Comprehensive Proteome Analysis of the Excretory/Secretory Proteins of Toxoplasma gondii

Abstract

Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8 species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/secretory proteins-GRA1, GRA7 and GRA2-were confirmed to be highly expressed in T. gondii using the spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the penetration and survival of T. gondii.

Introduction

Toxoplasma gondii is a member of Apicomplexa phylum and is the causative agent of toxoplasmosis.1 It is a widely distributed protozoan parasite that infects various warmblooded animals, including humans. Decoster et al. (1988) first described several excretory and secretory antigens (ESAs) of T. gondii in the sera of toxoplasmosis patients.2 ESA have been studied with regard to cell-mediated immunity,3-6 cell biology,7-10 and biochemical process.11,12 Most ESAs of T. gondii are released from highly specialized secretory organelles such as micronemes, rhoptries, and dense granules, which are involved in the penetrating process and the intracellular interactions within the parasitophorous vacuole (PV). The micronemes secrete a collection of adhesion proteins, termed microneme proteins (MICs) that mediate host cell entry.13-15 Rhoptry proteins (ROPs) and lipids form the junction moving during invasion,16 eventually resulting in a parasitophorous vacuole that enables the efficient procurement of nutrients and the evasion of host immune defenses.17,18 Dense granule proteins (GRAs) appear to facilitate the formation of specialized tubules that enable nutrient acquisition by the parasite, and some of these proteins, most notably GRA7, are also secreted into host cells.19 ROPs are also injected into the host cell, resulting in the extensive modification of host gene expression and signaling pathways.20 Eui-Sun Son and Ho woo Nam (2001), attempted to profile the production, subcellular localization, and exhaustion of excretory/secretory proteins (ESPs). This demonstrates that they play an essential role in providing the appropriate environment for the entry of the parasite into host cells.21 A couple of proteins have been identified in ESP including a 42 kD protease,22 GRA3 and GRA10.23 In a recent report on ESP, manipulation of the host PI3K/Akt signaling pathway and Nox4 gene expression is influenced by ESP, and this mechanism is involved in T. gondii survival and proliferation.24

Using two dimensional electrophoresis (2DE) and multidimensional protein identification technology, approximately 100 spots of ESPs related to host cell invasion could be identified. Many proteins were present in multiple spots, consistent with the presence of post-translational modifications.25,26 In a similar study, A23187 used to stimulate calcium mediated ESP from T. gondii. As a result, total of 213 protein spots were identified by 2DE.27 Purified microneme proteins, which play in long or short range interactions during parasite attachment and entry, have also been analyzed using proteomic techniques.28,29 Although there has been a substantial amount of T. gondii proteomics research, an extensive study on the identification of ESPs has not been reported.

In this study, we performed proteomic analysis of ESPs in the RH strain of T. gondii, identified 34 proteins, and analyzed their abundance.

 

Experimental

Chemicals. Acrylamide and bis-acrylamide cocktail solutions were obtained from Bio-Rad (USA). The chemicals 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and dithiothreitol (DTT) were purchased from GE Healthcare (USA). Urea, thiourea, iodoacetamide, and the other reagents for the polyacrylamide gel preparation were from Sigma (USA). All other chemicals were acquired from standard sources and were of molecular biology grade or higher.

Preparation of ESP. The RH strain of T. gondii was maintained by peritoneal passages in Balb/c mice. Tachyzoites were purified by centrifugation over 40% Percoll (GE Healthcare, USA) in PBS solution.21 Purified tachyzoites (3 × 108) were incubated at 37 °C for 1 h under mild agitation in 1.0 mL Hank’s balanced salt solution (Gibco BRL, USA). After centrifugation for 5 min at 6,000 rpm, the supernatant containing ESPs was recovered.

Western Blot Analysis. ESPs were separated in 12% SDS-PAGE gels and transferred onto nitrocellulose sheets (NC, Schlleicher and Shuell, Keene, USA). NC papers blocked by 5% skim milk in PBS/0.05% Tween-20 were incubated with total anti-T. gondii polyclonal antibody diluted 1:1,000, and then with 1:3,000 diluted HRP-conjugated goat antimouse IgG antibody (Cappel, Costa Mesa, USA). They were soaked in enhanced chemiluminescence (ECL) solution (Intron, Korea) for 1 min and exposed to an X-ray film for visualization (Konica, Japan).

Enzymatic In-gel Digestion. The proteins separated by SDS-PAGE were excised from the gel, and the gel pieces containing protein were destained with 50% acetonitrile (ACN) containing 50 mM NH4HCO3 and vortexed until CBB was completely removed. These gel pieces were then dehydrated in 100% acetonitrile and Vacuum-dried for 20 min in CentriVap® DNA Centrifugal concentrator. For the digestion, gel pieces were reduced using 10 mM DTT in 50 mM NH4HCO3 for 45 min at 56 °C, followed by alkylation by 55 mM iodoacetamide in 50 mM NH4HCO3 for 30 min in dark. Finally, each gel pieces were treated with 12.5 ng/μL sequencing grade modified trypsin (Promega, USA) in 50 mM NH4HCO3 buffer (pH 7.8) at 37 °C for overnight. Following digestion, tryptic peptides were extracted with 5% formic acid in 50% ACN solution at room temperature for 20 min. The supernatants were collected and dried by CentriVap® DNA Centrifugal concentrator. The samples were purified and concentrated in 0.1% formic acid using StageTip C18 (Thermo, German) before MS analysis.

