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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Host Microbe. Author manuscript; available in PMC Aug 19, 2011.
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PMCID: PMC2963626
NIHMSID: NIHMS225308

Integrative Genomic Approaches Highlight a Family of Parasite-Specific Kinases that Regulate Host Responses

Summary

Apicomplexan parasites release factors via specialized secretory organelles (rhoptries, micronemes) that are thought to control host cell responses. In order to explore parasite-mediated modulation of host cell signaling pathways, we exploited a phylogenomic approach to characterize the Toxoplasma gondii kinome, defining a 44 member family of coccidian-specific secreted kinases, some of which have been previously implicated in virulence. Comparative genomic analysis suggests that ‘ROPK’ genes are under positive selection, and expression profiling demonstrates that most are differentially expressed between strains and/or during differentiation. Integrating diverse genomic-scale analyses points to ROP38 as likely to be particularly important in parasite biology. Upregulating expression of this previously uncharacterized gene in transgenic parasites dramatically suppresses transcriptional responses in the infected cell. Specifically, parasite ROP38 down-regulates host genes associated with MAPK signaling and the control of apoptosis and proliferation. These results highlight the value of integrative genomic approaches in prioritizing candidates for functional validation.

INTRODUCTION

The phylum Apicomplexa includes thousands of obligate intracellular parasites, many of which are important sources of morbidity and mortality in humans and animals. Plasmodium parasites are responsible for malaria (World Health Organization 2009), while Toxoplasma is a leading source of congenital neurological birth defects and a prominent opportunistic infection in AIDS (Hill & Dubey 2002); T. gondii has also emerged as an experimentally tractable model system (Roos et al 1994; Roos 2005). These parasites have evolved novel mechanisms for invasion and intracellular survival, including an apical complex of specialized secretory organelles: ‘micronemes’ are associated with host cell attachment, while secretion from ‘rhoptries’ is associated with establishment of an intracellular ‘parasitophorous vacuole’ (Carruthers & Sibley 1997; Bradley & Sibley 2007). Several rhoptry (ROP) proteins contain kinase-like domains, although many lack an obvious catalytic triad (El Hajj et al 2006). Recent work on the active rhoptry kinases ROP16 & 18 (El Hajj et al 2007a), shows that the former is secreted into the infected cell and alters STAT 3/6 phosphorylation (Saeij et al 2007), while the latter is an important virulence determinant (Saeij et al 2006; Taylor et al 2006).

Eukaryote protein kinases (ePKs) are phylogenetically related (Hanks & Hunter 1995) and typically reside in the cytoplasm, where they play key roles in signal transduction (Manning et al 2002a). Genome sequencing has defined the complete kinome for various species (Hunter & Plowman 1997; Plowman et al 1999; Manning et al 2002b), including that of Plasmodium falciparum (Ward et al 2004), helping to elucidate molecular players that may be involved in signaling. We have exploited the T. gondii genome (Gajria et al 2008; ToxoDB.org) to define this parasite’s kinome, including 159 putative ePKs of which 108 are predicted to be active. The largest family of T. gondii kinases (ROPK) contains 44 members, including ROP16 and ROP18; orthologs of most ROPK proteins are also recognizable in the Neospora caninum genome. ROPK proteins have not been identified in Plasmodium, although these parasites possess another group of secreted kinases, the FIKK kinases (Ward et al 2004; Anamika et al 2005; Nunes et al 2007). This report, in conjunction with previous studies, indicates that ROPK proteins are secreted into the parasitophorous vacuole, trafficking to the intravacuolar membranous network, the vacuolar surface membrane, and/or the host cell.

Using comparative genomic approaches, we show that the ROPK family has been under positive selection since the divergence of Neospora and Toxoplasma. ROPK genes also exhibit an unusual degree of differential expression between strains and/or during differentiation. Integrating these genomic scale datasets highlights the previously uncharacterized kinase ROP38 as likely to be functionally important. Virulent RH strain T. gondii normally expresses virtually no ROP38, but this transcript is abundant in the relatively avirulent VEG strain parasites. Infection of mammalian cells with RH transgenics engineered to express VEG levels of ROP38 significantly alters the expression of ~1200 host genes (383 by >2-fold), usually manifested as a suppression of host genes induced by RH infection. Functional clustering shows that parasite expression of ROP38 exerts a potent effect on the expression of host transcription factors, signaling pathways, and the regulation of cell proliferation and apoptosis. Genes down-regulated >4 fold by ROP38 include c-fos, EGR2, and other early response genes such as CXCL1 and NAMPT, consistent with regulation of host-cell MAPK cascades (particularly ERK signaling).

