defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite...

Elizabeth Bromley1

Nicola Leeds2

Julie Clark1

Emma McGregor2

Malcolm Ward2

Michael J. Dunn3

Fiona Tomley1

1Institute for Animal Health,Compton, Berkshire, UK

2Proteome Sciences pIc,Institute of Psychiatry,King’s College,London, UK

3Institute of Psychiatry,King’s College,London, UK

Defining the protein repertoire of micronemesecretory organelles in the apicomplexan parasiteEimeria tenella

The apicomplexan pathogen Eimeria causes coccidiosis, an intestinal disease ofchickens, which has a major welfare and economic impact on the poultry industry.There is an urgent need to identify molecules that are rational targets for drug designand novel vaccines against coccidiosis. Apicomplexan secretory organelles, includ-ing micronemes and rhoptries, are essential for invasion of the host intestinal epithe-lium and establishment of parasitism. However, relatively little is known about theprecise molecular function of these organelles, partly because few organelle proteinshave been characterized. In this study, proteomics tools have been harnessed todefine the protein repertoire of micronemes. Purified microneme proteins fromEimeria tenella sporozoites were excised from two-dimensional (2-D) gels and ana-lyzed using matrix-assisted laser desorption/ionization-time of flight-mass spec-trometry (MALDI-TOF-MS) and chemically assisted fragmentation (CAF)-MALDIwith de novo sequencing. Peptide mass profiles were searched against the NCBInon-redundant (nr) database and against Eimeria-specific databases using the Mas-cot search algorithm, resulting in the identification of 37 of 96 spots excised from the2-D gels. In addition, we have found CAF-MALDI to be a useful adjunct for identifyingproteins, without the need for tandem MS. This global approach to protein charac-terization will be vital to gain greater understanding of the processes involved in api-complexan host cell invasion.

Keywords: Chemically assisted fragmentation / Eimeria / Microneme PRO 0479

1 Introduction

Protozoa of the phylum Apicomplexa include pathogensof medical and veterinary importance, such as Plasmo-dium, Cryptosporidium, Toxoplasma, Sarcocystis, Eim-eria, andNeospora. Seven species of Eimeria cause poul-try coccidiosis, an intestinal infection that is a serious wel-fare problem amongst intensively reared chickens. Ineconomic terms, this disease is one of the most importantin the poultry industry, costing £40 million per annumin the UK alone [1]. Although several live vaccines areavailable, there is keen interest in production of recombi-nant vaccines [2]. Study of invasion by Eimeria sporo-zoites into host intestinal epithelial cells will enable identi-fication of molecules with potential to act as new thera-peutic targets for coccidiosis.

During invasion, apicomplexan parasites attach to thehost cell via the apical complex, a characteristic structureat the anterior end (Fig. 1). The apical complex consists ofa conoid, a polar ring, subpellicular microtubules, and thesecretory organelles: micronemes, rhoptries, and densegranules. Micronemes and rhoptries are known to beessential for invasion, but only a limited number of organ-elle proteins have been identified and characterized.Eimeria is an excellent model for biochemical studies ofapicomplexan secretory organelles, as micronemes andrhoptries are particularly abundant and techniques fortheir purification are well established in comparison toother Apicomplexa [3, 4].

Invasion commences when the apical end of the sporo-zoite attaches to the host cell surface forming a tightadhesion zone. The parasite then passes through thiszone and is contained within a parasitophorous vacuolein the host cytoplasm. The invasion process is completedwithin 5–10 s [5], during which time there is sequentialsecretion from micronemes, rhoptries, and dense gran-ules [6]. Micronemes secrete their contents first and areinvolved in motility, host cell attachment, and recognition.This is followed by the release of the rhoptry proteins,

Correspondence: Elizabeth Bromley, Institute for Animal Health,Compton, Berkshire, RG20 7NN, UKE-mail: [email protected]: +44-1635-577263

Abbreviations: CAF, chemically assisted fragmentation; PMF,peptide mass fingerprint; TBP, tributylphosphine

Proteomics 2003, 3, 1553–1561 1553DOI 10.1002/pmic.200300479

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1554 E. Bromley et al. Proteomics 2003, 3, 1553–1561

Figure 1. Apical complex ofE. tenella showing secretoryorganelles involved in invasion:the micronemes, rhoptries, anddense granules. (a) Schematicdiagram, (b) electron micro-graph.

which are believed to aid parasite movement into the hostcell and the formation of the parasitophorous vacuolemembrane. Finally, dense granules secrete their proteins,which have roles in remodelling the parasitophorousvacuole following invasion.