Liquid Chromatography and Mass Spectrometric Analysis. The tryptic peptides were loaded onto a fused silica microcapillary column (12 cm × 75 μm) packed with C18 reversed phase resin (5 μm, 100 Å). LC separation was conducted under a linear gradient as follows: a 3-40% solvent B (0.1% formic acid in 100% ACN) gradient, with a flow rate of 400 nL/min for 60 min. The column was directly connected to an LTQ linear ion-trap mass spectrometer (Finnigan, USA) equipped with a nano-electrospray ion source. The electrospray voltage was set at 1.80 kV, and the threshold for switching from MS to MS/MS was 2000. The normalized collision energy for MS/MS was 35% of the main radio frequency amplitude (RF) and the duration of activation was 30 ms. All spectra were acquired in datadependent scan mode. Each full MS scan was followed by five consecutive MS/MS scans corresponding to the five most intense peaks in the full MS scan. Repeat count of peaks for dynamic exclusion was 1, and its repeat duration was 30 sec. The dynamic exclusion duration was set to 180 sec and the width of exclusion mass was ± 1.5 Da. The list size of dynamic exclusions was 50.

Database Searching. All MS/MS samples were analyzed using SEQUEST (Thermo Fisher Scientific, USA; version v.27, rev. 11). SEQUEST was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 3.0 Da. Carbamidomethylation of cysteine was specified in SEQUEST as a fixed modification. Oxidation of methionine was specified in SEQUEST as a variable modification. Scaffold (version Scaffold_4.2, Proteome Software Inc., USA) was used to validate MS/MS based peptide and protein identifications (Peptide/Protein FDR 1%) which contained at least 2 identified peptides. SEQUEST was used to search the TgondiiRH_ORFs_Anotated_GRA_ROP_MIC_Uniprot _Mouse_Contaminants.fasta database (248,268 entries including decoy sequences) assuming that the peptide fragments were resulted from trypsin digestion. The T. gondii RH protein database was reconstructed with TgondiiRH_ORFs fasta (from http://toxodb.org/toxo/showApplication.do), GRA/ROP/MIC.fasta, and mouse.fasta from UniportKB (http://www.uniprot.org). The protein abundance and rank estimation were defined by the previously described Fabb index.30,31

The putative function of proteins, which were not listed in UniProtKB, was further analyzed using Pfam (http://pfam.xfam.org) and gene ontology databases (http://www.ebi.ac. uk/ego/).

Criteria for Protein Identification. Scaffold (version Scaffold_4.3.2, Proteome Software Inc., USA) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 81.0% probability to achieve a false discovery rate (FDR) less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 98.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.32 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony

 

Results and Discussion

Western Blot Analysis. When detected using an anti T. gondii polyclonal antibody, about 20 bands appeared in the total ESP from T. gondii (Fig. 1). We confirmed proper separation of ESP using western blot analysis results compared with a reference.21 The molecular mass of 15 ESP were estimated as 110, 97, 86, 80, 70, 60, 54, 42, 40, 36, 30, 28, 26, 22, and 19 kD, which were compatible with the previously described ESA of T. gondii,3,4 with minor differences. The protein samples identified by western blot analysis were stored at −80 °C until analyzed.

Figure 1.Western blot analysis of the ESPs of the Toxoplasma gondii (RH) strain. The ESP bands on nitrocellulose membrane were reacted with a reference serum from a mouse infected with RH tachyzoites. ESP bands were detected by anti T. gondii polyclonal antibody. M, size marker; lane 1 and 2, two-batch samples of ESP; lane 3, Total T. gondii lysate.

Identification of ESP by LC/MS/MS Analysis. The ESPs were digested by trypsin and the resulting peptides were analyzed by LC/MS/MS. A total of 314 proteins were identified by MS analysis. Among them, 280 proteins were identified as mouse proteins and only 34 proteins were confirmed to be derived from T. gondii. The reason was that the ESP were obtained from the abdominal cavity of mice. Mice proteins outnumbered T. gondii by far, therefore ESP identification was difficult, relatively. The 34 identified proteins are listed in Table 1 along with protein name, molecular weight, number of unique peptides, number of total spectra, number of estimated protein copy and rank by abundance. Among them, 16 proteins registered in the UniProtKB were listed in Table 2 along with protein name, alternative name, UniProtKB ID, function by Gene Ontology database, and reference. A total of 18 species of protein, including TgESP-1, were newly identified in this study. The amino acid sequences of these 18 proteins were described in Support Information 1.