RESULTS

The T. gondii kinome contains 108 putative kinases and 51 pseudokinases

Analysis of the T. gondii genome (Methods) identifies a total of 159 ePKs, including 108 predicted to be active based on the presence of twelve complete kinase subdomains, Pfam domain PF0069, and three conserved amino acids constituting the catalytic triad (Lys30, Asp125, Asp143; Hanks & Hunter 1995). Representatives of previously-defined human (Manning et al 2002b), yeast (Hunter & Plowman 1997) and P. falciparum (Ward et al 2004) ePK subfamilies were used as seeds for phylogenetic classification of all T. gondii kinases predicted to be active, most of which could readily be assigned to established ePK groups (Figs 1 & S1, Tables SI & SII).

Figure 1
The T. gondii Kinome

The active T. gondii kinome includes 10 cyclic nucleotide regulated kinases (AGC), 20 cyclin dependent kinases and close relatives (CMGC, including CDK, MAPK, GSK), 20 calcium/calmodu-lin regulated kinases (CAMK), 3 casein kinase-like proteins (CK1), 7 tyrosine kinase-like proteins (TKL) and 1 MAP kinase kinase (MAPKK, STE). Additional kinases (‘Other’ in Table SI) include 9 Nima/NEK, 4 ULK, 1 Aurora, 2 Wee, and 3 PIK3R4 (two display architecture distinct from their animal/fungal homologs). More than half of the ‘Other’ kinases identified in T. gondii are restricted to the phylum apicomplexa. The T. gondii genome is also predicted to include 51 pseudokinases, defined as inactive based on the absence of a complete catalytic triad and/or extremely low HMM scores. Only 4 of these could be classified into major kinase groups (2 CMGCs, 1 CAMK, 1 AGC), and most are specific to the apicomplexa. To facilitate cross-species comparison for functional analysis, a combined kinome for P. falciparum and T. gondii kinome (including representative human and yeast orthologs) is presented as Fig S1 (see also Table SII).

Apicomplexans have evolved a unique repertoire of secreted kinases

In order to understand how kinases have evolved in the protozoa, the T. gondii and P. falciparum kinomes were compared with previously published analyses of the amoeba Dictyostelium discoideum (Goldberg et al 2006) and the kinetoplastid parasites Leishmania major, Trypanosoma brucei, and T. cruzi (Parsons et al 2005), in addition to plants (Arabidopsis thaliana, Oryza sativa; Dardick et al 2007), fungi (Saccharomyces cerevisiae; Plowman et al 1999), and animals (Caenorhabditis elegans, Drosophila melanogaster, Homo; Plowman et al 1999; Manning et al 2002b), as shown in Table SI. While the complete proteomes of Toxoplasma and Plasmodium are estimated to differ by <25% in size (EuPathDB.org), the kinome of T. gondii is almost double the size of the P. falciparum kinome. Parasite kinases exhibit similar distribution among the major groups, with many CMGC and CAM kinases and no tyrosine kinases (TK) or receptor guanylate cyclases (RGC) (Table SI). Only a single STE kinase (MAPKK) was identified, in T. gondii. Consistent with the secondary endosymbiotic history of the apicoplast (Foth & McFadden 2003), three T. gondii kinases exhibit probable plastid origin: two CAMKs and 1 AGC (Table SII).

The most striking feature of the apicomplexan kinome is the large fraction of kinases that do not fall within traditional groups: ‘Other’ represents ~55% of the apicomplexan kinome, vs. 30–37% for other unicellular species and ~20% for metazoa. Most are parasite-specific, usually at the species level: 11 are shared between apicomplexa (including the FIKK family), 24 are unique to P. falciparum; (Ward et al 2004; Nunes et al 2007), and 51 are unique to T. gondii, including the virulence factor ROP18 (Saeij et al 2006; Taylor et al 2006; El Hajj et al 2007a). Interestingly, while ePKs are typically cytosolic, an unusual number of apicomplexan kinases are predicted to contain secretory signal sequences (red dots in Fig 1), including the FIKK family and several T. gondii kinases from the ROP18 clade.

