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Cell Host Microbe. Author manuscript; available in PMC Oct 20, 2012.
Published in final edited form as:
PMCID: PMC3254672
NIHMSID: NIHMS332451

The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries

Summary

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either “intracellular” or purified away from host material. Using liquid chromatography and tandem mass-spectrometry we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly both parasites have phosphorylated tyrosines and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This dataset provides important information on the role of phosphorylation in the host-pathogen interaction, and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.

Introduction

The intracellular apicomplexan parasites, Plasmodium falciparum, the causative agent of malaria, and Toxoplasma gondii, the causative agent of toxoplasmosis, rely on specialized pathways to enter, live within and leave a host cell. Host cell invasion is a rapid process that entails complex signaling events within the parasite. Once within a host cell, the parasites reside within a parasitophorous vacuole that is extensively modified, likely for the acquisition of nutrients and evasion of the host’s immune response. All of these processes are driven by parasite proteins, many of which are stored within specialized organelles, the rhoptries, micronemes and dense granules. Secretion of these organelles during exit from the old cell and invasion of a new cell is highly regulated and apparently conserved between the two organisms (Boothroyd and Dubremetz, 2008; Cowman and Crabb, 2006).

While the detailed mechanisms underlying this regulation are not yet known, mounting evidence implicates phosphorylation as critical to these events. For example, protein kinases such as protein kinase A (PKA), protein kinase G (PKG), calcium-dependent protein kinases (CDPKs) and rhoptry kinases (ROPs) injected into the host cell have been shown to be critical in invasion, host cell remodeling and/or host cell manipulation (Billker et al., 2009; Dvorin et al., 2010; Fentress et al., 2010; Leykauf et al., 2010; Lourido et al., 2010; Moon et al., 2009; Saeij et al., 2006; Steinfeldt et al., 2010; Sugi et al., 2010; Tewari et al., 2010). Despite their importance, however, we know little about how parasite kinases and their targets are dynamically regulated and which proteins they phosphorylate, either within, or beyond the parasite’s boundaries. Phosphorylation of some parasite proteins has been shown to be essential for survival (Green et al., 2008; Joyce et al., 2010; Leykauf et al., 2010; Treeck et al., 2009; Zhang et al., 2010) and the identification of three Toxoplasma proteins (ROP2, ROP4, GRA7) that are phosphorylated after injection into the host cell (Carey et al., 2004; Dunn et al., 2008) further implicates phosphorylation as a possible means for regulation after secretion. To date, however, only a few phosphorylated parasite proteins have been described and the exact localization of the phosphorylated residues is generally not known. As a result, targeted approaches to understand the importance of phosphorylation for the parasites have largely been precluded.

Recent advances in phosphopeptide enrichment followed by liquid chromatography and tandem mass-spectrometry (LC-MS/MS) have enabled systems-level studies of proteins regulated by phosphorylation and allowed detailed analysis of the phosphoproteomes of many uni- and multicellular organisms (Bodenmiller et al., 2007; Villen and Gygi, 2008). We have used this technology and performed phosphopeptide enrichment starting with P. falciparum schizont stages and tachyzoites of T. gondii that are either “intracellular” (i.e., still within the host cell) or that have been purified away from host material. We report here an extensive inventory of phosphorylation sites in these parasites, including the identification of phosphorylated tyrosines, as well as a method to predict phosphorylation events that occur beyond the parasites’ boundaries. This dataset will help further our understanding of the role of phosphorylation in host-pathogen interactions and our ability to target protein modifications to inhibit infection.

Results

Identification of phosphorylation sites from P. falciparum schizont stages and T. gondii tachyzoites

To perform a comparative analysis of the phosphoproteome of Plasmodium and Toxoplasma asexual forms, we chose to analyze P. falciparum schizonts (40 +/- 8 hours post-infection (hpi)) and T. gondii tachyzoite stages just prior to egress (28 hpi). These stages were chosen because: 1) they represent the fast-growing forms of each parasite; 2) they are responsible for much of the pathogenesis associated with the infections; 3) they are fully or near fully developed and so contain most, if not all of the proteins needed for efficient host cell invasion; and 4) they grow intracellularly, enabling us to also collect parasite proteins that have been introduced into the host cells. Because of the potential importance of the latter class of proteins, we divided tachyzoite-infected cultures into one sample where the parasites were first separated away from the host cell material (“purified”) and another in which the entire infected host cell was used (“intracellular”). The purified parasites are fully viable and invasion-competent and represent the form used by many investigators as a starting point for studies on invasion and pathogenesis. In addition to enabling us to tentatively identify parasite proteins that are phosphorylated within the host cell, the purified fraction also had the benefit of removing the excess of host proteins that otherwise decreased the sensitivity of our analyses (Figure 1A).

Figure 1
Generation of phosphoproteome and proteome data of P. falciparum and T. gondii parasites

To prepare the P. falciparum samples, schizont-containing red blood cells (RBCs) were purified away from uninfected cells using the magnetic properties of hemozoin that accumulates in this life cycle stage. Visual inspection of Giemsa-stained parasites showed >95% late trophozoite/schizont stages, <1% gametocytes, <3% merozoites and <1% uninfected RBCs.