Table 1.The list of identified proteins found in ESP of Toxoplasma gondii

Table 2.The list of reported protein on UniProtKB

Among the identified proteins, proteins related to the hostparasite interaction were of particular interest. All components of ESP are known to be released from highly specialized secretory organelles of T. gondii, micronemes (Micronemal protein, MIC), rhoptries (Rhoptry protein, ROP), and dense granules (Dense granule protein, GRA), which function in the penetrating process and during subsequent intracellular interactions within the parasitophorous vacuole (PV). From these proteins, 8 species of MIC protein, 2 species of ROP protein, and 6 species of GRA protein were identified (Table 2). The Gene Ontology database functions of these proteins are antigen presenting in the extracellular region, the factors of transport vesicles, and pathogenesis. The ROP2 protein has a kinase domain, and it is believed to be involved in protein phosphorylation.

The frequencies of the 34 proteins from T. gondii were further analyzed using the spectral count method and the abundances of the proteins are listed in Table 1. GRA1 is the most abundant protein followed by GRA7 and GRA2. Interestingly, the top 3 abundant proteins are all GRA proteins. The GRA1 protein is considerably more abundant than the other 33 kinds of identified proteins, and is more than double the abundance of GRA7 or GRA2. GRA1 has been reported to be useful as a diagnostic marker for chronic toxoplasmosis and vaccine development.33 In the case of GRA7, the T. gondii actively recruits host microtubules, resulting in the selective attraction of endo-lysosomes to the PV. GRA7 acts like a lasso that sequesters host endocytic organelles in the vacuolar space.19 The expression of GRA1 and GRA7 is much higher than the other proteins, and the function of these proteins shows their considerable importance for interactions with the host system. Some of these GRAs have been shown to be targeted to the parasitophorous vacuolar space, the parasitophorous vacuole membrane or the intravacuolar tubular network where they play significant roles in the biogenesis and modification of the parasitophorous vacuole where it interfaces with the host cell.33 TgESP-1, the 4th most abundant protein out of our identified ESPs, it has a similar sequence to elongation factor 1-alpha, suggesting it is involved in protein synthesis. We can infer that ESPs, including TgESP-1, are involved in the synthesis or modification of host proteins. GRA proteins are potent antigens that can induce strong parasite-directed T-cell and B-cell responses.34 Because of the abundance of their expression, the proteins GRA1, GRA2, GRA7 and TgESP-1 deserve to be considered as promising candidates for vaccine development.

Functional Estimation of Unknown Proteins. The cellular function of 18 newly identified proteins were not listed in the UniProtKB database (Table 3). The functions of these proteins were estimated from their structural homologues using the Pfam database, UniProt database, and Gene Ontology database. The putative functions indicated that TgESP- 1 and TgESP-4 were involved in protein synthesis; TgESP- 2, TgESP-3, TgESP-5 and TgESP-10 were involved in the cytoskeleton; TgESP-7 was involved in blood coagulation and proteolysis; TgESP-9 was involved in the oxydoreductation reaction; TgESP-11 was involved in carbohydrate metabolism; TgESP-12, TgESP-14, and TgESP-15 were involved in nucleotide synthesis; and TgESP-13 was involved in protein translation. TgESP-6 and TgESP-8 have a domain of the Kazal-type serine protease inhibitor, but the function of this domain is not recorded in the GO database. TgESP-16 has a domain common to Cytochrome C and Quinol oxidase polypeptide I, but the function of this domain is not recorded in the GO database. TgESP-17 and TgESP-18 do not have a known structural domain. TgESP-1 and TgESP-4 are structurally similar to elongation factor 1-alpha, TgESP-2 and TgESP-3 are structurally similar to profilin, TgESP-6 and TgESP-8 are structurally similar to the tachyzoite serine proteinase inhibitor, and TgESP-5 and TgESP-10 are structurally similar to actin. The function of these 18 kinds of proteins, inferred from the structural information, indicates that they are involved in the cytoskeleton, protein synthesis, proteolysis and translation. It can be considered that ESPs from T. gondii, including these 18 proteins, are actively involved in protein metabolism.

Table 3.The list of novel reported proteins

 

Conclusion

We have analyzed ESPs from T. gondii using LC/MS/MS analysis techniques and estimated their abundance using spectral counting methods. A total of 314 proteins were identified, and among those there were 34 proteins identified that are ESPs of T. gondii and 18 of those proteins are novel and reported in this study for the first time. The putative functions of the 18 proteins indicate they are involved in protein metabolism, oxidoreductation, carbohydrate metabolism, and nucleotide binding and protein translation. The 34 proteins identified are listed by abundance, and among the top 3 most abundant proteins are GRA proteins. These quantitative results revealed that GRA proteins are important for the penetration and survival of T. gondii. In addition, these high abundant proteins deserve to be considered as promising candidates for anti-parasite drug or vaccine development.