Five microneme proteins have been identified in Eimeriatenella [7–11] and a further five putative micronemeproteins have been found by homology with proteinsfrom other apicomplexans (Tomley et al., unpublished).Many of the apicomplexan microneme proteins charac-terized so far contain adhesive domains, includingthrombospondin type I (TSP-1) domains, integrin inser-tion (I) domains, epidermal growth factor (EGF) domains,and Apple domains [7, 9–12]. The presence of thesedomains indicates that these microneme proteins mayhave key roles in attachment and invasion. Micronemeproteins have also been implicated in the motility ofE. tenella sporozoites [13]. After secretion in vitro, micro-neme proteins were capped backwards over the para-site surface and released from the posterior of the cell.Capping was prevented by cytochalasin D, an inhibitorof actin polymerization. Thus it was hypothesized that,following secretion, microneme proteins on the parasitesurface form a connection with the sub-pellicular actin-myosin network, which acts as the motor for the cell[13].

Many microneme proteins have been identified usingtechniques such as immunoscreening. However, progresshas been slow as each protein was studied on an individ-ual basis. A global approach is necessary to define theprotein repertoire of these secretory organelles in order toallow workers to concentrate on assigning functions toproteins. We intend to define the repertoire of organellarproteins in Eimeria tenella using cell-map proteomics. In

this paper we outline our approach to this problem,demonstrate some of the techniques involved and presentdata on the identification of some microneme proteins.

For a proteomics approach to succeed it is essential toscreen the data generated against Eimeria sequencedatabases. The Eimeria tenella genome project was setup recently in a collaboration between the Institute forAnimal Health, Compton, UK and the Sanger Institute,Cambridge, UK. Fivefold coverage of the genome of theE. tenella Houghton (H) strain is now complete and thelatest assembly of contigs is publicly available (http://www.sanger.ac.uk/Projects/E_tenella/). In addition, ESTprojects at Washington University and University Kebang-saan Malaysia have released �14 000 E. tenella ESTs(http://www.ncbi.nlm.nih.gov/dbEST), derived from spo-rozoite and merozoite invasive stages. These have beenassembled into a clustered database (http://ftp.sanger.ac.uk/pub/databases/E. tenella/EST_clusters).

2 Materials and methods

2.1 Parasites

Oocysts of the H strain of E. tenella were harvested fromchicken caeca, isolated and sporulated, as describedpreviously [14, 15]. Sporozoites were purified by pas-sage through a DE52 cellulose column [16].

2.2 Isolation of micronemes

Purified sporozoites were sonicated and subcellular frac-tionation carried out over sucrose gradients as describedpreviously [3, 4]. Material was drawn off from the visiblemicroneme band in the sucrose gradient using a needleand syringe.


Proteomics 2003, 3, 1553–1561 Microneme proteins of Eimeria tenella 1555

2.3 Sample preparation

Approximately 100 �g protein in solution (measured usingthe Bradford reagent assay; Bio-Rad, Hercules, CA, USA)was precipitated by the addition of an equal volume ofacetone and incubation for 1 h on ice. The sample wascentrifuged at 13 000�g for 10 min at 4�C and the pelletallowed to air-dry for 2 min. The pellet was resuspendedin either 150 �L lysis buffer A (9.5 M urea, 2% CHAPS,1% DTT, 0.8% Pharmalyte pH 3–10) or 150 �L lysis bufferB (5 M urea, 2% CHAPS, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulphonate (SB3–10), 2 mM tributyl-phosphine (TBP), 0.8% Pharmalyte pH 3-10) with pro-tease inhibitors (Sigma # P2714, St. Louis, MO, USA).Each sample was then diluted with 300 �L rehydrationbuffer A (8 M urea, 2% CHAPS, 0.5% IPG buffer (Amers-ham Biosciences, Uppsala, Sweden), 0.21% DTT, tracebromophenol blue; for use with lysis buffer A) or 300 �Lrehydration buffer B (5 M urea, 2 M thiourea, 2% CHAPS,2% SB3-10, 2 mM TBP, 0.2% Pharmalyte pH 3–10, tracebromophenol blue; for use with lysis buffer B).