Phosphopeptides and non phosphorylated peptides were preliminary seperated by SCX (strong cation exchange) chromatography (Figure 1B), enriched by IMAC (immobilized metal affinity chromatography) (Villen and Gygi, 2008) and analyzed by LC-MS/MS on a LTQ-Velos Orbitrap. Peptide identification was performed using SEQUEST (Eng et al., 1994). Phosphorylation site localization was performed using the Ascore algorithm (Beausoleil et al., 2006) which scores the relative likelihood that a given phosphorylation site was correctly assigned versus a neighboring candidate site. A site is described as confidently localized in this study if it was assigned an Ascore of 19 or higher, corresponding to a 99% relative likelihood of correct phosphate localization. All identified peptides, phosphorylation sites, the associated Ascore and other scores used for data filtering can be found in the supplemental information (supplementary Tables S1A and S1B).

P. falciparum and T. gondii possess a complex phosphoproteome, including phosphorylation of tyrosines

All datasets in this study were filtered for a peptide false discovery rate (FDR) of <1% and a protein FDR of <3% using the target-decoy method (Elias and Gygi, 2007) which resulted in the datasets presented in Figure 1C. Importantly, filtering was also performed on the phosphorylation site level (that is, we calculated the FDR separately for all phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) residues) to a site FDR of <1% as we observed a higher number of decoy-database hits for pY (27.7%) than in pS (5.4%) and pT (13.8%) in the unfiltered datasets. By treating the phosphorylated residue separately we could greatly increase the sensitivity of the dataset as a whole while maintaining highly stringent filtering criteria.

For P. falciparum, we identified 7,835 phosphopeptides with 8,463 phosphorylation sites that matched to 1,673 proteins. For Toxoplasma, “intracellular” samples (i.e., tachyzoites and their associated host cells) we could identify 11,822 phosphopeptides with 12,793 phosphorylation sites matching to 2,793 proteins. We observed almost a 2-fold increase of identified parasite phosphopeptides in the purified tachyzoite samples (21,498) with 24,298 phosphorylation sites matching to 3,506 Toxoplasma phosphoproteins (for a complete list of the identified phosphorylation sites see supplementary Table S1A). We also analyzed the flow-through material containing predominantly non-phosphorylated peptides and identified 1,964 P. falciparum proteins (10,408 peptides), 2,971 Toxoplasma proteins in intracellular tachyzoites (15,456 peptides) and 4,395 proteins in purified tachyzoites (30,563 peptides) (see supplementary Table S1B). Since we did not generate data from uninfected host cells, their phosphoproteomes will not be further discussed here.

Considering the relative frequency of localized pS, pT and pY, our data recapitulates observations made from other organisms: from all sites with an Ascore ≥19 (Figure 1D), we found that pS was the most abundant phosphorylated residue followed by pT. Phosphotyrosines, on the other hand, represented a much smaller fraction of the detected phosphorylation sites (0.51%, 0.25% and 0.24% for P. falciparum, “intracellular” T. gondii and “purified” T. gondii, respectively). The fact that pY is less abundant than pT or pS is not unexpected but the values are considerably less than seen in other organisms where pY is typically ~1-4% of all phosphorylated residues without specific enrichment of pY (Huttlin et al., 2010, Tan et al., 2009). To determine if these measured differences reflect a biological reality, the result of excessive filtering or other technical artifacts, we analyzed the phosphotyrosine content of human proteins in the intracellular tachyzoite sample. The results showed that ~1.2% of the total phosphorylation sites in the human proteins with an Ascore >19 were pY (data not shown). This is close to previously published estimates indicating that the lower percentages in the parasites are real.

Given the absence of recognizable genes encoding tyrosine-specific kinases in both parasite genomes (and an absence of apparent tyrosine-specific phosphatases in P. falciparum; (Peixoto et al., 2010; Tewari et al., 2010; Ward et al., 2004; Wilkes and Doerig, 2008)), the presence of any pY is an important finding. Tyrosine-phosphorylation is often involved in critical signaling pathways such as the MAP kinase pathway (Lim and Pawson, 2010; Mok et al., 2010; Tan et al., 2009).

Because of both the importance of this finding and their small number, we examined individual pY sites in detail. Several were in the expected positions in highly conserved proteins involved in signaling and metabolic processes (e.g. GSK-3, DYRK-kinase, MAP-kinase) strongly suggesting they were correctly identified as phosphotyrosines. In addition, manual inspection of a subset of the MS/MS spectra from all 67 pY-containing peptides indicated that at least 15 (5 from P. falciparum and 10 from T. gondii) appeared unambiguous; i.e., the observed peaks in the spectra matched the theoretical fragmentation pattern for the phosphotyrosine peptide substantially better than the theoretical fragmentation pattern of a peptide in which an alternative residue was phosphorylated (e.g., a phosphoserine or phosphothreonine; see supplementary Table S1A and supplementary Figure S1 for a representative spectrum).

