THE OPPORTUNISTIC PATHOGEN TOXOPLASMA GONDII DEPLOYS A DIVERSE LEGION OF INVASION AND SURVIVAL PROTEINS

Abstract

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins using two complementary methodologies, 2-DE/MS and LC/ESI-MS-MS (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by 2-DE revealed ∼100 spots, most of which were successfully identified by protein microsequencing or MALDI-MS analysis. Many proteins were present in multiple species suggesting they are subjected to substantial posttranslational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. The findings provide a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.

The protozoan Toxoplasma gondii is a human pathogen that causes severe opportunistic disease (toxoplasmosis) in congenitally infected babies and immuno-compromised individuals (e.g., AIDS) (1,2). While foodborne transmission via ingestion of infected meat products contributes to the steady rise in age dependent seroprevalence, waterborne transmission of the highly infectious, feline-derived oocyst stage has led to recent outbreaks (3,4). The efficiency of waterborne transmission and the availability of drug resistant strains have raised awareness of toxoplasmosis as a threat to public health from natural outbreaks or the potential malicious contamination of public water sources. Since effective control of outbreak situations will be enhanced by having multiple options for the diagnosis and treatment, the identification and exploitation of novel parasite targets is of acute importance.

T. gondii belongs to phylum Apicomplexa, which also includes several other notable pathogens such as Plasmodium (the agents of malaria), Cryptosporidium (the cause of cryptosporidiosis), and Eimeria (the cause of coccidiosis). For several aspects of apicomplexan biology, Toxoplasma is emerging as a important model organism (5) because it exhibits many of the features and capabilities that define the phylum, yet it is more amenable to experimental manipulation than many of its kin.

Toxoplasma is an obligate intracellular parasite and must invade a vertebrate host cell for survival and replication. Invasion initiates the lytic cycle leading to the cell and tissue destruction that is a hallmark feature of Toxoplasma pathology. Toxoplasma invasion is a rapid (<30 seconds), dynamic, and complex process that relies on the secretion of numerous secretory proteins from specialized secretory organelles including micronemes, rhoptries, and dense granules (6). Previous studies revealed that sequential secretion of these secretory proteins are critical events in parasite invasion and establishment of infection (7,8). Micronemal proteins (MICs) are released first upon the parasite apical attachment to a host cell and function in host cell attachment and penetration (reviewed in (9)). Next, the contents of rhoptries are discharged and are thought to be critical for biogenesis of the parasitophorous vacuole (PV) that envelops the parasite during invasion and its interaction with host cellular organelles (10,11). Finally, dense granules proteins (DGs) are exocytosed both during and after invasion and are thought to be function in intracellular survival and replication (12-14). Recent studies have also suggested that the parasite releases products capable of manipulating the host immune response (15,16). Also, secretory products and surface antigens (SAGs) have received scrutiny as promising diagnostic markers (17,18). Despite this progress, to date only a limited number of secretory proteins have been discovered. This point is underscored by the recent bioinformatic identification of >800 genes encoding proteins with a putative secretory signal peptide (www.toxodb.org), yet fewer than 30 Toxoplasma secretory proteins have been described to date. While many of the putative secretory proteins may be retained in internal compartments such as the ER/Golgi or apicoplast (a plastid like organelle), a significant fraction is probably exported to external sites for interaction with the host.

The recent coupling of high-resolution protein separation techniques (2-DE or multi-chromatography) with high-throughput identification strategies has permitted the wide-scale analysis of protein identity, expression, modification, localization, and interactions within whole cells or sub-cellular fractions (19,20). With these tools, it is possible to analyze a multitude of cellular proteins from a more global perspective. The recent completion of parasite genome projects (21-23) has fueled proteomic studies of Plasmodium (24,25), Eimeria tenella (26), Trypanosoma cruzi (27) and Leishmania major (28). The Toxoplasma genome is nearing completion and two studies have described preliminary mapping and partial identification of the tachyzoite (a rapidly dividing form of the parasite) proteins, either by 2-DE immunoblotting (29) or by 2-DE coupled with MALDI-MS (30). While these studies provided a valuable initial view of the parasite’s protein complement, a relatively small subset of the Toxoplasma proteome was examined.

Here we use complementary approaches to examine a cohort of excreted/secreted antigens (ESA) freely released by Toxoplasma tachyzoites, the lifestage responsible for the tissue pathology observed in toxoplasmosis. Our findings reveal that Toxoplasma mobilizes a rich assortment of putative effector molecules that may interact with the host during infection. These include adhesins that presumably contribute to attachment and penetration, proteases that may facilitate invasion or migration into host tissues, metabolic enzymes potentially involved in nutrient acquisition, and series of hypothetical proteins of unknown function. These findings significantly widen the viewing aperture of the molecular players contributing to Toxoplasma survival and pathogenesis during infection.

EXPERIMENTAL PROCEDURES

Reagents and chemicals - Porcine trypsin (sequencing grade) was from Promega (Madison, WI, USA). MS calibration mixture II (peptide mass standard kit) was from Applied Biosystems (Framingham, MA, USA). 1-D Electrophoresis and 2-DE reagents and immobilized IEF strips were from Bio-Rad (Hercules, CA, USA) or Amersham Biosciences (Piscataway, NJ, USA). The MALDI matrix, α-cyano-4-hydroxycinnamic acid (CHCA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All the organic solvents are HPLC grade. All other reagents and chemicals were obtained from either Fisher (Pittsburgh, PA, USA) or Sigma-Aldrich and were of the highest purity available.

Cell Culture and Large-scale ESA Preparation - Parasite culture and ESA (excretory/secretory antigen) preparation was performed according to (31). Briefly, T. gondii strain 2F was propagated in human foreskin fibroblast (HFF) cells. Freshly egressed tachyzoites were harvested by passage twice through a 20-gauge needle followed by filtration through a 3-μm pore size membrane to remove host cell debris. Parasites were washed twice by centrifugation in D0 medium (DMEM, 2 mM glutamine, 10 mM HEPES). Large-scale preparation of ESA proteins was performed by incubating ∼4×10⁹ filter purified tachyzoites in 15 ml of D0 plus 1% (v/v) ethanol at 37°C for 20 min followed by cooling on ice 5 min. Parasites were removed by centrifugation (1000 × g, 10 min, 4°C). The supernatant, which was supplemented with a cocktail of proteinase inhibitors, was concentrated to ∼400 μl using C-20 concentrators according to the manufacturer’s instructions (Millipore; Billerica, MA, USA).