2.4 Isoelectric focusing

Samples were pipetted into 24 cm Immobiline StripHolders (Amersham Biosciences) and a 24 cm ImmobilineDryStrip of the required pH range (pH 3–10 or pH 4–7) wasapplied. The strips were rehydrated then focused at0.05 mA/strip for 61.5 kVh at 20�C. The strips were equili-brated in equilibration buffer (6 M urea, 50 mM Tris-ClpH 8.8, 30% w/v glycerol, 2% SDS, trace bromophenolblue) with 1% w/v DTT. Equilibration was repeated with2.5% w/v iodoacetamide in place of DTT.

2.5 Second-dimensional SDS-PAGE

Each strip was loaded into an Ettan DALT II gel cassettecontaining a precast 12.5% gel (Amersham Biosciences).The gel cassettes were loaded into an Ettan DALT II sys-tem (Amersham Biosciences) and electrophoresis com-menced according to the manufacturer’s instructions, at60 W at 25�C for 3–8 h, until the dye front was 5 mmfrom the bottom of the gel.

2.6 Silver staining

The 2-D gels were silver-stained using PlusOne silverstaining kit (Amersham Biosciences) omitting glutaralde-hyde from the protocol for MS compatibility, accordingto [17].

2.7 MALDI-MS

The gels were scanned using ImageScanner and LabScansoftware (Amersham Biosciences). Protein spots on thegels were detected either manually or using ImageMastersoftware (Amersham Biosciences). Spots were pickedfrom the gel using a robotic Ettan Spot Picker (AmershamBiosciences) and each gel plug (2 mm diameter) placedinto a well of a 96-well plate. The gel pieces were reducedwith DTT and alkylated using iodoacetamide, prior toovernight digestion with trypsin [18]. When conventionalMALDI analysis provided insufficient data for protein iden-tification, genetic information was obtained by modifyingthe digest supernatant using a chemically assisted frag-mentation (CAF)-MALDI sequencing kit (Amersham Bio-sciences).

2.8 Data analysis

Peptide mass fingerprint (PMF) data were searched usingMascot (Matrix Science) against NCBInr, E. tenella ge-nome, and E. tenella clustered EST databases, with fixedcarbamidomethyl modification, variable oxidized methio-nine modification, and 50 ppm mass tolerance.

3 Results

3.1 Screening PMF data

Micronemes were isolated from sonicated E. tenella spo-rozoites by sucrose gradient ultracentrifugation (Fig. 2).Figure 3a demonstrates that microneme fractions areenriched for certain polypeptides in comparison withunfractionated sporozoites (see also [3]). For example,the level of EtMIC1 was enhanced in the micronemefraction compared to the sporozoite sample (Figs. 3a andc). Although sucrose gradient ultracentrifugation is themethod of choice for separation of organelles [19], some

Figure 2. Electron micrograph of purified micronemesisolated from sucrose gradient.



Figure 3. (a) Coomassie-stained SDS-PAGE gel, (b) West-ern blot probed with mouse anti-rhoptry serum, (c) Westernblot probed with rabbit anti-EtMIC1 serum. Lanes: S, un-fractionated sporozoite; M, microneme fraction; all sam-ples from E. tenella H strain. Blots developed using alka-line phosphatase system with 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium substrate.

non-microneme contamination does occur and themicroneme fraction in this case was also found to containa trace of rhoptry protein (Fig. 3b).