It is well established that phosphorylation sites are often enriched in regions of disorder and, within these, in secondary structures predicted to be in coils (Huttlin et al., 2010; Iakoucheva et al., 2004). We thus analyzed all phosphorylation sites with high-confidence localization scores and all non-phosphorylated S, T or Y residues of all proteins identified in this study for the predicted secondary structure in the local region containing them. As observed in other organisms (Dunker et al., 2002; Huttlin et al., 2010; Iakoucheva et al., 2004; Jimenez et al., 2007), we found phosphorylation sites are preferentially found in regions of disorder and, to a lesser degree, appear somewhat enriched in secondary structures classified as coils (Figure 1E).

Opposing evolutionary forces appear to operate on phosphorylation sites in P. falciparum

The datasets described here enabled us to identify parasite-specific phosphorylation motifs. To do this, we compared the amino acid sequences surrounding P. falciparum and T. gondii phosphorylation sites (6 amino acids N- and C-terminal of the phosphorylated residue) to those found in the mouse (Huttlin et al., 2010). The results showed a very distinct amino acid context for P. falciparum (Figure 2A). Specifically, amino acids encoded by A:T-rich codons (i.e., K, N, I, F, M and Y) were over-represented relative to phosphopeptides in the Toxoplasma or murine datasets. A similar over-representation was previously noted for the P. falciparum proteome as a whole (Gardner et al., 2002) and has been presumed to reflect forces driving the P. falciparum genome toward an overall A:T content of ~80.6%.

Figure 2
Comparative analysis of phosphorylation sites

To explore this trend further, we compared the amino acid representation in the phospho-13mers compared to all 13mers, phosphorylated or not, containing a serine or threonine residue from all proteins identified in this study (Figure 2B); for a comparison to the whole predicted proteome see supplementary Figure S2A and B. Unlike T. gondii and M. musculus, there was no tendency in P. falciparum for proline-enrichment in the phopho-13mers and, similarly, essentially no depletion for the extremely high asparagine-content (~14%). T. gondii and P. falciparum both have a decrease of serine residues nearby the phosphorylation site that is not seen in the mouse data. To visualize any bias in the precise location of a given amino acid relative to the phosphorylated residues, we generated position-specific intensity maps for all three organisms (Figure 2C, supplementary Figure S2C). In P. falciparum, we observed a strong enrichment of proline in the P+1 position of both pS- and pT-containing peptides, reminiscent of classical proline-directed motifs, despite very low proline frequencies overall (Figure 2A). Furthermore, whereas asparagine (N) was enriched in the -2 and -1 positions in pT-peptides, we observed an additional enrichment of this amino acid in the pS-peptides in the +1 position. In Toxoplasma and mouse (supplementary Figure S2C), asparagine appears to be tolerated or slightly enriched in the -1 position but depleted in the +1 position. Other amino acids enriched in a position-specific manner in both parasites were, as seen in other eukaryotic phosphoproteomes, basic residues in the -3 position as well as acidic residues in the +1-+3 positions in all datasets, arguing for a strong conservation of basic and acidic kinase phosphorylation motifs.

We validated and refined the intensity map data by generation of significant phosphorylation motifs (p <0.000001), using the motif-identification algorithm Motif-X (Schwartz and Gygi, 2005) (Figure 2C; and supplementary Table S2 for further information on P. falciparum and Toxoplasma-specific motifs). As expected from the position-specific intensity maps for P. falciparum, we identified several motifs including some that are unknown in their human hosts. We divided all motifs into 6 broad classes: “acidic”, “basic”, “proline”, “hydrophobic”, “asparagine” and “other” (Figure 2D). We included hydrophobic and asparagine-directed classes to account for the substantial number (~10% in both cases) of these motifs in P. falciparum. Proline-directed phosphorylation motifs are commonly among the most prevalent phosphorylation motifs (~30% in the mouse and >35% in Toxoplasma tachyzoites). P. falciparum showed a strongly reduced number of proline-directed phosphorylation motifs (5.6%) compared to Toxoplasma and mouse. Acidic and basic phosphorylation site motifs were slightly enriched in both parasites; however, the number of motifs that could not be classified as any of the three classical motifs (“basic-“, “acidic- and “proline-directed”) was specifically increased in P. falciparum. We also identified several hybrid motifs with acidic stretches in the P+1-+6 positions and also R/K in the -3 position. We searched the identified motifs against the human protein reference database that contains all currently known human phosphorylation motifs (www.hprd.org/PhosphoMotif_finder) and identified 12 specific phosphorylation motifs from P. falciparum that are not present in the human phosphoproteome (Figure 2C, red asterisk).