Two-dimensional Gel Electrophoresis - ESA proteins were separated in the first dimension on 11 cm or 18 cm immobilized dry strips (pH 3-10, pH 4-7) using an isoelectric focusing system (Amersham Bioscience or Bio-Rad). 120 μg of ESA protein was mixed with rehydration buffer containing 8 M urea, 2% CHAPS, 0.5% carrier ampholytes Buffer, and 65 mM DTT. Rehydration and isoelectric focusing were performed according to the manufacturer’s instructions. Following isoelectric focusing, proteins were reduced and alkylated by successive 15 min treatments with equilibration buffer containing 2% dithiothreitol followed by 2.5% iodoacetamide. Proteins were then resolved in the second dimension on a SDSPAGE gel (12.5% homogenous gel for the Amersham system, 10-20% gradient gel for Bio-Rad). Resolved proteins were either transferred directly to PVDF membrane for N-terminal sequencing or stained with colloidal Coomassie Blue stain (32) for MALDI-MS analysis.

N-terminal Sequencing - Proteins on a 2-DE gel were transferred to Immobilon^® P^SQ membranes (Millipore) and stained with Coomassie Brilliant Blue R-250. Bands were excised and subjected to Edman degradation on a Perkin-Elmer Biosystems Model 477A Gas-Phase Protein Sequencer.

In-gel digestion, peptide extraction, MALDI-MS analysis and database searching - Protein spots from 2-DE gels were excised, in-gel digested with trypsin (12.5 ng μl^-1 in 50 mM NH₄HCO₃), and solvent-extracted as described previously (33). After extraction, peptide mixtures were dried down and re-dissolved in 2 μl of 50% acetonitrile and 0.3% TFA, mixed with saturated CHCA (matrix) solution and deposited on the MALDIMS target plate according to a general two-layer method (34). The MS analysis of digested peptides was performed with a Voyager DE-STR MALDITOF mass spectrometer (PerSeptive Biosystems, Framingham, MA. USA). The spectra were acquired in the reflection positive mode with delayed extraction and, external peptides or proteins adjacent to the sample spots were used for MS calibration. Internal calibration was refined using selected trypsin autolysis ions. Peptide monoisotopic masses were used to search against Toxoplasma NCBI database via the MS-Fit algorithm (http://prospector.ucsf.edu) or to search Toxoplasma protein/EST/genome databases via an in-house installed Mascot program (35). In both cases, a mass tolerance of ±50 ppm was allowed in the database searches. Generally, a minimum number of four peptide matches, sequence coverage of at least 15%, and a match to a T. gondii entry were required for true positive match when using MS-fit algorithm, while a score higher than the Mascot significant score were regarded as positive match in Mascot database searches.

MudPIT analysis and protein identification - Lyophilized ESA proteins were reconstituted in 1 ml of 50 mM ammonium bicarbonate solution. The proteins were reduced by adding 10 μl of DTT (1 M) and incubation at 37 °C for 2 h. Cysteines were alkylated by adding 10 μl of iodoacetic acid (1 M in 1 N NaOH) and incubated in the dark for 30 min at room temperature. The sample was spun through a Microcon YM-3 concentrator until dry and resuspended in 1 μl of 50 mM ammonium bicarbonate buffer for trypsin digestion.

The digested ESA peptide mixtures were buffer exchanged into 5% acetonitrile, 0.1% acetic acid prior to analysis. The MudPIT experiments were performed according to the manufacturer’s instructions on a ProteomeX workstation (Thermo-Finnigan, San Jose, CA), which is comprised of an autosampler, two HPLC pumps, a 10-port column-switching valve, and a Deca-XP ion-trap mass spectrometer with a micro-ESI interface. For 1D MudPIT, HPLC separation of peptide mixtures were conducted through RP chromatography only, while for 2D MudPIT, the HPLC separation were performed through ion exchange chromatography in the first dimension then RP chromatography in the second dimension. Specifically, in the case of 2D MudPIT, peptides were bound to a strong cation exchanger (BioBasic SCX, 0.32 mm x 10 cm, Thermo Hypersil, PA) and eluted stepwise with five NH₄Cl concentrations (10 mM, 50 mM, 100 mM, 200 mM, 500 mM, respectively). Peptides eluted from each salt step were further separated on a reverse phase capillary column (BioBasic C18, 300Å, 5 μm silica, 180 μm x 10 cm) using an acetonitrile gradient (0%-60% solvent B in solvent A, A=0.1% formic acid; B=100% acetonitrile containing 0.1% formic acid). The mass/charge (m/z) ratios of eluted peptides and fragmented ions were analyzed by an LCQ Deca XP ion-trap mass spectrometer. Following each full scan mass spectrum, two MS/MS spectra of the top two most intense peaks were acquired. The dynamic exclusion feature was enabled to obtain MS/MS spectra of co-eluted peptides.

Protein identification was performed by searching the T. gondii protein subset of NCBI ‘nr’, the amino acid TgTwinScan gene predictions, and the clustered EST databases using the TurboSequest algorithm (36) in the Bioworks 3.1 software package (Thermo Finnigan). The identified peptides were further evaluated using charge-state versus cross-correlation number (Xcorr). The criteria for positive identification of peptides were Xcorr > 1.5 for singly charged ions, Xcorr > 2.0 for doubly charged ions, Xcorr > 2.5 for triply charged ions (37). Low scoring peptide matches (Xcorr < 1.9/2.2/3.1 for +1/+2/+3, respectively) were verified by manual inspection of MS/MS spectra. Additionally, only proteins with at least two peptides of distinct sequence identified were accepted.

In silico analysis of identified proteins - All proteins identified by any of the three proteomic approaches were subjected to multiple in silico analyses. Predicted localization of proteins was assigned primarily through previously published localization and subsequently by several algorithms: signal peptide (SignalP 3.0) (38), apicoplast targeting (PATS) (39), mitochondria targeting (PlasMit) (40), and nuclear localization sequence (PredictNLS) (41). Proteins with predicted signal peptides were also analyzed for the presence of transmembrane domains (TMHMM) (42) and GPI anchor sites (DGPI) (http://129.194.185.165/dgpi/index_en.html). Protein function was assigned based on published results. Novel proteins were assigned putative function if strong homology over long stretches of the protein (based on BLASTP with default settings against NCBI non-redundant database) was found to a protein of known function.