Proteins were solubilized with lysis buffers A or B, andseparated by 2-D electrophoresis on gels with pH rangespH 4–7 or pH 3–10 (Fig. 4). Although lysis buffer A gavebetter spot separation (compare Figs. 4a and 4b), lysisbuffer B solubilized the sample more efficiently. A total of96 spots were picked and analyzed by MALDI-TOF. PMFswere obtained for 68 of these spots. The 68 PMFs werescreened initially against the NCBInr database, leadingto positive identifications for 26 spots (Table 1). Five ofthese are derived from non-Eimeria proteins viz. soybeantrypsin inhibitor and bovine serum albumin, both of whichare potential contaminants from the parasite purificationand subcellular fractionation processes. The remaining21 spots are Eimeria-specific, with 20 PMFs giving signif-icant matches to 6 different E. tenella proteins and 1 giv-ing a significant match to an Eimeria acervulinaDNAK-like

Figure 4. Example of E. tenella microneme proteins separated by 2-D electrophoresis. All gels were silver-stainedaccording to [17]. All picked spots are ringed, but only those mentioned in the text are labelled. (a) pH 4–7, lysis bufferB. Spots 1–56 were picked from this gel. (b) pH 4–7, lysis buffer A (spots 57–87) (c) pH 4–7, lysis buffer B (spots 88–91)(d) pH 3–10, lysis buffer B (spots 92–96).



Table 1. Spots from 2-D gels of microneme proteins which were identified by screening NCBInr with PMF data, usingMascot software

SpotNo.

SpotMW(Da)

SpotpI

Protein ID ProteinMW(Da)

ProteinpI

No.peptidesmatched

%Cover-age

M6 95 250 5.0 Immunoglobulin heavy chain binding protein (E. tenella)Z66492 gi1037176

77 345 5.22 27 43

M7 95 250 5.0 Immunoglobulin heavy chain binding protein (E. tenella)Z66492 gi1037176

77 345 5.22 18 34

M11 73 590 4.9 Beta tubulin (E. tenella) U19268 gi624292 49 921 4.78 24 59

M15 64 250 4.3 Microneme protein EtMIC2 (E. tenella) AF111702 gi4164596 35 093 4.44 14 46

M18 66 020 5.0 Actin (Toxoplasma gondii) gi1703160 41 881 5.05 10 36



M24 64 250 5.8 Aspartyl protease (E. tenella) AJ293829 gi12054066 50 341 5.91 12 33



M32 39 410 4.8 Sporulated oocyst antigen TA4 precursor fragment (E. tenella)gi627043

24 631 4.79 16 76


24 631 4.79 12 54


24 631 4.79 10 57

M44 28 840 4.5 Trypsin inhibitor (soybean) gi3891584 20 082 4.61 19 92





24 631 4.79 8 44


24 631 4.79 9 56


24 631 4.79 11 55

M62 93 820 5.1 Dnak-type molecular chaperone (E.acervulina) gi480678 70 415 5.16 20 31

M63 93 820 5.6 Serum albumin (Bos taurus) X58989 gi2190337 69 278 5.82 16 31



24 631 4.79 15 64


24 631 4.79 8 45


24631 4.79 7 41

molecular chaperone, a housekeeping protein that wouldbe expected to be highly conserved between the twoEimeria species. Only one Eimeria protein, EtMIC2, is apreviously described microneme protein [8]. The remain-der (immunoglobulin heavy chain binding protein, DNAK-

like chaperone, beta tubulin, actin, aspartyl proteinaseand antigen TA4) are derived from the parasite cytoskele-ton or from membrane-associated compartments of theparasite such as the endoplasmic reticulum, refractilebody organelle, and plasma membrane.



Table 2. Screening PMF data against E. tenella clustered EST database: significant hits only

SpotNo.

Clustered ESTcontig

No.peptides

%Coverage

Putative identity from NCBI

M38 Contig 591 13/38 29% No matchM39 Contig 591 12/60 39% No matchM56 Contig 314 14/33 30% E. acervulina antigen M86228M79 Contig 530 7/30 45% Putative hsp70 domainM83 Contig 1326 7/25 28% NcMic1 (AF421187) (Neospora)M84 Contig 1326 8/57 30% NcMic1 (AF421187) (Neospora)M85 Contig 314 13/51 28% E. acervulina antigen M86228M86 Contig 11 11/60 18% Plasmodium calmodulin (NC_004317)M91 Contig 205 7/31 28% T. gondii depolymerizing factor (U62146)M94 Contig 530 7/49 45% Putative hsp70 domainM95 Contig 571 19/60 32% T. gondii protein disulphide isomerase

(AJ496761)

Spots already identified by direct comparison of PMF data with NCBInr (given in Table 1) have beenexcluded from the table. The EST contig which matched PMF data in each case was then screenedagainst NCBInr to ascertain putative identity for protein spot.