The ROPK family: a coccidian specific family of secreted kinase related proteins

In order to further define the T. gondii rhoptry kinase (ROPK) family, the monophyletic group of active kinases highlighted in Fig 1, plus ROP16 (but excluding 49.m05665 and 20.m03646, which harbor large insertions interrupting the kinase domain) was used to construct a family-specific profile HMM (see Methods). Applying this ROPK HMM to the entire T. gondii genome identifies 34 unique genes (Fig 2); application to the T. gondii kinome alone yielded identical results. ROPK genes were also identified in Neospora caninum, and assigned as orthologs based on synteny. Degenerate ROPK genes (pseudogenes and inaccurate gene models) were detected based on sequence similarity (see Methods), identifying 10 additional family members: 3 with insertions >3 kb in the kinase domain (ROP33, 34, 46), and 7 with truncated kinase domains (5 of which are not recognized by PF00069; Table SIII). In aggregate, the T. gondii ROPK family contains at least 44 genes, including all previously reported kinase-like rhoptry proteins and more than doubling the number of previously described family members (Boothroyd & Dubremetz, 2008).

Figure 2
The T. gondii ROPK family

24 of the 33 T. gondii kinases and pseudokinases predicted to be secreted outside of the parasite are members of the ROPK family. Among the 34 non-degenerate ROPK family members (Fig 2), 22 contain an obvious N-terminal signal peptide (red), two (ROP26 & 28) are predicted to contain a signal anchor (Table SIII), and experimental reanalysis of two (ROP4 & 7) identifies signal sequences previously missed (Carey et al 2004). Signal sequence identification depends on accurate 5′ end prediction, which is notoriously difficult in large eukaryotic genomes (Liu et al 2008). Four more ROPKs (ROP45, 29, 30 & 41) were found to contain signal peptides based on 5′ rapid amplification of cDNA ends (RACE) and/or comparison with syntenic orthologs in N. caninum genome. It appears likely that all ROPK family members encode a secretory signal.

In order to evaluate the accuracy of these predictions, seven novel ROPKs (ROP19, 20, 21, 22, 23, 25 & 38) and four proteins found in the rhoptries by proteomic analysis (ROP17, 24, 39 & 40; (Bradley et al 2005) were engineered as recombinant HA-tagged fusion proteins for expression in T. gondii. Transient transfections (Figs 3 & S2) demonstrate that at least nine of these ROPK proteins colocalized with a rhoptry marker (Figs 3 top & S2A), and all traffic to the parasitophorous vacuolar membrane or network (a tubular membrane complex within the vacuole; Coppens et al 2006), although many lack the predicted amphipathic helix known to facilitate membrane association of some ROPK proteins (Reese & Boothroyd 2009). ROP21 & 22 did not colocalize with the rhoptry marker (Fig S2B), but were nevertheless observed in the parasitophorous vacuole; ROP21 was also observed in the host cell cytoplasm (Fig 3 bottom). It is unlikely that these patterns of distribution are attributable to overexpression using a heterologous promoter, as overexpression of secretory proteins more commonly results in staining of the parasite cytoplasm or endoplasmic reticulum, rather than promiscuous secretion (Nishi et al 2008).

Figure 3
ROPK Localization

The ROPK family is under diversifying selection

Multiple sequence alignment of the ROPK family (Fig S3) shows a high degree of divergence (ave 16% pairwise identity). Conservation is concentrated within the N-terminal portion of the kinase domain encompassing the activation loop and substrate-binding site. Considerable degeneracy was observed at the initial position of the ‘KDD’ catalytic triad, accounting for the large number of pseudokinases. With the exception of ROP16, the activation loop of all active ROPKs includes the Ser/Thr whose phosphorylation is responsible for regulation in other ePKs. Recent work indicates that ROPK phosphorylation is important mechanisms for regulation (Qiu et al 2009).

The ROPK phylogenetic tree contains two main clades (Fig 4). One accommodates most previously identified rhoptry proteins (ROP2, 4, 5, 7, 8 & 18), including many recent duplications. ROP18 is the only active member of this clade, half of which have degenerated into pseudogenes in Neospora. A second clade contains most of the active kinases, including ROP16 and most of the novel ROPKs described in this report. Several derive from recent duplications, including ROP38, 29, 19 and two degenerate ROPKs on chromosome VI, and ROP42, 43 & 44 on chromosome Ib. The N-terminal portion of rhoptry kinases has been implicated in secretory targeting (Reese & Boothroyd 2009) and this domain is highly conserved in the ROP2/ROP18 group, contributing to confidence in the monophyly of this clade. The ROPK tree retains the same basic two clade structure even when the N-terminal domain is excluded from analysis, however (not shown).