Ortholog identification - To identify orthologs to ESA proteins we followed the approach used to generate the TIGR orthologous gene alignments (TOGA) (43). ESA amino acid sequences were BLASTed against available apicomplexan TIGR gene indices (44) using WUBLAST tblastn and for sequence matches with E-values < 10^-5 the percent conservation of the best high-scoring pair (HSP) was recorded. Putative orthology was assigned if the reciprocal BLAST (blastx) using identical stringency against the Toxoplasma TgTwinScan amino acid gene predictions identified the original query as the top hit. As an alternate approach we identified the protozoan OrthoMCL (45) ortholog clusters containing identified ESA proteins and retransformed the relative similarity scores among proteins into the original BLAST E-values.

Expression and analysis of YFP fusion proteins - Several novel ESA predicted to be in the secretory pathway were cloned using primers designed against the TgTwinScan gene predictions and Cterminally fused to YFP (in destYFP/sagCAT, (46)) using the Gateway cloning system (Invitrogen). Both extracellular and intracellular wild-type parasites were transiently transfected and grown in HFF cells. Extracellular parasites were isolated by membrane filtration. Parasites were fixed in solution for 15 min with 2.5% formaldehyde/0.02% gluteraldehyde, washed three times with PBS for 5 min, and extracellular parasites were permitted to settle on poly-lysine coated chamber-slides for 30 min. Slides were blocked with PBS/10% FBS for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. For mitochondrial staining isolated extracellular tachyzoites were incubated for 15 min in media containing Mitotracker Red (1:10,000, Molecular Probes/Invitrogen, Carlsbad, CA, USA) before fixation. For immunofluorescence, slides were incubated with primary antibody in PBS/1%FBS/1%NGS for 60 min, washed, stained with the appropriate Alexa-594 conjugated secondary antibody (1:500, Molecular Probes/Invitrogen, Carlsbad, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 5 μg ml^-1, Sigma), washed again, and mounted using mowiol. The following primary antibodies were used at the indicated dilutions: mouse monoclonal anti-GRA2 (Tg17-179; 1:400), rabbit anti-GRA7 (kind gift of Isabelle Coppens; 1:400), rabbit anti-ROP2 (kind gift of Con Beckers; 1:2000) for extracellular parasites, mouse monoclonal anti-ROP2,3,4 (T34A71C8; 1:1000), and mouse monoclonal anti-AMA1 antibody (B3.90; 1:1000). Images were captured under 600X or 1000X magnification using a Nikon Eclipse E800 microscope equipped with a Spot RT CCD camera. Images were deconvoluted using SimplePCI (C-Imaging Systems, Cranberry Township, PA, USA).

RESULTS

Toxoplasma secretory products have been implicated in parasite entry, intracellular survival, and interaction with the host (reviewed in (47)). Regardless of whether they are initially released in a soluble or membrane associated form, many of these products ultimately accumulate in ESA as a result of proteolytic shedding from the parasite surface (48-50).

ESA proteins were recovered from the medium after stimulating parasite secretion with 1% ethanol treatment (51). To maximize ESA product discovery, we employed several strategies that are outlined in Fig. 1. The first approach was to separate proteins by 2-DE coupled with identification either by N-terminal sequencing (Fig. 1A), which was primarily used for alkaline proteins, or by MALDI-MS and peptide mass fingerprinting (PMF) (Fig. 1B). The second approach was to use multi-dimensional liquid chromatography to separate tryptic peptides inline with ESI-MS/MS to acquire tandem MS data (Fig. 1C). ESA proteins were identified by matching the acquired N-terminal sequence, MALDI/PMF, or ESI-MS/MS data with sequences in the T.gondii subset of the NCBI non-redundant database, T. gondii EST, or gene prediction (TgTwinScan) databases using several search algorithms including BLAST, ProteinProspector (52)(51), Mascot (35), and SEQUEST (36). As subset of the novel putative secretory proteins identified in these screens was then expressed in a YFP fusion construct to test for localization within the parasite secretory pathway.

Fig. 1.

Schematic diagram showing the proteomic strategies used in this study.As described in Experimental Procedures, purified tachyzoites were treated with 1% ethanol to induce secretion and parasite products were collected from culture supernatants (ESA). ESA proteins were concentrated in a 10 kDa retentate and used for each of three analysis schemes. (A) ESA proteins were resolved on pH 3-10 2-DE gels and spots in the alkaline region were excised for N-terminal sequencing (after transfer to PVDF membranes) or for in-gel trypsin digestion and analysis by MALDI-MS. The acquired PMF data were used to search against T. gondii databases using ProteinProspector or Mascot. (B) ESA proteins were separated on pH 4-7 2-DE gels and analyzed by MALDI/PMF as above. (C) For MudPIT analysis, ESA protein mixtures were digested by trypsin and the peptide mixtures were separated by 2-dimensional chromatography using a strong cation exchange (SCX) column in tandem with a reverse phase (RP) column. Eluted peptides were analyzed by ESI MS-MS. The tandem mass spectra generated were correlated to theoretical mass spectra generated from T. gondii EST or predicted gene sequences using TurboSEQUEST. Finally, intracellular localization of a subset of the novel putative secretory proteins was assessed by expression of YFP fusion proteins in Toxoplasma tachyzoites.

To view a wide profile of extracellular products, ESA proteins were initially resolved on broad pH range IEF strips (pH 3-10). Since only a minority of species occupied the alkaline region of the gel, these proteins were identified by a combination of N-terminal sequencing (Fig. 2A) and MALDI/PMF (Fig. 2B). Note that different electrophoresis systems were used in Fig. 2A and 2B/C. Twelve spots were subjected to N-terminal microsequencing and matched to their cognate genes by BLAST (Table 1). Seven of the twelve spots were surface antigen (SAG) proteins; a family of glycosylphosphatidyl inositol (GPI) anchored proteins that abundantly occupy the parasite surface. SAG1 and SAG2 each migrated as three distinct species. Each of the SAG1 species possessed the same N-terminal sequence suggesting that the charge heterogeneity is not due to differential cleavage of the N-terminus. On the other hand, N-terminal sequences of the SAG2 species were slightly offset in a manner that would alter both the size and charge of the protein, potentially resulting in the distinct migration of these species. Their presence in the ESA suggests that SAG proteins are shed from the parasite surface, possibly contributing to their well known hyper-immunogenicity and pro-inflammatory properties (53). One novel protein, termed P40, was identified by N-terminal sequencing. This product is discussed in greater detail below.