Assessment of phosphorylation status of orthologous protein groups

The MS analysis of phosphorylated peptides and the flow-through that predominantly contains non-phosphorylated proteins, enabled us to determine whether phosphorylation is more common in certain functional classes of parasite proteins. We identified the fraction of proteins that are phosphorylated and associated with the nucleus, surface, mitochondrion, apicoplast, inner-membrane complex, “invasome” (a network potentially involved in host-cell invasion), “exportome” (proteins that are exported to the host cell beyond the parasite boundaries in P. falciparum), or dense granules and rhoptries (which contain the “exportome” of Toxoplasma, i.e., proteins exported into the PV and into the host cell); (all shown in supplementary Table S3). To enable this analysis, we determined the total inventory of detected proteins and phosphoproteins (see also Figure 1 and supplementary Tables S1A and S1B). We defined the total proteome identified in this study as the pool of all identified proteins (i.e., the enriched phosphopeptides and the non-phosphorylated flow-through peptides) and analyzed the overlap between this and the total predicted proteome (all proteins predicted to be encoded by the entire genome of each parasite). We identified a total of 47.3% of all predicted proteins in P. falciparum. Within these identified proteins, 66.5% were identified as phosphoproteins. For T. gondii, we identified 50.9% of the total predicted proteome, 63.9% of which were found to be phosphorylated. These numbers are comparable to what was found in a large-scale study of yeast (Olsen et al., 2010).

Analysis of the various functional classes of proteins from each parasite revealed several to be overrepresented within the detected proteome relative to all identified parasite proteins (Figure 3A, B). This is not unexpected as most of the chosen groups comprise proteins with an important role in intracellular development. In contrast, the P. falciparum exportome and the surface proteins of Toxoplasma were significantly underrepresented in the overall detected proteome, likely due to stage- and/or variant-specific expression of these proteins: a large proportion of the predicted exportome in P. falciparum belongs to multigene families (e.g. rifins, var, stevor, phist), many of which are expressed in a mutually exclusive manner. In Toxoplasma, many of the proteins on the surface of the zoites themselves are expressed exclusively in life-cycle stages other than tachyzoites (e.g., in bradyzoites or sporozoites; (Jung et al., 2004; Lyons et al., 2002). In terms of representation within the phosphoproteome, as opposed to the overall proteome, the surface and mitochondrion were significantly underrepresented in both parasites, as was the apicoplast in Toxoplasma and maybe in P. falciparum where the result was just below the threshold of significance. Conversely, we found a significantly higher proportion of the “kinome” in both parasites’ phosphoproteomes. We also found many proteins of the inner membrane complex and the invasome to be phosphorylated, although not significantly overrepresented. These groups contain proteins that potentially are highly regulated during parasite maturation and invasion. Unfortunately, a large-scale computational comparison of the two phosphoproteomes to identify the most important phosphorylation sites was not successful due to the generally poor conservation between Plasmodium and Toxoplasma proteins; only the most highly conserved proteins yielded unambiguously orthologous phosphorylation sites and these did not reveal any new information not already evident from the extreme conservation of these proteins.

Figure 3
Relative representation of functional protein groups in the detected proteome and phosphoproteome

The parasite plasma membrane is not a boundary to parasite protein phosphorylation

Toxoplasma and Plasmodium secrete proteins into the parasitophorous vacuole (PV) as well as into the infected host cytoplasm where they can continue on to other compartments (e.g., the nucleus or infected cell’s surface for cells infected with Toxoplasma and Plasmodium, respectively; (Crabb et al., 2010; Gilbert et al., 2007; Haase and de Koning-Ward, 2010; Saeij et al., 2007; Spielmann and Gilberger, 2010)). Once within the PV or host cell, these proteins fulfill a range of functions essential for parasite survival, including active remodeling of the host cell. We found a large fraction of the predicted “exportome” in both parasites is phosphorylated (Figure 3A and B), suggesting that these proteins may be subject to regulation after release.

To more fully explore the degree to which exported proteins of Toxoplasma are phosphorylated after release from the parasite, we compared the phosphoproteome and proteome of “intracellular” vs. purified tachyzoites, the latter being where host-cells and secreted material have been removed by gel filtration. In most cases, purification of tachyzoites away from host cell material should increase parasite protein identification, because of the substantial decrease in sample complexity. We found this to be true, both in terms of the number of proteins detected (see above) as well as in the average number of spectral counts per protein which were 1.9-fold (phosphoproteins) and 2.0-fold (proteins) higher in the purified vs. “intracellular” preparations (Figure 4A).

Figure 4
Identification of proteins that are phosphorylated after host cell invasion

To identify candidate Toxoplasma proteins that are introduced into and phosphorylated within the host, we looked for phosphoproteins that, against the trend of a 1.9-fold increase, showed a reduction of at least 2-fold in the number of spectral counts in the purified vs. “intracellular” parasite preparations. Of 123 phosphoproteins that emerged as outliers (Figure 4B, red dots, and supplementary Table S4), we found many proteins known to be secreted into the host cell. We identified two of the three proteins previously known to be phosphorylated in the host cell (GRA7 and ROP2); the third (ROP4) fell below the threshold necessary for inclusion in this group (a minimum number of 5 spectral counts was required in “intracellular” parasites). We identified the non-phosphorylated forms of most peptides of this outlier group within the total, nonphosphorylated proteome dataset (93 non-phosphorylated versions out of 123 phosphoproteins), arguing against a general loss of these proteins in the “purified” parasite sample (Figure 4C).