Figure 4
Evolution of the ROPK family of secreted kinases

Amplification of the ROPK family clearly preceded the divergence of T. gondii and N. caninum, as most genes are represented by orthologs in both species (Fig 4A; tick marks indicate branch points of Neospora orthologs). Comparing these genes indicates that all but one exhibit nucleotide sequence identity equal to or greater than the observed amino acid conservation, suggesting diversifying selection (Table SIII). The ratio of nonsynonymous to synonymous substitutions (dN/dS) is commonly used as a marker of evolutionary pressure, with values >1 indicating positive selection. Because selection is only expected to apply to a small subset of amino acids, we employed a likelihood ratio test to assess whether the observed data is better explained by a models including or excluding sites with dN/dS > 1, i.e. site-specific positive selection (PAML models M7 and M8; Yang 1997). 16 ROPK proteins show signs of site-specific positive selection (p < 0.01), including all T. gondii specific duplications (Fig 4A, Table SIII). None of the ROPK show signs of positive selection over the entire gene (average dN/dS), suggesting that selection occurs at a few specific sites, although the available sequence data precludes identification of specific site, as partitioning the protein leaves too few independent sites in the alignment for reliable estimation (it will be interesting to revisit this question as sequences for other coccidian ROPK genes become available). It has long been known that expanded gene families show relaxed constraints and are likely to show rapid divergence. This may be the case for ROP38/29/19, where nucleotide alignments reveal independent triplication in both T. gondii and N. caninum, but amino acid alignments suggest functional conservation (or convergence) between TgROP38 and NcROP19.2 (Fig 4B).

Taking advantage of the complete genome sequence available for three representative lineages (GT1, ME49, VEG; ToxoDB.org), single nucleotide polymorphisms (SNPs) were identified in 30 ROPK genes (Table SIII). The ROPK family is significantly more polymorphic than the genome, the secretome (SignalP+), or the kinome as a whole (p = 0.09; Fig 5A), and several ROPKs (ROP5, 16, 17, 18, 19, 24, 26, 39 & 40) also show a high ratio of non-synonymous to synonymous polymorphisms (Table SIII), although more extensive sampling will be required to determine whether any of these genes is under selection at the population level.

Figure 5
Functional genomics of the ROPK family

The ROPK family is differentially regulated among T. gondii strains, and during tachyzoite-to-bradyzoite transition

As previous studies on ROP18 showed that expression levels are an important for virulence (Saeij et al 2007), we exploited genome-wide expression profiling to identify other differentially expressed ROPK genes. RNA from the rapidly growing tachyzoite stage of five T. gondii isolates (representing all three major lineages common in the US: type I, RH & GT1; type II, PRU & ME49; type III, VEG) was hybridized to an Affymetrix microarray (ToxoDB.org) and analyzed as described in ‘Methods’. 90% of ROPKs are expressed in tachyzoites (Fig 5B) – a significantly higher fraction than observed for the entire genome (75%), kinome (75%), or secretome (67%); ROP4/7, 11 & 40 are among the most highly expressed genes in the genome. Others ROPK genes display dramatic differences in expression relative to the RH reference (Fig 5C), including ROP18 (>100X lower in VEG), ROP38 (up >64X in VEG; >8X in ME49), and ROP35 (up >16X in VEG).

Differentiation of tachyzoites into bradyzoite ‘tissue cysts’ is among the most biologically and clinically significant events in T. gondii biology (Dzierszinski et al 2004). To explore changes in gene expression during this conversion, Prugniaud strain parasites were subjected to alkaline conditions or CO2 starvation (Bohne et al 1999), following known bradyzoite markers as controls (see Methods). Only 6% of the entire T. gondii genome (8% of the secretome, 5% of the kinome) was differentially expressed, vs 48% of the ROPK family (Fig 5D; Table SIII). Most of these ROPK genes were down-regulated during differentiation, but ROP28 and 38 were induced ~5-fold.