Fig. 2.

2-DE profiles of Toxoplasma ESA protein.The gels were stained by colloidal Coomassie Blue the identified spots are marked directly with the name of the protein. (A) First dimension, pH 3-10 IEF strip (Bio-Rad); second dimension, 10-20% gradient SDSPAGE gel. (B) First dimension, pH 4-7 IPG strip (Amersham); second dimension, 12.5% SDS-PAGE gel. (C) First dimension, pH 3-10 IPG strip (Amersham); second dimension, 12.5% SDS-PAGE gel.

Table 1.

N-terminal sequences of alkaline ESA proteins

Protein	Empirical Mr/pI	Theoretical Mr/pI	N-term. Seq.
SAG1	30/6.6	30/6.3	SDPPL
SAG1	30/6.3	30/6.3	SDPPLVANQVVT
SAG1	30/5.9	30/6.3	SDPPLV
SAG2	13/8.0	14/7.6	TPAPIECTAGATKTV
SAG2	14/6.3	14/6.4	ETPAPIEXTAGA
SAG2	15/6.1	15/6.4	STXETPAPIEXT
SAG3	39/6.0	35/6.5	XKSXSKITYF
MIC11 C-term.	9.0/8.2	6.7/8.2	KFVDTLKTLAKGALK
P40	34/8.4	40/8.4	KMPAVVKKVK
Cyp18	13/6.0	18/6.1	ENAGVRKAYMDI
Cyp20	15/8.4	19/8.7	XNPXVFFDI
SOD	23/7.5	23/6.9	VFTLPPLPYAHDALAPXISS

As shown in Figure 2B, several additional proteins in the alkaline region were identified by MALDI/PMF including GRA2, a dense granule protein necessary for maintaining the intravacuolar network of membranes within the parasitophorous vacuole (13). Also present in the alkaline region was ROP9 (p36), a protein of unknown function (54) and the only rhoptry derived protein identified in this study. A parallel study of the rhoptry proteome by Bradley and colleagues (this issue) provides a comprehensive analysis of this unique subset of secretory products.

Since the migration of ESA proteins was strongly skewed toward the acidic region of the broad pH range gels, we used high-resolution narrow pH range IEF strips (pH 4-7) to further separate the protein mixture (Fig. 2C). These narrow range gels revealed ∼100 distinct spots, of which 55 were successfully matched to 35 distinct proteins by MALDI/PMF analysis (Table 2). Identified proteins were classified into several main categories including MIC proteins (MIC1, 2, 4, 5, 6, 8, 10, 11, M2AP, AMA1, and SUB1), dense granule proteins (GRA1, 2, 5, 7, TgPI-1), surface antigens (SAG1, 2, 3, SRS1), and other secretory proteins (PDI, Cyp18). Also, 2-DE/MS revealed the extensive proteolytic modifications that proteins such as M2AP, MIC2, MIC11, MIC4, and SUB1 undergo. In addition to previously identified proteins, several novel hypothetical proteins were discovered as a result of searching against the Toxoplasma databases using PMF data (Table 2). In general, this gel based approach provided the advantage of visualizing the complexity and heterogeneity of ESA products but was limited by relatively low sensitivity since many of the low abundance species were not successfully identified.

Table 2.

Proteins identified by 2-DE and N-terminal microsequencing or MALDI/PMF

Localization	Name	NCBI GI No.	Methoda	Putative function	Reference
Microneme	MIC1	CAA96466	P	Lactose-binding lectin	(57,89)
	MIC2	AAB63303	P	Glycosaminoglycan binding	(59,68,90)
	MIC4	AAD33906	P	Host cell binding	(58)
	MIC5	CAA70921	P	Unknown	(60)
	MIC6	AAD28185	P	Escorter	(65)
	MIC8	AAK19757	P	Host cell binding	(50)
	MIC10	AAG32024	P	Unknown	(62)
	MIC11	AAN16379	P,N	Unknown	(91)
	M2AP	AAK51546	P	MIC2 accessory protein	(64,66)
	AMA1	AAB65410	P	Unknown	(61)
	SUB1	AAK94670	P	Unknown	(63)
Dense	GRA1	P13403	P	Unknown	(92)
granule	GRA2	P13404	P	Biogenesis of intravacuolar membranes	(13,93)
	GRA5	Q07828	P	Unknown	(94)
	GRA7	AAC48314	P	Immune protection and parasite virulence	(14,95)
	TgPI-1	AAF32427	P	Serine protease inhibitor	(71,72)
Rhoptry	ROP9	CAC04420	P	Unknown	(54)
Surface	SAG1(P30)	CAA32245	P,N	Immune modulation	(81,96)
	SAG2(P22)	A44968	P,N	Unknown	(97)
	SAG3	AAO72428	P,N	Host cell attachment	(78)
	SUS1	AAC47762	P	N/A	(98)
Others (ER, cytosolic, etc)	PDI	CAC28361	P	Immunodominant antigen in mucosal immune response	(82,99)
	Cyp18	AAA17997	P,N	PPIase; Immune response modulator	(83,100)
	Cyp20	AAA17998	P,N	PPIase	(83)
	Enolase	AAG60329	P	Glycolytic enzyme	(101)
	14-3-3	BAA25996	P	Signal transmitter	(102)
	Mul adaptin	AAM77470	P	Vesicular transport	(103)
	HSP70	AAC72001	P	Chaperone; protection against cell damage associated with invasion	(104,105)
	SOD	AAC63943	N	antioxidant	(106)
	Peroxiredoxin	AAG25678	P	antioxidant	(107)
	Aldolase	AAN75043	P	glycolysis; motility	(108)
	Catalase	Q9XZD5	P	antioxidant	(109)
Novel Proteins	P40	27723986	N	unknown	N/A
	A30/9.0	TgEST_1001 21304; TgTwinScan_2359	P	Host-cell binding?	N/A
	A70/6.0	Tg9x_99429 0_gs_1 TgTwinScan_0069	P	unknown	N/A
	A40/4.5	Tg9x_99463 5_gs_1	P	unknown	N/A
	A50/7.0	Tg9x_99425 0_gs_3	P	unknown	N/A
	A11/6.5	TgTwinScan_1327	P	unknown	N/A
	A25/6.8	TgTwinScan	P	unknown	N/A