All 68 PMFs were next screened against E. tenella ge-nome and clustered EST databases. Only three gave sta-tistically significant hits against the genome databaseindicating that, in its current state of assembly and anno-tation, the 5� shotgun is not useful for screening PMFs.In contrast, PMFs from 28 spots gave statistically signi-ficant hits to contigs in the clustered EST database. Ofthese, 17 were amongst the 21 Eimeria-specific spotsthat had already been identified by screening PMFs di-rectly against NCBInr (Table 1). The remaining 11 PMFs,which gave matches to 7 different contigs in the clusteredEST database, were from spots that had not been identi-fied by screening NCBInr indicating that these are novelE. tenella proteins (Table 2). When the 7 contigs wereBLASTed against the NCBInr database, 6 were found tohave significant homologies to known proteins, includingseveral that have been described in other apicomplexans(Table 2). Of particular interest, spots M83 and M84 arehomologous to the microneme protein NcMIC1 of Neo-spora caninum, spots M56 and M85 are homologous toa surface antigen from Eimeria acervulina and spots M91and M95 are homologous to two different membrane-bound proteins from Toxoplasma gondii.

In summary, screening 68 PMFs against NCBInr and theEimeria clustered EST database resulted in positive iden-tifications for 37 spots (26 from direct NCBInr screeningplus an additional 11 from clustered EST screening). The37 spots correspond to just 15 different proteins, two ofwhich are exogenous, non-Eimeria contaminants. Of the13 Eimeria proteins, one is definitely from the microneme(EtMIC2), one is likely to be from the microneme (homolo-gous to NcMIC1) and one is entirely novel, so may be

from the microneme (spots M38 and M39). The remaining10 proteins are probably not micronemal in origin but arederived from other parts of the parasite, such as the cyto-skeleton, plasma membrane, endoplasmic reticulum andrefractile body organelle.

3.2 CAF-MALDI

As many spots could not be identified by screening PMFdata against NCBInr, a pilot study of CAF-MALDI wasundertaken. Tryptic peptides from three spots (M15, M38and M56) were analyzed further by post-source decay(PSD) with CAF-MALDI, to obtain sequence information[20]. PSD can be used directly to generate sequencefrom peptides, but the spectra generated are typicallycomplex and difficult to interpret. CAF improves this frag-mentation by introducing a negatively charged (sulphonic)group (�136 Da) to the N-terminus of the tryptic peptide(Fig. 5, Step 2). Lysine side chains are protected fromsulphonation by the prior conversion to homoarginine(�42 Da) (Fig. 5, Step 1). Arginine side chains are alreadyprotected. The addition of a negative charge means that

Figure 5. Mechanism of CAF-MALDI.



two protons are required to create a net positive chargeon the peptide. One of these is situated at the C-terminusand the second is present on the backbone where itassists fragmentation. The other advantage is that afterfragmentation, only the y-ions retain a positive chargeand are therefore detected by the mass spectrometer,creating a simplified fragmentation spectrum.

Previously, sample M15 gave no significant hits againstthe clustered EST or NCBInr databases. However, follow-ing CAF-MALDI, the sequence RPDVPG[I/L]V[Q/K] gener-

ated for peptide m/z 1525.7 was found to match EtMIC2(AF111839), a 50 kDa microneme protein which dispersesover the parasite surface either during or shortly afterinvasion [8]. Sample M38 hit contig 591 when the clus-tered EST database was searched with PMF data, al-though no identity could be assigned to this contig byscreening NCBInr. Figure 6 shows two CAF-MALDI spec-tra obtained from spot M38 for peptides m/z 1812.8 and1401.6, along with the de novo sequence manually deter-mined from this data. These sequences were found tomatch to a protein that was independently under study

Figure 6. CAF spectra and de novo sequence for two peptides withm/z 1812.8 and 1401.6, detected from spot M38.



as a putative microneme component, tentatively assignedas EtMIC6. However, it is now established that this proteinis not in the microneme but is instead a GPI-linked sur-face antigen (Tabares and Tomley, manuscript in prepa-ration).