Signal peptides are a prerequisite for protein secretion into the host cell. We found that only ~14% of all identified phosphoproteins in Toxoplasma contained a predicted signal peptide compared to ~22% of all predicted proteins, suggesting that most phosphorylation events occur within the parasites’ cytosol (Figure 4D). In contrast, however, ~40% of proteins within the outlier group showing decreased phosphorylation in the “purified” parasite sample contained a predicted signal peptide. This group is enriched in proteins that derive from the rhoptries (ROPs) or dense granules (GRAs), the two Toxoplasma subcellular organelles known to export their contents into the PV and/or host cell. Our data suggest these proteins may be phosphorylated within the PV or cytosol of the infected host cell, after release from the parasite.

Discussion

We have generated large-scale phosphoproteomic datasets for two related parasites, P. falciparum and T. gondii, greatly augmenting the sparse information on phosphorylation sites previously known for obligate intracellular eukaryotes. The only comparable study in a parasitic protist was performed on the extracellular Trypanosoma brucei (Nett et al., 2009b) where a much smaller number of sites were identified: 491 phosphoproteins and 1,204 phosphorylation sites from an estimated total proteome of ~9,000 proteins. Methods for analyzing phosphoproteomes have increased in sensitivity in recent years and the greater extent of observed phosphorylation described here could reflect such advances rather than true biological differences between Apicomplexa and Trypanosoma. In the more exhaustively studied Saccharomyces cerevisiae, with a predicted proteome size (~6,000 proteins) similar to that of P. falciparum, a total of ~2,000 phosphoproteins (www.Phosphopep.org) were identified further suggesting that the relatively small extent of phosphorylation observed in Trypanosoma brucei was due to technical limitations. Interestingly, the fraction of proteins that are phosphorylated in P. falciparum and Toxoplasma (~29% and ~43% of the predicted proteome, and ~63% and ~68% of the detected proteome, respectively) is well within the range reported for multicellular eukaryotic organisms (Olsen et al., 2010; Tan et al., 2009).

We observed that removing host cell proteins from Toxoplasma samples greatly increased the identification of parasite peptides and phosphopeptides. This suggests that our P. falciparum findings are likely to be an underestimate of the total phosphoproteome in this organism.

The observed numbers and localization of phosphorylated amino acids within the predicted disordered regions of proteins was largely as expected; however, the presence of phosphotyrosines in our datasets was not a certain outcome given the absence of genes for known or predicted tyrosine kinases in both parasites’ genomes (Peixoto et al., 2010; Tewari et al., 2010; Ward et al., 2004). Among the phosphotyrosines, we found sites conserved in the orthologous proteins of eukaryotes ranging from trypanosomes to humans (Nett et al., 2009a; Tan et al., 2009). DYRK kinases and GSK-3 have been shown to be able to autophosphorylate at the critical residue and BLAST searches identified MAPK15 (ERK7) as the human protein that is most closely related to the tyrosine-phosphorylated MAP-kinases found in this study (PF11_0086 and TGGT1_114510, respectively). In other systems, MAPKs are typically phosphorylated by an upstream MAPK-kinase, but HsMAPK15 has been shown to be capable of autophosphorylation of the essential activation loop tyrosine in vitro (Abe et al., 2001). These results leave open the possibility that a substantial number of phosphotyrosines in signaling pathways in the parasites derive from autophosphorylation events. Other pY sites in kinase-unrelated proteins however indicate the presence of tyrosine phosphorylation per se. Studies such as those described here help provide the evidence and motivation to explore these key pathways further. If the parasites’ tyrosine kinases are clearly distinct from the host’s enzymes, these might be exploited as targets for highly specific kinase inhibitors. Interestingly, at least one secreted kinase in Toxoplasma has been shown to be capable of tyrosine-phosphorylation of host cell proteins (Ong et al., 2010; Yamamoto et al., 2009).

The extremely AT-rich P. falciparum genome results in an unusual amino acid composition in its proteome, prompting us to investigate whether this parasite might have distinct phosphorylation motifs. Our analysis provided evidence that phosphorylation within P. falciparum exploits motifs that are distinct from those used by Toxoplasma and mammals to direct kinase specificity. These changes are likely due to the A:T-richness and whatever the force that has driven P. falciparum to such an extreme of A:T richness, it appears stronger than that operating in other systems that favor proline (and, to some extent, disfavors asparagine) at phosphorylation sites. Whether the enrichment of asparagine and hydrophobic amino acids in the P+1 position in P. falciparum is a compensation for the loss of proline and is required for a specific subset of kinases, or if these residues are simply tolerated is unknown. Regardless, it seems likely that these differences in motifs are associated with structural differences in the active site of P. falciparum serine-threonine kinases. Such a relationship between an asparagine in the phosphorylation motif and specific structural properties of the kinase active center has recently been argued for in yeast, which also has a somewhat A:T-rich (~60%) genome (Mok et al., 2010).