^aIdentified either by MALDI/PMF (P) or N-terminal microsequencing (N)

As a complementary approach to 2-DE/MS, the chromatography-based proteomic method MudPIT was used to analyze ESA proteins. ESA proteins were trypsin-digested, subjected to two-dimensional LC-MS/MS analysis and identified by searching the un-interpreted product ion spectra against T. gondii clustered ESTs, the predicted genes (TwinScan), or the T. gondii subset of the NCBI non-redundant database using the TurboSequest algorithm (36). A total of 62 proteins were identified (Table 3). Strikingly, this approach resulted in the identification of additional proteins that were not detected on 2-DE gels such as GRA3, NTP1, NTP2, NTP3, SRS2, and others. Moreover, several novel proteins emerged including most notably a secreted metalloprotease of the insulinase family (TgTwinScan_4000), one or more of a family of Apple/PAN domain containing proteins (TgTwinScan_2357, 2358, 2359, 2361) similar to E. tenella MIC5 and T. gondii MIC4, and three proteins (TgTwinScan_0203, 3857, 6350) with significant homology to hypothetical proteins of the human malaria parasite, P. falciparum. Also detected were several additional proteins (TgTwinScan_1114, 1327, 2489, 3416) without homology to proteins or domains in the public databases. These proteins represent novel Toxoplasma-specific products that may contribute to the unique biological features of this parasite.

Table 3.

Proteins identified by MudPIT

				Predicted
Sequence ID	Name/Best NCBI BLAST Hit (E<10−5)	Putative Function	Reference	Targeting#	nr	Twin-scan	EST
Micronemes
gi|2062142	MIC1	Lactose-binding lectin	(57,89)	S	10	9	10
gi|1923217	MIC2	Glycosaminoglycan binding	(59,68,90)	S	10	9	10
gi|4929214	MIC4	Host cell binding	(58)	S	11	11	9
gi|1806541	MIC5	Unknown	(60)	S	5	5	5
gi|4704627	MIC6	Escorter	(65)	S	5	5	5
gi|11141615	MIC10	Unknown	(62)	A(S)	5	7	7
gi|23267147	MIC11	Unknown	(91)	S	9	9	10
gi|13991884	M2AP	MIC2 accessory protein	(64,66)	S	5	5	6
gi|2293476	AMA1	Unknown	(61)	S	10	11	11
gi|15419013	SUB1	Proteolysis	(110)	S	23	2	22
Rhoptries
gi|9909994	ROP9	Unknown	(54)	C(S)	3	2	4
Dense Granules
gi|129322	GRA1	Unknown	(92)	S	8	4	4
gi|2506908	GRA2	Biogenesis of intravacuolar membranes	(93) (13)	S	3	3	2
gi|2498422	GRA3	Unknown	(111)	S	2	2	3
gi|3023908	GRA7	Unknown	(14,95)	S	7	6	8
gi|2499220*	NTP1	Unknown	(112,113)	S	16*	16*	16*
gi|2499221	NTP2	Unknown	(112,113)	S	16	9	16
gi|2702317*	NTP3	Unknown	(112,113)	S	15*	9*	15*
Surface
gi|37778533	SAG1	Immune modulation	(81,96)	S	9	6	9
gi|37778535	SAG2	Unknown	(97)	S	4	-	4
gi|37778537	SAG3	Host cell attachment	(78)	S	5	2	3
gi|1718389	SRS1	Unknown	(98)	S	2	2	2
gi|2305260	SRS2	Unknown	(98)	S	2	2	2
Secretory
gi|11125362	Ferredoxin NAPD oxidoreductase	Unknown	(114)	Api	2	-	-
gi|14530190	PDI	Chaperone, Mucosal Antigen	(82)	S	2	2	2
gi|22035892	G6PDH	Glycolytic enzyme		S	5	2	5
Cytoplasm
gi|1695772	LDH1	Glycolytic enzyme	(115)	C	4	4	4
gi|13377044	G3PDH	Glycolytic enzyme	(116)	C	2	2	3
gi|1703160	Actin	Cytoskeleton/Motility	(117)	C	3	3	3
gi|44889871	eIF2a kinase A	Translation/Stress response	(118)	C	3	2	-
gi|27817640	Ser/Thr Phosphatase 2C	Cytoskeleton/Motility	(119,120)	C	2	2	-
gi|3746358	SOD	Antioxidant	(106)	C	2	2	2
Secretory (predicted)
TgTwinScan_2357*	Microneme protein 5 [E.t.]	Adhesion/Invasion	Novel Protein	S	-	2*	2*
TgTwinScan_2358*	Microneme protein 5 [E.t.]	Adhesion/Invasion	Novel Protein	S	-	2*	-
TgTwinScan_2359*	Microneme protein 5 [E.t.]	Adhesion/Invasion	Novel Protein	S	-	2*	2*
TgTwinScan_2361*	Microneme protein 5 [E.t.]	Adhesion/Invasion	Novel Protein	S	-	2*	-
TgTwinScan_4000	Secreted insulinase-like peptidase [C.p.]	Proteolysis	Novel Protein	S	-	10	2
TgTwinScan_1683	Amine Oxidoreductase [H.s.]	Unknown	Novel Protein	S	-	2	-
TgTwinScan_2661	P40	Unknown	Novel Protein	S	-	12	11
TgTwinScan_0203	Hypothetical protein [P.f.]	Unknown	Novel Protein	S	-	2	2
TgTwinScan_6350	Hypothetical protein [P.f.]	Unknown	Novel Protein	S	-	7	4
TgTwinScan_2489	No Hit in ‘nr’	Unknown	Novel Protein	S	-	2	2
TgTwinScan_1114	No Hit in ‘nr’	Unknown	Novel Protein	S	-	2	2
TgTwinScan_1327	No Hit in ‘nr’	Unknown	Novel Protein	S	-	4	5
TgTwinScan_3416	No Hit in ‘nr’	Unknown	Novel Protein	S	-	2	2
Cytoplasmic (predicted)
TgTwinScan_0458*	Elongation factor 1-alpha [C.p.]	Translation/Cytoskeleton/Motilty	Novel Protein	C	-	7	7
TgTwinScan_3553*	Elongation factor 1-alpha [C.p.]	Translation/Cytoskeleton/Motilty	Novel Protein	C	-	7	7
TgTwinScan_3857	Perforin-Like Protein [P.f.]	Unknown	Novel Protein	C(S)	-	6	-
TgTwinScan_0397	Chitinase-like protein [G.h.]	Unknown	Novel Protein	C(S)	-	2	-
TgTwinScan_0436	Similar to Dynein heavy chain [D.d.]	Cytoskeleton	Novel Protein	C	-	2	-
TgTwinScan_1519	No Hit in ‘nr’	Unknown	Novel Protein	C(TM)	-	2	-
TgTwinScan_2092	LepA homolog [A.t.]	Unknown	Novel Protein	C	-	2	-
TgTwinScan_6565	RAD50 [C.p.]	DNA Repair	Novel Protein	Nuc	-	2	-
TgEST_100120118	Insulinase-like peptidase [C.p.]	Unknown	Novel Protein	C	-	-	2
TgTwinScan_2426	Hypothetical protein [C.p.]	Unknown	Novel Protein	C	-	2	-
TgTwinScan_5841	Hypothetical protein [P.y.]	Unknown	Novel Protein	Nuc	-	2	-
TgEST_95058115	Hypothetical protein [P.y.]	Unknown	Novel Protein	C	-	-	2
TgTwinScan_4526	No Hit in ‘nr’	Unknown	Novel Protein	Nuc	-	2	-
TgTwinScan_3652	No Hit in ‘nr’	Unknown	Novel Protein	Mito/Nuc	-	2	2
TgTwinScan_6494	No Hit in ‘nr’	Unknown	Novel Protein	Nuc	-	2	-
TgTwinScan_5465	No Hit in ‘nr’	Unknown	Novel Protein	C	-	2	-
Unknown
TgEST_95059642	No ‘nr’ Hit	Unknown	Novel Protein	?	-	-	3