ROP38 dramatically alters host-cell responses to infection

The evolutionary and functional characterization outlined above demonstrates that the ROPK family exhibits various attributes – stage- and strain-specific expression, secretion, positive selection – likely to be associated with important aspects of parasite biology. ROP18 emerges as being of interest, and this gene has previously been shown to play an important role in regulating parasite virulence (Taylor et al 2006). ROP38 also emerges from this analysis: its ancestor gene was triplicated independently in T. gondii and N. caninum, and ROP38 exhibits hallmarks of selection (Fig 4). ROP38 is also among the most profoundly regulated genes in the parasite genome (ToxoDB.org), and it is the only ROPK gene that is both differentially expressed between strains and induced during differentiation (Fig 5C & D).

In order to explore the biological significance of ROP38, RH strain parasites (which normally express this gene at very low levels) were engineered to express an HA-tagged transgene under control of the β-tubulin promoter (RH-ROP38), and a parallel mutant was engineered to overexpress HA-tagged ROP21, which lacks indicators of selection or differential expression (Figs 4 & 5), but traffics into the host cell (Fig 3, bottom). The tagged products of both transgenes were found to associate with parasitophorous vacuole membranes (PVM or PV network), and microarray analysis demonstrated upregulation by >25-fold (Table SIV), raising ROP38 expression to the levels typically observed in wild-type VEG strain parasites. Parallel changes were also observed in steady-state RNA abundance for several T. gondii genes, including TGME49_116390, a coccidian gene of unknown function that is strongly up-regulated in both the RH-ROP21 and RH-ROP38 transgenics. Overall, however, these parasites are more notable for their similarities than their differences. In vitro replication of the RH-ROP38 line was comparable to wild-type RH (doubling time ~6.8 hr), as was virulence in mice (100% morbidity by 10d after intraperitoneal inoculation of Balb/c mice with 100 tachyzoites), in contrast to VEG strain parasites, which replicate slowly and are relatively avirulent (Jerome et al 1998; Saeij et al 2005).

Illumina arrays were used to examine the effects of parasite ROPK expression on host cell transcript levels (Fig 6A), providing a far more detailed picture than previously available for T. gondii infection (Blader et al 2001). VEG strain parasites significantly increase transcript levels of 400+ host cell genes, and reduce levels of >250, while RH parasites exert a far more dramatic effect, reproducibly up-regulating >5000 genes and down-regulating >1000. Transcription factors (c-fos, EGR2), cytokines (NAMPT, CXCL1), and kinases (especially those associated with MAPK signaling) are prominent among host genes up-regulated by RH infection (Table SV). Modulation of MAPK signaling by T. gondii has been reported previously (Kim et al 2004; Molestina et al 2008), and increased c-fos and CXCL1 expression was confirmed by quantitative RT-PCR (not shown).

Figure 6
The Effect of ROP38 on host cell gene expression

Remarkably, expression of the ROP38 transgene suppressed most of the transcriptional changes induced by RH strain parasites (Table SV, Fig 6). Infection with RH-ROP38 parasites significantly altered the expression of only ~400 host cell genes – an effect more similar to the impact of infection with the avirulent VEG strain than with the RH parental parasite line. For example, c-fos was induced >16X by RH infection, but <4X by RH-ROP38 infection, and not at all by the VEG strain; similar effects were observed for CXCL1, EGR2, NAMPT, and many other genes. Quantitative RT-PCR showed a 2.8-fold lower CXCL1 levels in RH-ROP38 parasites relative to RH controls (average of two replicate experiments on each of two independent ROP38 transgenics), validating the 4.5-fold reduction observed on Illumina arrays (first line in Table SV).

Genome-wide, the impact of ROP38 on expression of host genes constitutes an approximately two-fold suppression of the effects caused by infection with RH strain parasites (Fig 6B). Observed differences in c-fos and CXCL1 expression were confirmed by quantitative RT-PCR, and modulation of MAPK signaling pathways were confirmed by phosphoERK immunoblots (Fig S4). Increased expression of ROP21 did not significantly affect host gene expression (Fig S5B, which also serves to illustrate the reproducibility of independent biological experiments), demonstrating that the profound effect of ROP38 on host cell transcription is not simply the consequence of expressing any ROPK protein in the host cell cytoplasm (although note that ROP21 differs from ROP38 in its localization within the parasite and infected host cell; Figs 3 & S2B).