The ability to compare phosphorylated proteins in two related organisms that share similar (intracellular) lifestyles not only increased the confidence in the individual data concerning specific proteins but also the trends that were seen in both. For example, ontological analysis of proteins and phosphoproteins involved either in general or parasite-specific processes revealed phosphorylated surface proteins were underrepresented in both cases, suggesting that these proteins might not require regulation by phosphorylation. We also observed significant underrepresentation of phosphorylation in mitochondrial and apicoplast proteins which could reflect their relatively recent bacterial origin as proposed by Gnad and colleagues (Gnad et al., 2010).

Surprisingly, we identified a substantial number of phosphorylated parasite proteins that are found outside the parasites boundaries. Both parasites export or inject proteins beyond their own plasma membrane, either to facilitate acquisition of nutrients, remodel the host cell for immune-evasion, or perform other, as yet unknown functions. How the parasites regulate these processes has not been determined, but within the Toxoplasma dataset, 50 proteins with a predicted signal peptide showed substantially more phosphorylation in the infected cell preparations than in purified parasites, strongly suggesting that their phosphorylation occurs upon or soon after secretion into the host cell. Some of these secreted phosphoproteins are predicted kinases whose activity might be regulated by phosphorylation; for the majority, however, their function is unknown although their likely introduction into the host cell clearly argues for a role in the cross-talk between the infecting organism and the host.

The kinases responsible for the phosphorylation of secreted proteins could be host cell enzymes, perhaps acting as a mechanism of defense against infection. Alternatively, the parasites could be using their own or host kinases to actively regulate the function of their exportome. Toxoplasma also is known to export several active kinases into the host cell cytosol (Boothroyd and Dubremetz, 2008) and one or more of these could be responsible for at least some of the phosphorylation described here, in addition to their role in modifying host proteins (Fentress et al., 2010; Ong et al., 2010; Steinfeldt et al., 2010; Yamamoto et al., 2009).

For parasite phosphoproteins that occupy the lumen of the parasitophorous vacuole (PV), it is most likely that parasite kinases are responsible for the phosphorylation as host cell proteins are generally thought to be excluded from this compartment (Mordue et al., 1999). ROP kinases, which localize to the PV membrane, are candidates for this activity although recent evidence suggests that they are facing into the host cytosol, not the PV lumen (El Hajj et al., 2007; Reese and Boothroyd, 2009). One of Toxoplasma’s “ROP” kinases (ROP21) has been shown to be targeted to the PV independent of rhoptry trafficking and thus could have a different orientation and catalyze phosphorylation in the PV lumen (Peixoto et al., 2010).

We also identified a large number of exported proteins of P. falciparum that are phosphorylated. The presence of at least 20 P. falciparum kinases that are thought to be exported into the host cell (Nunes et al., 2007) suggests that this parasite, too, may have evolved the ability to use phosphorylation as a means of regulating protein function beyond its own plasma membrane. This is likely to be a feature of many organisms, parasitic and otherwise, that have adopted an intracellular lifestyle and the datasets described here represent an important entrée into determining molecular details underlying such functions.

Experimental procedures (see also supplementary experimental procedures)

Sample preparation and Mass spectrometry

All parasite samples were lysed in 8m urea lysis buffer, followed by trypsin digestion, strong-cation-exchange (SCX) chromatography and IMAC phosphopeptide enrichment (Villen and Gygi, 2008). Samples were analyzed in duplicate via LC-MS/MS and the resulting spectra were searched against the respective parasite protein database concatenated with reversed decoy sequences using the SEQUEST algorithm (Eng et al., 1994). Phosphorylation sites were localized using the AScore algorithm (Beausoleil et al., 2006) and the dataset was filtered using the target-decoy strategy (Elias and Gygi, 2007, 2010) for phosphorylation site-FDRs <1% and a protein FDR <3%. The proteome datasets were filtered for peptide FDRs <1% and protein FDRs <3%.

Phosphorylation motif analysis

The ratio of frequencies was calculated by (frequency of residues in the Phospho13mers/frequency of residue in total proteome) or (frequency of residues in Phospho13mers/frequency of residues in the Nonphospho13mers) and plotted as the log2 ratio. Data used for calculating the amino acid frequency around mouse phosphorylation sites was retrieved from (Huttlin et al., 2010). The position-specific heatmap was generated by plotting the log10 of the ratio of frequencies. Motif-X search was performed to identify statistically significant phosphorylation motifs (Schwartz and Gygi, 2005).

Identification of secreted phosphoproteins

Peptide-spectrum matches (PSMs) were combined on the protein level and used for comparing abundance of a given protein or phosphoprotein. Proteins were filtered for a minimal number of spectral counts of 5 for intracellular phosphoproteins. The ratio of intra- vs. extracellular counts was calculated and a minimum fold-change of 2 (intracellular/extracellular) was set as the threshold for inclusion into the outlier list.

Highlights

  • Proteome-wide analysis of phosphorylation sites in P. falciparum and T. gondii.
  • Evidence for tyrosine-phosphorylation in both parasites.
  • Insights into unusual phosphorylation-site motif usage in P. falciparum.
  • Identification of a method to identify phosphorylation beyond a parasites’ boundary.