^#S, signal; A, anchor; C, cytoplasmic; Mito, mitochondrial; Api, apicoplast. () indicates conflicting information from other sources

^*due to high sequence similarity between genes a unique hit cannot be designated based on peptide hits

? incomplete ORF

A.t., Arabidopsis thaliana; C.p., Cryptosporidium parvum; D.d., Dictyostelium discoideum; E.t., Eimeria tenella; G.h., Gossypium hirsutum; H.s., Homo sapiens; P.y., Plasmodium yoelii; P.f., Plasmodium falciparum.

As shown in Figure 3, although MudPIT yielded the highest number of proteins identified, the combination of approaches promoted maximum coverage and provided validation for those products identified by more than one strategy.

Fig. 3.

Comparative distribution of secretory proteins identified by N-terminal sequencing, 2-DE-MS and MudPIT proteomics.In total, 76 distinct proteins were identified by 2-DE-MS and MudPIT approaches. The Venn diagram shows that 17 Proteins (22%) were identified by both 2-DE proteomics and MudPIT approaches, whereas 12 Proteins (16%) were exclusively identified by 2-DE proteomics and 39 Proteins (51%) were exclusively identified by MudPIT. N-terminal microsequencing of 2-DE spots also identified 8 of the proteins found by the other approaches.

To assess localization and potential function, ESA protein sequences were analyzed by a series of algorithms designed to detect targeting signals and orthologous proteins. The absence of targeting signals in 30% of the ESA proteins (Fig. 4) suggests that some cytosolic proteins are released into the ESA as a result of inadvertent parasite lysis. As expected, the majority (58%) of ESA proteins contained a putative signal sequence or a signal anchor sequence, many of which have been previously shown to localize to the either parasite’s surface or secretory organelles.

Fig.4.

Predicted localization of identified ESA proteins. 58% percent of identified proteins were predicted to enter the secretory pathway, 8% to target to the nucleus, 3% to target to the mitochondrion, and for 30% no targeting motifs were found thus predicting cytoplasmic localization. Among the secretory proteins, 15%, 1%, and 12% have been demonstrated to reside in the micronemes, rhoptries, and dense granules, respectively. The ferredoxin oxidoreductase has been shown to reside in the apicoplast and PDI is an ER resident protein. 21% have predicted secretory signal sequences but no other targeting information was found. Localization was assigned based on published reports and available targeting prediction algorithms (Signal Sequence: SignalP, Apicoplast: PATS, Mitochondrion: PlasMit, Nucleus: PredictNLS).

As an initial screen to determine the intracellular locations of novel secretory proteins, we selected five genes (TwinScan_0203, 1327, 2359, 2489, 2661) predicted to lack a transmembrane segment or GPI anchor and cloned them in frame with a C-terminal YFP encoding expression plasmid. After transient transfection into tachyzoites, expression and localization was assessed by fluorescence microscopy of formaldehyde fixed parasites (Fig. 5). Despite multiple attempts, no expression of TwinScan_2489-YFP was observed. Together with the observation that transfection of this construct yielded morphologically defective parasites not seen with the other constructs, we conclude that expression of TwinScan_2489-YFP was toxic or otherwise poorly tolerated. TwinScan_0203-YFP localized to tubular structures resembling the parasite mitochondrion, a finding that was confirmed by co-localization with Mitotracker Red. Although this protein was predicted to contain a secretory signal sequence, it also possesses a putative mitochondrial targeting sequence which appears to be the dominant sorting element, at least in the context of this reexpression system. TwinScan_1327-YFP showed a punctuate pattern distributed throughout the parasite, which showed only limited co-localized with the dense granule markers GRA2, GRA4 (data not shown), and GRA7 in extracellular parasites. In intracellular parasites TwinScan_1327-YFP accumulated in the vacuole at the convergence of parasites’ distal ends, where it partially co-localized with the dense granule markers secreted into the vacuole. Precisely how this protein reaches the parasitophorous vacuole remains to be determined. TwinScan_2359 (encoding the only member of the novel Apple/PAN family represented by tachyzoite ESTs) and TwinScan_2661 (encoding P40) showed similar localization in the apical region. This distribution was apical to the Golgi (GRASPRFP; data not shown) and showed partial overlap with rhoptries (ROP2) and with micronemes (AMA1). This staining pattern may reflect inefficient trafficking to one of these apical organelles, with much of the protein accumulating in an intermediate compartment along the pathway. Imperfect targeting has previously been after overexpression of tagged secretory proteins in T. gondii (50). Collectively, these data confirm that several of the novel ESA products are expressed in the secretory system where they traffic to shared and distinct exocytic organelles within the parasite.

