by mAb 1209 decreased in most of the sporozoites.
B
aRB Apex pRB pRB
Apex aRB
Fig. 4. Trafficking of SO7 0 refractile body (RB) protein prior to sporozoite invasion. The mAb 1209 was used to detect SO7 0 protein in sporozoites. (A) Immunolabelling of the RB protein SO7 0 on freshly purified sporozoites. (B) Immunolabelling on sporozoites activated by incubation with FCS 5% at 41 C. (aRB) anterior Refractile Body. (pRB) posterior Refractile Body.
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CM DMSO
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Fig. 5. Secretion of SO7 protein. (A) Sporozoites were incubated either in PBS at 4 C or at 41 C in medium containing FCS (CM). Excretory/secretory antigens were collected by centrifugation and analysed by immunoblot probed with mAb 1209 or with sera raised against EtMic2 protein. Secreted proteins were quantified using GeneTool analysis software. The results are an average of three independent experiments. (B) Sporozoites were incubated at 41 C in CM in the presence of staurosporine (STS). Inhibition of secretion of SO7 0 and EtMic2 by staurosporine was observed when immunoblots were compared with solvent controls.
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To determine whether the relocalised protein SO7 0 was secreted in the culture medium, sporozoites were incubated in CM and the presence of SO7 0 was looked for in ESA (Fig. 5A). As for the EtMIC2 protein used as microneme secretion control, the SO7 0 protein was secreted only when incubated in CM at 41 C. This secretion was inhibited by the kinase inhibitor STS known to prevent microneme release (Fig. 5B). 4. Discussion RB is a structure unique to the Eimeriidae family whose composition is still unknown. In an attempt to define the function of this structure, its sub-proteome was compared with the sporozoite proteome that we previously established (de Venevelles et al., 2004). The definition of the RB sub-proteome by a proteomic approach needed to purify RB with a protocol compatible with 2-DE and mass spectrometry analysis. This protocol required the fixation of RB during purification. Acetone appeared to be the most valuable chemical product to maintain RB integrity and is compatible with subsequent steps of precipitation and solubilisation (Jacobs et al., 2001; Jiang et al., 2004). Nevertheless, it remains difficult to determine if all purified RB proteins have been solubilised compared with sporozoite protein solubilisation. The different controls performed during RB purification show that enrichment was sufficient to carry on the subproteomic approach. Analysis of enriched fractions with Ab directed against eimerian proteins strongly suggested that RB were separated from cytoplasm or small organelles. After separation of RB proteins by 2-DE, 16 proteins were enriched on RB gels corresponding with three known E. tenella proteins and three newly identified proteins. Other proteins were without significant homology with proteins present in databases. Analysis could be improved in future with complete genome annotation. Numerous proteins were also found in gels with an enrichment factor less than 2.75 times. These proteins are probably not exclusively located in RB. Among eimepsin and SO7 0 , this work confirmed the RB localisation of these already known proteins. Nevertheless, transhydrogenase, which has been shown to be localised in RB by immunolabelling (Vermeulen et al., 1993), was impoverished here by a factor of 1.56 times on RB gels compared with sporozoite gels. The RB proteome analysis suggests different functions for these structures. Indeed, eimepsin and SO7 0 , the two most abundant RB proteins, have been described to take part in invasion since mAb directed against these proteins can inhibit this step (Augustine, 1999, 2001; Jean et al., 2000). The exact role of these proteins remains to be determined. Eimepsin is an aspartyl protease like the Plasmodium falciparum plasmepsins that are localised in the food vacuole and participate in heme degradation (Liu et al., 2005). A serine protease was also detected in RB. This pro-
tein was homologous to subtilase 2, a P. falciparum protein localised in the secretory organelles of merozoites just before invasion (Withers-Martinez et al., 2004), which is also essential for invasion of the host cell (Uzureau et al., 2004). The presence of all these proteins in RB seems to confirm the proposed function of this structure as a reservoir for proteins involved in invasion. Other RB proteins identified here are implicated in the maintenance of cellular homeostasis. A lactate dehydrogenase involved in energetic metabolism was detected. A group of proteins involved in redox mechanisms was also found with a protein homologous to a haloacid dehalogenase type hydrolase, an enzyme implicated in detoxification in bacteria (Munro et al., 2000), a carbonyl reductase and one isoform of the 2-cys peroxiredoxin. An ubiquitin family protein was also detected. This protein is present in intracellular stages of P. falciparum and its corresponding transcripts are increased in trophozoites and schizonts and in response to heat shock (Horrocks and Newbold, 2000). The comparison of RB with other parasitic organelles remains difficult, because no lipidic membrane seems to delimitate RB. Fat inclusion bodies have been described in Toxoplasma gondii and P. falciparum. These structures, devoid of membrane, are proposed to be an energetic and membrane biogenesis reservoir for the parasite (Vielemeyer et al., 2004). Moreover, these lipid inclusion bodies play a role in the G3P pathway and participate in the diacyl and triacylglycerol synthesis in apicomplexan parasites (Vielemeyer et al., 2004). The labelling of RB with ‘‘Nile red’’ suggests that RB are lipid rich or contain highly hydrophobic proteins. Several peptides of RB proteins presented homologies with an acylCoA synthase by MS/MS analysis. These results suggest that RB could be related to lipid bodies but studies of their biochemical composition by other approaches are required. The particular fate of RB during the asexual stage of Eimeria sp. Life-cycle remains unknown. Eimepsin is localised in RB and in cytoplasmic granules and just before invasion is relocalised at the apex of sporozoites (Jean et al., 2000). In this study, we show that SO7 0 is also localised at the apex after sporozoite activation and secreted in the culture medium. Eimerian EST database analysis revealed that only a few EST corresponding to SO7 0 are detected in sporozoites, suggesting that SO7 0 is not actively translated before invasion and consequently SO7 0 at the apex could result from the traffic of the protein from RB. All these observations strongly suggest that a part of the RB content is redirected to the sporozoite apex during the invasion processes. We compared all the sequences of the proteins detected in RB in order to underline a signal for trafficking to the RB and secretory pathway. No common sequence was detected between these proteins but all RB proteins contain a signal peptide. Analysis by MS/MS of SO7 0 shows that isoforms containing a peptide signal are enriched in RB.
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