Supplementary Material

04

06

Acknowledgments

We thank Matt Bogyo for providing laboratory space and reagents for Plasmodium falciparum cell culture. Many thanks to Dominique Figueroa for excellent technical assistance and Bernd F. Bodenmiller and all members of the Elias and Boothroyd labs for helpful discussions. This work was supported by NIH (AI21423). MT is supported by the Deutsche Forschungsgemeinschaft. JLS is supported by a NIH Training grant.

Footnotes

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References

  • Abe K, Aoyagi A, Saito H. Sustained phosphorylation of mitogen-activated protein kinase is required for basic fibroblast growth factor-mediated axonal branch formation in cultured rat hippocampal neurons. Neurochem Int. 2001;38:309–315. [PubMed]
  • Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol. 2006;24:1285–1292. [PubMed]
  • Billker O, Lourido S, Sibley LD. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe. 2009;5:612–622. [PMC free article] [PubMed]
  • Bodenmiller B, Mueller LN, Mueller M, Domon B, Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nature methods. 2007;4:231–237. [PubMed]
  • Boothroyd JC, Dubremetz JF. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nature reviews. 2008;6:79–88. [PubMed]
  • Carey KL, Jongco AM, Kim K, Ward GE. The Toxoplasma gondii rhoptry protein ROP4 is secreted into the parasitophorous vacuole and becomes phosphorylated in infected cells. Eukaryotic cell. 2004;3:1320–1330. [PMC free article] [PubMed]
  • Cowman AF, Crabb BS. Invasion of red blood cells by malaria parasites. Cell. 2006;124:755–766. [PubMed]
  • Crabb BS, de Koning-Ward TF, Gilson PR. Protein export in Plasmodium parasites: from the endoplasmic reticulum to the vacuolar export machine. International journal for parasitology. 2010;40:509–513. [PubMed]
  • Dunker AK, Brown CJ, Lawson JD, Iakoucheva LM, Obradovic Z. Intrinsic disorder and protein function. Biochemistry. 2002;41:6573–6582. [PubMed]
  • Dunn JD, Ravindran S, Kim SK, Boothroyd JC. The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4. Infection and immunity. 2008;76:5853–5861. [PMC free article] [PubMed]
  • Dvorin JD, Martyn DC, Patel SD, Grimley JS, Collins CR, Hopp CS, Bright AT, Westenberger S, Winzeler E, Blackman MJ, et al. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science. 2010;328:910–912. [PMC free article] [PubMed]
  • El Hajj H, Lebrun M, Arold ST, Vial H, Labesse G, Dubremetz JF. ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii. PLoS pathogens. 2007;3:e14. [PMC free article] [PubMed]
  • Elias JE, Gygi SP. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature methods. 2007;4:207–214. [PubMed]
  • Elias JE, Gygi SP. Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol Biol. 2010;604:55–71. [PMC free article] [PubMed]
  • Eng JK, Mccormack AL, Yates JR. An Approach to Correlate Tandem Mass-Spectral Data of Peptides with Amino-Acid-Sequences in a Protein Database. J Am Soc Mass Spectr. 1994;5:976–989. [PubMed]
  • Fentress SJ, Behnke MS, Dunay IR, Mashayekhi M, Rommereim LM, Fox BA, Bzik DJ, Taylor GA, Turk BE, Lichti CF, et al. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence. Cell host & microbe. 2010;8:484–495. [PMC free article] [PubMed]
  • Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419:498–511. see comment. [PMC free article] [PubMed]
  • Gilbert LA, Ravindran S, Turetzky JM, Boothroyd JC, Bradley PJ. Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryotic cell. 2007;6:73–83. [PMC free article] [PubMed]
  • Gnad F, Forner F, Zielinska DF, Birney E, Gunawardena J, Mann M. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria. Mol Cell Proteomics. 2010;9:2642–2653. [PMC free article] [PubMed]
  • Green JL, Rees-Channer RR, Howell SA, Martin SR, Knuepfer E, Taylor HM, Grainger M, Holder AA. The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. The Journal of biological chemistry. 2008;283:30980–30989. [PMC free article] [PubMed]
  • Haase S, de Koning-Ward TF. New insights into protein export in malaria parasites. Cellular Microbiology. 2010;12:580–587. [PubMed]
  • Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME, Gygi SP. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell. 2010;143:1174–1189. [PMC free article] [PubMed]
  • Iakoucheva LM, Radivojac P, Brown CJ, O’Connor TR, Sikes JG, Obradovic Z, Dunker AK. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 2004;32:1037–1049. [PMC free article] [PubMed]
  • Jimenez JL, Hegemann B, Hutchins JR, Peters JM, Durbin R. A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database. Genome Biol. 2007;8:R90. [PMC free article] [PubMed]
  • Joyce BR, Queener SF, Wek RC, Sullivan WJ., Jr Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:17200–17205. [PMC free article] [PubMed]
  • Jung C, Lee CY, Grigg ME. The SRS superfamily of Toxoplasma surface proteins. International journal for parasitology. 2004;34:285–296. [PubMed]