Fig. 5.

Preliminary localization of novel putative secretory products expressed as YFP fusion proteins. TgTwinScan_0203-YFP (green) co-localizes with Mitotracker Red dye in the single mitochondrion of extracellular tachyzoites. TgTwinScan_1327-YFP (green) shows punctuate localization, which overlaps to a small degree with dense granule markers (GRA4 or GRA7, red) in extracellular tachyzoites. In intracellular parasites it accumulates in the vacuole at the convergence of the parasites distal ends, potentially the residual body. TgTwinScan_2359-YFP and TgTwinScan_2661-YFP (both in green) localize to the apical end of extracellular tachyzoites with partial overlap with the rhoptries (ROP2, red) and micronemes (AMA1, red). All samples were stained for DNA with DAPI (blue). Micrographs were captured under 1000X magnification, with the exception of Twinscan_0203-YFP (600X). Scale bar 5 μm.

Signal-containing proteins were assigned to one of seven categories according to function based on previous studies or on their similarity to proteins of known function. Strikingly, proteins of unknown function constitute the largest category (54%), a reflection of the incomplete understanding of the roles fulfilled by Toxoplasma secretory products. The next largest category (24%) comprise proteins involved in adhesion/invasion, followed by proteins associated with immune evasion (7%), proteolysis (7%), and protein folding (4%), intracellular survival (2%), and glycolysis (2%).

Since apicomplexan parasites share many core biological features including an obligate intracellular lifestyle, we were interested to what degree the Toxoplasma secreted ESA proteins were conserved across the phylum. Emulating the approach used to generate the TIGR orthologous gene alignments (TOGA), we assigned reciprocal BLAST hits against the TIGR gene indices for 7 apicomplexan parasites as putative orthologs, identifying orthologs to 33 ESA proteins in one or more Apicomplexa (Fig. 6). As expected, the very closely related coccidian Neospora caninum showed the highest degree of conservation both in number of orthologs and their sequence homology. In general, after normalization with respect to the number of unique sequences in each TIGR gene index (Lowest: C. parvum 910, Highest: P. yoelli 9812), the number of orthologs identified decreased according to their phylogenetic distance from T. gondii (55). Since representation in EST collections like the TIGR gene indices is skewed by stage-specificity and abundance of transcripts, the false negative rate of this analysis is expected to be high, making a failure to detect orthologs in the other Apicomplexa less informative. To compensate for this and to expand our analysis to additional protozoa we consulted a collection of orthologous clusters generated using OrthoMCL developed by David Roos’ lab at the University of Pennsylvania. Using OrthoMCL, which utilizes a wider array of sequences, we identified dozens of additional orthologs, particularly in more distantly related organisms. Using both of these approaches we successfully identify orthologs in one or more apicomplexan parasites for 80% (35/44) of the predicted secretory ESA proteins, indicating that a number of ESA proteins are conserved within the phylum.

Fig. 6.

Phylogenetic conservation of secretory ESA proteins among apicomplexan parasites. Only proteins with at least one putative ortholog are listed. Reciprocal BLAST hits against the TIGR gene indices of six other apicomplexan parasites with E-value < 10^-5 were assigned as putative orthologs with percent positive (identical or chemically similar) conservation in the best high-scoring pair (HSP) shown and indicated in color code. The corresponding OrthoMCL ortholog clusters were used as comparison and to expand ortholog identification to additional protozoa. For the OrthoMCL clusters the negative logarithm of the BLAST expect values are color-coded to indicate the extent of conservation. Additionally, the number of genes in each species (i.e., paralogs) belonging to the orthologous gene cluster is shown. Putative orthologs identified by one but not the other method are indicated by (X). Predicted localization is also specified: microneme (Mic), rhoptry (Rho), dense granule (DG), surface (Sur), Apicoplast (Api), other secretory (OSec), unknown secretory (USec), and mitochondrial (Mito). Species abbreviations: Neospora caninum (Nc), Sarcocystis neurona (Sn), Eimeria tenella (Et), Cryptosporidium parvum (Cp), Theileria annulata (Ta), Plasmodium berghei (Pb), Plasmodium yoelli (Py), Plasmodium falciparum (Pf), Plasmodium chabaudi (Pc), Plasmodium knowlesi (Pk), Tetrahymena tetraurelia (Tt), and Cyanidioschyzon merolae (Cm).

DISCUSSION

In this study we substantially widen the perspective of Toxoplasma proteins that form the parasite’s interface with its host during infection. The efficiency with which Toxoplasma initiates infection, avoids clearance by the host’s innate immune system, and persists indefinitely in host tissues is determined in large part by the parasite’s surface and secretory products. Although the precise function of most ESA products remains poorly understood, the current expanded view of these components is an important prelude to understanding mechanisms underlying Toxoplasma:host interactions contributing to infection and disease.

A number of cytosolic proteins were identified in the ESA. Since it was necessary to prepare ESA samples in defined, serum-free medium, some inadvertent parasite lysis was expected. While most of these proteins are likely authentic cytosolic proteins, it is also possible that a subset may be incorrectly assigned due to inaccuracies of the gene assembly programs used to analyze the Toxoplasma genome, particularly the difficulty of correctly predicting the initial exon containing the signal sequence.