Sample M56 matched an EST contig which was foundto be similar to an E. acervulina antigen when screenedagainst NCBInr. It is likely that this antigen is conservedin E. tenella. Following CAF-MALDI on sample M56,the sequence F[I/L][I/L]DG[Q/K]ET[I/L]R was obtainedfor peptide m/z 1192.4 by de novo sequencing. Thissequence was found to match another putative micro-neme protein, EtMIC7, that was coincidentally underinvestigation. In this case, a definitive cellular locationhas not yet been assigned and characterization of theprotein is ongoing.

4 Discussion

We have begun to use proteomic approaches to definemicroneme proteins in E. tenella, and early data areencouraging. The preliminary analysis of 2-D gel-sepa-rated proteins by MALDI-TOF and the screening of PMFsagainst NCBInr are powerful tools to identify exogenouscontaminants (trypsin inhibitor and serum albumin), aswell as a small number of Eimeria proteins. Most of theEimeria proteins identified in this way would not be pre-dicted to occur in the microneme, but reside in other loca-tions within the parasite. This indicates that the micro-neme fractions prepared by sucrose-gradient ultracentri-fugation are not completely pure, although preparationsappear clean when observed by electron microscopy(Fig. 2). This was further supported when screening ofthe PMF data against the clustered EST database identi-fied seven contigs, only two of which potentially encodemicroneme proteins, one homologous to the Neosporacaninum MIC1 protein and another with no significanthomologies. Screening of PMFs has thus proved usefulas a means to eliminate spots of non-microneme originfrom further, more time-consuming and expensive analy-ses. In addition, a small number of potentially novelE. tenella microneme proteins have been identified. The5� random shotgun sequence of the E. tenella genome,which is currently assembled into �10 000 contigs, wasnot very useful for screening PMFs. This is probably be-cause many Eimeria genes are interrupted by introns,which make the raw data unsuitable for PMF screening:for example, the aspartyl proteinase, eimepsin, is organiz-ed over 18 exons [21]. However, over the next few months,as the E. tenella genome data are analyzed in more depth,rescreening the PMFs against catalogues of predictedE. tenella genes is likely to be much more fruitful.

In choosing spots to pick from the 2-D gels, we triedto avoid knowingly picking spots that correspond to thepreviously characterized E. tenella microneme proteins(EtMIC1-5). Nevertheless, the PMF screening readilyidentified several spots corresponding to EtMIC2 [8]. Thescreening identified several other instances of multiplespots that appeared to be derived from a single protein.There are several potential explanations for this phenom-enon. First, despite the use of protease inhibitors in allprocedures, proteolysis may still occur and multiple spots,of different molecular mass, may represent degradedforms of the same protein. Second, different charge iso-forms of the same protein may be expressed by the para-site and, therefore, be present in the sample. Third, novelproteins, perhaps products of a multi-gene family, couldpotentially be very similar to previously characterizedproteins and contain many of the same tryptic peptides,resulting in very similar PMFs. In this case, the true iden-tity of such novel proteins could only be verified using LC-MS/MS and de novo sequencing. Finally, since spotswere selected from four different gels, it is possible thatthe same protein was picked from more than one gel,although efforts were made to avoid this.

Although it was essential to begin this study with MALDI-TOF analysis, full definition of the microneme proteinrepertoire will not be achieved without the use of othertools. CAF-MALDI with de novo sequencing on a pilotgroup of three spots was very successful, giving identitiesfor all three spots. Thus, CAF-MALDI is potentially a veryuseful tool for characterizing proteins without resorting toexpensive LC-MS/MS techniques and will be adoptedmore extensively as an intermediate technique for theidentification of new proteins from E. tenella.

We would like to thank Al Ivens of the Sanger Institute,Cambridge, for compiling the clustered EST databaseand Patricia Bland (IAH) for providing electron micro-graphs. We also wish to thank our colleagues at ProteomeSciences plc for their support and assistance during thiswork. Particular thanks to James Campbell for preparationof some index files required for Mascot. This project wassupported by BBSRC research grants 201/S15358 and28/S15362.

Received December 20, 2002

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