To further explore the effects of T. gondii ROP38 on human host cells, we examined the enrichment of functional annotation (GO terms, KEGG pathways, Interpro domains, UniProt keywords) associated with parasite-induced or -repressed genes (Dennis et al 2003; Huang et al 2009). Terms associated with host genes up-regulated by RH infection include: transcriptional control, signaling, proliferation/apoptosis, sterol biosynthesis, and cell cycle regulation (Table SVI, Fig 7). Organellar functions, including mitochondrial metabolism and vesicular transport, were enriched among transcripts down-regulated by RH infection (Table SVI). VEG parasites also induced genes related to cell cycle control and sterol biosynthesis, but not those associated with transcriptional control, signaling or negative regulation of apoptosis, and did not down-regulate mitochondrial metabolism. As noted above, ROP38 expression counteracts many of the effects of RH infection, specifically by down-regulating RH-induced transcription factors and signaling molecules associated with proliferation/repression of apoptosis, and up-regulating mitochondrial function (Fig 7; Table SVI).

Figure 7
Effects of T. gondii infection (+/− ROP38) on host cell transcripts

DISCUSSION

The T. gondii kinome and the ROPK family of secreted kinases

The 108 predicted active T. gondii kinases in Fig 1 (20% more than Plasmodium, but <25% the size of the human kinome) constitute the largest apicomplexan kinome described to date (Table SI). This classification confirms previous experimental evidence, while expanding our knowledge of signaling cascades likely to be present in apicomplexans. Receptor kinases, including tyrosine kinases and receptor guanylate cyclases are completely absent (although tyrosine kinases have been found in some unicellular species; (Shiu & Li 2004). The T. gondii genome is predicted to encode a single STE kinase, and P. falciparum appears to lack MAPKK orthologs entirely, suggesting limited ability to exploit traditional MAPK cascades. A large fraction of the T. gondii kinome lacks the canonical catalytic triad, although we note that mounting evidence suggests that at least some such ‘pseudokinases’ are able to phosphorylate subtrates (Kannan & Taylor 2008; Kornev & Taylor 2009).

Even where parasite enzymes can be classified into one of the major kinase groups, they are often highly divergent; 78 of the 108 ‘active’ T. gondii kinases lack an obvious ortholog in human or yeast (Table SII). Far more apicomplexan kinases share orthologs between Toxoplasma and Plasmodium (Fig S1, Table SII). While secreted protein kinases are rare in eukaryotes, ~15% of apicomplexan kinases are predicted to be secreted outside of the parasite (Fig 1), suggesting that they may affect host-pathogen interactions. Most of these kinases belong to parasite-specific families, including the P. falciparum FIKKs and the T. gondii ROPKs. Two ROPK proteins (ROP16 & 18) have been reported as the only active members of a 9–12 member family dominated by kinase-like proteins lacking catalytic activity (El Hajj et al 2006; Sinai 2007). Our analysis reveals that the ROPK family contains at least 44 members, including 16 predicted to be active (Fig 2). Several ROPK genes are tandemly duplicated in the T. gondii genome (ROP2/8, ROP4/7, ROP 42/43/44, ROP19/29/38), and comparison with the genome assembly (ToxoDB.org) reveals that many lie at contig breaks, suggesting the presence of additional tandemly-duplicated ROPK genes.

ROP proteins were originally defined by their association with the rhoptries – part of the distinctive apical complex of secretory organelles defining the phylum apicomplexa. Rhoptries facilitate the interaction of T. gondii with host cells, including establishment of the parasitophorous vacuole within which these obligate intracellular parasites survive and replicate (Boothroyd & Dubremetz 2008). Subcellular localization using tagged transgenes demonstrates that most ROPK proteins target to the rhoptries and are secreted into the parasitophorous vacuole, where they associate with the vacuolar membrane and/or tubulovesicular network (Figs 3, ,4,4, S2). A recent report (Reese & Boothroyd 2009) described an N-terminal amphipathic alpha-helix associated with some ROPK genes (particularly the ROP2/18 clade; Fig 4A) that facilitates trafficking to the parasitophorous vacuole membrane when expressed in the cytoplasm of infected mammalian cells. We find that ROPK proteins lacking this domain (e.g. ROP38, formerly known as ROP2L5; Fig 3, top) are also able to associate with the tubulovesicular network, via unknown mechanisms.

Leveraging genomic-scale datasets to prioritize ROPK genes for further analysis

Because advantageous traits frequently emerge through gene duplication and functional divergence (Ohno et al 1968), expanded gene families can provide useful insights into organismal biology. Several members of the ROPK family have previously been shown to regulate T. gondii virulence and host-pathogen interactions: ROP18 was identified as a virulence factor by genetic mapping (Saeij et al 2006; Taylor et al 2006), and ROP16 alters phosphorylation of host STAT3/6 (Saeij et al 2007). Having defined the full spectrum of ROPK genes in the T. gondii genome, can we identify those that are most likely to play important roles in parasite biology?