  • Leykauf K, Treeck M, Gilson PR, Nebl T, Braulke T, Cowman AF, Gilberger TW, Crabb BS. Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite. PLoS pathogens. 2010;6:e1000941. [PMC free article] [PubMed]
  • Lim WA, Pawson T. Phosphotyrosine signaling: evolving a new cellular communication system. Cell. 2010;142:661–667. [PMC free article] [PubMed]
  • Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature. 2010;465:359–362. [PMC free article] [PubMed]
  • Lyons RE, McLeod R, Roberts CW. Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends in Parasitology. 2002;18:198–201. [PubMed]
  • Mok J, Kim PM, Lam HY, Piccirillo S, Zhou X, Jeschke GR, Sheridan DL, Parker SA, Desai V, Jwa M, et al. Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs. Science signaling. 2010;3:ra12. [PMC free article] [PubMed]
  • Moon RW, Taylor CJ, Bex C, Schepers R, Goulding D, Janse CJ, Waters AP, Baker DA, Billker O. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite. PLoS pathogens. 2009;5:e1000599. [PMC free article] [PubMed]
  • Mordue DG, Hakansson S, Niesman I, Sibley LD. Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp Parasitol. 1999;92:87–99. [PubMed]
  • Nett IR, Davidson L, Lamont D, Ferguson MA. Identification and specific localization of tyrosine-phosphorylated proteins in Trypanosoma brucei. Eukaryotic cell. 2009a;8:617–626. [PMC free article] [PubMed]
  • Nett IR, Martin DM, Miranda-Saavedra D, Lamont D, Barber JD, Mehlert A, Ferguson MA. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness. Mol Cell Proteomics. 2009b;8:1527–1538. [PMC free article] [PubMed]
  • Nunes MC, Goldring JP, Doerig C, Scherf A. A novel protein kinase family in Plasmodium falciparum is differentially transcribed and secreted to various cellular compartments of the host cell. Mol Microbiol. 2007;63:391–403. [PubMed]
  • Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Science signaling. 2010;3:ra3. [PubMed]
  • Ong YC, Reese ML, Boothroyd JC. Toxoplasma rhoptry protein 16 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. The Journal of biological chemistry. 2010;285:28731–28740. [PMC free article] [PubMed]
  • Peixoto L, Chen F, Harb OS, Davis PH, Beiting DP, Brownback CS, Ouloguem D, Roos DS. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe. 2010;8:208–218. [PMC free article] [PubMed]
  • Reese ML, Boothroyd JC. A helical membrane-binding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole. Traffic. 2009;10:1458–1470. [PMC free article] [PubMed]
  • Saeij JP, Boyle JP, Coller S, Taylor S, Sibley LD, Brooke-Powell ET, Ajioka JW, Boothroyd JC. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science. 2006;314:1780–1783. [PMC free article] [PubMed]
  • Saeij JP, Coller S, Boyle JP, Jerome ME, White MW, Boothroyd JC. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature. 2007;445:324–327. [PMC free article] [PubMed]
  • Schwartz D, Gygi SP. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol. 2005;23:1391–1398. [PubMed]
  • Spielmann T, Gilberger TW. Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways? Trends in Parasitology. 2010;26:6–10. [PubMed]
  • Steinfeldt T, Konen-Waisman S, Tong L, Pawlowski N, Lamkemeyer T, Sibley LD, Hunn JP, Howard JC. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 2010;8:e1000576. [PMC free article] [PubMed]
  • Sugi T, Kato K, Kobayashi K, Watanabe S, Kurokawa H, Gong H, Pandey K, Takemae H, Akashi H. Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step. Eukaryotic cell. 2010;9:667–670. [PMC free article] [PubMed]
  • Tan CS, Bodenmiller B, Pasculescu A, Jovanovic M, Hengartner MO, Jorgensen C, Bader GD, Aebersold R, Pawson T, Linding R. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases. Sci Signal. 2009;2:ra39. [PubMed]
  • Tewari R, Straschil U, Bateman A, Bohme U, Cherevach I, Gong P, Pain A, Billker O. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe. 2010;8:377–387. [PMC free article] [PubMed]
  • Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, et al. Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process. PLoS pathogens. 2009;5:e1000322. [PMC free article] [PubMed]
  • Villen J, Gygi SP. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc. 2008;3:1630–1638. [PMC free article] [PubMed]
  • Ward P, Equinet L, Packer J, Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics. 2004;5:79. [PMC free article] [PubMed]
  • Wilkes JM, Doerig C. The protein-phosphatome of the human malaria parasite Plasmodium falciparum. BMC Genomics. 2008;9:412. [PMC free article] [PubMed]
  • Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama H, Kubo E, Ito H, Takaura M, Matsuda T, et al. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J Exp Med. 2009;206:2747–2760. [PMC free article] [PubMed]
  • Zhang M, Fennell C, Ranford-Cartwright L, Sakthivel R, Gueirard P, Meister S, Caspi A, Doerig C, Nussenzweig RS, Tuteja R, et al. The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. The Journal of experimental medicine. 2010;207:1465–1474. [PMC free article] [PubMed]
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