As expected, the largest sub-group of secretory proteins identified in the ESA is derived from the micronemes. In fact, the only known tachyzoite MIC protein not detected in this study was MIC3. This high coverage rate suggests that many of the novel ESA proteins may also be derived from MICs, a notion supported by the partial localization of recombinant TgTwinScan_2359 and TgTwinScan_2661 within these organelles. MICs are thought to participate in parasite adhesion and invasion of host cells (reviewed in (56)). Consistent with their putative role in host cell attachment, many of the MIC proteins possess recognizable adhesive domains found in vertebrate proteins such as thrombospondin, integrins, and epidermal growth factor (9). Moreover, several studies have documented the cell binding activities of adhesive MICs including MIC1, MIC2, MIC4 and MIC3 (49,57-59). However, several other MIC proteins (MIC5, MIC10, M2AP, SUB1 and AMA1) do not contain obvious adhesive sequences, suggesting these proteins have alternative or accessory functions (60-64). Another feature of Toxoplasma MICs is they often assemble into protein complexes and work in concert. For example, MIC6 partners with MIC1 and MIC4 (65), MIC8 escorts MIC3 (50), and MIC2 accompanies M2AP (64,66,67). Among these, the MIC2-M2AP complex appears to be the most abundant on the 2DE map of the ESA based on the Coomassie-stained 2-DE profile (Fig. 2), a finding that is consistent with recent evidence that this adhesion complex plays a central role in parasite adhesion and invasion (59,66,68).

In the present study, ten DG proteins (GRA1, GRA2, GRA3, GRA5, GRA7, GRA8, TgPI-1 and NTPase I, II, and III) were recovered from the ESA fraction (Table 2 and 3). DG proteins are secreted at the highest rate soon after the parasite is fully within the vacuole (69,70). In most cases, the precise functions of DG proteins remain unclear, partly because they lack significant homology with proteins of known function. Despite this, several observations strongly suggest that DGs participate in the modification of PV and its interaction with the host cytosol. For example, almost all of the known DG proteins are membrane associated proteins, which is in accordance with recent protein genetic evidence that GRA2 and GRA6 play important roles in the biogenesis of intravacuolar membranes (13). GRA7 was suggested to be involved in immune protection and a decrease in GRA7 synthesis paralleled the loss of parasite virulence (14). The T. gondii serine protease inhibitor (TgPI-1) (71) was recently confirmed as a DG protein, presumably playing an important role in protecting against the proteolytic enzymes and modulating immune response during the acute phase of infection (72). NTPase isoforms are apyrases, which were demonstrated to function in intracellular replication of the parasite based on antisense interruption of expression (73). Also, the full-scale activation of NTPase with dithiols may contribute to parasite exit from the host cell (74). It is likely that several of the novel secretory products identified herein are also derived from the DGs. Among these may be proteins of low antigenicity that would have been invisible to the antibody based screens previously used to identify and characterized DG products.

It was somewhat unexpected to find significant amounts of SAGs in the ESA, given that these proteins are normally tethered to the parasite surface via a GPI anchor (75). At this time it is unclear how these SAGs are released into the ESA and several possible explanations can be made. We speculated that the SAGs are soluble in the ESA because a small fraction of these proteins may have failed to acquire a GPI anchor, were liberated by a parasite phospholipase, or were proteolytically released from the surface. Alternatively, it is possible that SAGs are released by surface membrane blebbing, as has been observed for Toxoplasma (76) and other pathogenic protozoa (77). As for the function of SAGs in invasion, a SAG3-null mutant (78) showed an approximate two-fold reduction in host cell invasion and partially attenuated infectivity in mice. This result was explained by a reduction in the capacity to attach the host cell. In addition, SAGs are one of the main targets for humoral and cell-mediated immune responses during initial infection (79-81).

The ER is the site of nascent secretory protein folding, which is facilitated by a collection of enzymes and molecular chaperones including protein disulfide isomerase (PDI), heat shock protein (HSP70) and secretory cyclophilin (Cyp). Although T. gondii PDI has not been definitively localized to the parasite ER, it displays a Cterminal sequence (GEEL) resembling an ER retention/retrieval element (K/HDEL) (82). Interestingly, T. gondii PDI was recently shown to be a major target of mucosal IgA antibodies (82), thus its excretion in a soluble form could contribute to its elevated immunogenicity. Cyp18 is a secretory cyclophilin that was initially identified as one of two major immunophilins in T. gondii (83). Cyp18 is mainly located in the ER, but a subset of intracellular parasites show significant Cyp18 staining in the PV (V.B. Carruthers, unpublished). In addition to its role as a foldase, Sher and co-workers (16) recently demonstrated that Cyp18 stimulates dentritic cells to secrete IL-12 thereby contributing to the robust pro-inflammatory response often seen during Toxoplasma infection.

Several novel secretory proteins were identified in our analysis of ESA fractions. The sequences of some of these proteins are highly conserved in the Apicomplexa, implying that they fulfill parallel roles in the biology of these parasites. For example, we identified a novel insulinase-like metalloprotease (TwinScan_4000), which may contribute to tissue damage or migration/dissemination during infection. Also, MudPIT revealed a family of proteins containing adhesive cysteine-rich Apple/PAN domains that are highly analogous to that of E. tenella MIC5 (84). Although genes encoding this family are tandemly linked in the genome, analysis of EST sequences suggests that they are stage regulated since only one member (TwinScan_2359) is expressed in tachyzoites. A YFP fusion to this tachyzoite specific product localizes to an apical compartment partially overlapping with micronemes and rhoptries. Additional studies will be required to determine its precise localization and whether this protein is mobilized to the parasite surface during invasion where it could contribute to host receptor binding via one or more of its Apple/PAN domains.

Apicomplexan specific proteins that are conserved across the phylum likely play fundamentally important roles the biology of these intracellular parasites. Our phylogenetic analysis of ESA products revealed extensive conservation of AMA1 and MIC2 (TRAP family), which is consistent with recent studies (67,68,85,86) strongly implicating these proteins in cell invasion. Interestingly, ROP9 is also well conserved implying that this protein may play a central, albeit still shrouded, role in the Apicomplexa. Among the novel ESA products that are particularly well conserved is TgTwinScan_3857, which encodes a protein containing a membrane attack complex (MAC)/perforin domain. Although similar proteins are expressed by Plasmodium and have been recently reported to be required for migration through cells and tissue in the mosquito and mammalian hosts (87,88), the precise molecular mechanisms by which they fulfill this role remains to be determined.

In summary, the current study illuminates a diverse legion of secretory proteins that contribute to parasite survival and pathogenesis during infection. Novel parasite-specific proteins identified in this screen significantly expand the number of potential targets for therapeutic intervention or diagnostic gain. We anticipate that ongoing in-depth functional analysis of identified novel proteins would further unravel the invasive and pathogenic mechanisms used by Toxoplasma during infection.