Although the ROPK family forms a single clade distinct from previously-characterized kinase families (Fig 1), it is quite diverse: the subtree defined by ROP16 (Fig 4) exhibits greater protein sequence diversity than the entire human AGC family (PKA, PKC, etc), and the subtrees defined by ROP16 and ROP18 are as different from each other as PKA vs CAM kinases. Many ROPK proteins lack the complete catalytic triad required for kinase activity, but even inactive ‘pseudokinases’ may play important roles in substrate binding and/or allosteric interactions (Boudeau et al 2006).

Comparative genomics shows that the ROPK family is restricted to coccidia: the ROPK HMM identifies family members in Eimeria, Neospora, and Toxoplasma, but not Plasmodium, Babesia or Theileria. The apicomplexa also possess another family of secreted kinases (FIKK; Nunes et al 2007), which is expanded in P. falciparum only. Neospora and Toxoplasma share a recent common ancestor (Frenkel & Smith 2003), and comparison of their ROPK families is particularly informative (Fig 4). Most ROPK genes display a 1:1 correspondence between species, but there is evidence for rapid diversification. ROP 4, 7, 18 & 20 correspond to N. caninum pseudogenes, and NcROP46 corresponds to a pseudogene in T. gondii (Table SIII). Independent tandem duplications are also evident (Fig 4). It is interesting to note that DNA sequence is more highly conserved than protein sequence for most ROPK genes, and there is evidence for positive selection in 16 family members (Fig 4A, Table SIII).

Functional genomics data may also be exploited to identify genes of likely biological interest. Transfection studies have shown that the importance of ROP18 in parasite virulence is mediated through the regulation of transcript abundance. (Saeij et al 2006). Analysis of expression in the parasite kinome, secretome, and genome shows that a disproportionate number of ROPK genes are highly expressed in parasite tachyzoites (Fig 5B), differentially expressed in different strains (Fig 5C), and/or transcriptionally regulated during differentiation (most are down-regulated in bradyzoites; Fig 5D). Considerable difference is observed even between recently-duplicated ROPK genes (cf. ROP 19 vs 38; Table SIII).

Integrating these comparative and functional genomics analyses, ROP18 and ROP38 display the most striking indicators of biological significance (Table SIII): both show evidence of evolutionary selection at the population or species level (Khan et al 2009; Fig 4), both display >16-fold differences in expression level between strains (Fig 5C), and both are differentially regulated during bradyzoite differentiation (Fig 5D). ROP38 is particularly intriguing, as this clade has been independently expanded in both Toxoplasma and Neospora (Fig 4B), with NcROP19.2 and TgROP38 retaining (or converging upon) similar sequence while the other paralogs have diverged. Preliminary efforts to delete this gene have been unsuccessful, perhaps arguing for essentiality, as suggested by its high degree of conservation among T. gondii strains and between coccidian species.

ROP38 is a potent regulator of host-cell transcription

Previous studies showed that infection with T. gondii induces transcriptional changes in the host cell (Blader et al 2001; Saeij et al 2007) but the extent of change has not been accurately defined. We identified ~700 genes whose expression is significantly up- or down-regulated 24 hr after infection with VEG strain parasites (~400 >2-fold). RH-strain parasites exhibit a strikingly different pattern, significantly up- or down-regulating the expression of >6000 host cell genes (~1200 >2-fold;), including all previously-described examples of induction by RH infection (CXCL1, EGR2, HIF1α, etc (Blader et al 2001; Spear et al 2006; Phelps et al 2008; Fig 6, Table SV). Functional analysis reveals that infection with any T. gondii strain induces transcription of host genes associated with cell cycle control, DNA replication/repair, RNA processing, and sterol biosynthesis (Table SVI), but RH infection specifically induces transcriptional control and signaling pathways (including inhibitors of apoptosis, specially MAPKs), and represses organellar pathways (Table SVI). The striking changes in host cell transcription induced by this strain may be responsible for some of its unusual biological characteristics (Saeij et al 2005).

As noted above, several lines of evidence suggest that ROP38 is functionally important for T. gondii biology, and indeed, transgenic expression in RH strain parasites dramatically diminishes the impact of infection on host expression profiles. Increasing ROP38 expression to levels normally observed in VEG strain parasites significantly alters transcript levels of >1200 host genes (Fig 6B, Table SV). Although there is no evidence that significant quantities of ROP38 leave the parasitophorous vacuole (Fig 3 top), overexpression specifically down-regulates host transcription factors and genes associated with regulation of signaling and apoptosis/proliferation (all strongly induced by RH infection, but not altered by infection with VEG strain parasites). These genes include the transcription factors c-fos and EGR2 (known to be induced in a rhoptry-dependent manner; Phelps et al 2008). Functional clustering implicates host MAPK cascades, and preliminary results show that the kinetics of ERK phosphorylation in RH-ROP38-infected cells is distinct from the response to the parental RH line (Fig S4). We note, however, that the dramatic changes of parasite ROP38 expression on host transcript levels do not appear to affect parasite replication in vitro or virulence in vivo.

This report highlights the potential of integrating multiple, diverse genomic-scale datasets to aid in the discovery of biologically important molecules. Comparative genomic approaches defined the parasite kinome, revealing the full diversity of rhoptry kinases, and evolutionary genomic analysis of positive selection indicates the importance of this family. Functional genomics datasets facilitated the prioritization of ROPK family members for further exploration, and transcriptional profiling of the host cell, coupled with functional clustering, highlights pathways likely to be regulated by parasite infection and a role for ROP38 in the regulation of host transcription. In aggregate, integrating phylogenetics with functional genomic analysis and experimental manipulation of transgenic parasites has expanded our understanding of secreted kinases and their role as effector molecules during host cell infection. It will be interesting to determine the targets of these kinases, and whether they act directly or indirectly on host cell signaling pathways.

METHODS

T. gondii protein kinase phylogeny and classification

The T. gondii proteome (ToxoDB.org) was searched for protein kinase domains using Pfam Hidden Markov Model (HMM) PF00069 (pfam.sanger.ac.uk) and the HMMer package (Eddy 1998; cutoff −150, E <1). Matches were expanded to include orthologs identified by OrthoMCLv1 (OrthoMCL.org). Id entical results w ere obtained using the SMART H MMs SM00219 & 220 and Interpro IPR017442. Active kinases were defined based on a putative KDD catalytic triad and HMM score >−100; proteins scoring from −100 to −150 or lacking a complete triad were designated as pseudokinases.

Experimentally validated representatives from all major kinase groups were selected from the published kinomes of Homo sapiens (Manning et al 2002b), Saccharomyces cerevisiae (Hunter & Plowman 1997) and Plasmodium falciparum (Ward et al 2004), and aligned with active T. gondii kinase domains using HMMer, with PF00069 as a reference. Kinase subdomains were assessed by manual inspection, removing other regions from the alignment. PHYML 3.0 (Guindon & Gascuel 2003; Guindon et al 2005) was used for phylogeny reconstruction; 100 and 1000 bootstrap replicates yielded comparable results. Each pseudokinase was classified independently by constructing a new alignment and ML tree (100 bootstrap replicates), using the set of active kinases noted above.

Identification of the ROPK family and analysis of divergence

A ROPK-specific HMM was constructed based on kinase domain alignment of all active ROPKs without insertions: ROP16, 17, 18, 19, 21, 25, 27, 29, 30, 31, 32, 35, 38 & 41. Truncated genes were identified based on higher sequence identity to the ROPK HMM than any other sequence in the kinome. Neospora orthologs were identified by conceptual translation of syntenic regions from ToxoDB.org. MUSCLE (Edgar 2004) was used for multiple sequence alignment of full length ROPK proteins (excluding columns with gaps in >90% of sequences), and PHYML 3.0 was used for phylogeny reconstruction. For analysis of site-specific positive selection, full length orthologous protein sequence pairs from T. gondii and N. caninum (along with recent paralogs) were aligned with MUSCLE, and the underlying nucleic acid sequence alignments used as input for PAML codeml (Yang 1997), using nested models M7 and M8 to generate a likelihood ratio test; sequences under positive selection were determined based on a standard Chi-square probability distribution.

Parasite transfection and immunolabeling

ROP16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 38, 39, and 40 were amplified from an RH strain T. gondii cDNA library using gene specific primers, subcloned into the NheI/BglII cloning sites in ptub-HA/sagCAT (Nishi et al 2008), and sequenced to confirm fidelity. All gene specific primers amplified a single gene product. Transfections were performed as previously described (Roos et al 1994), and examined by immunofluorescence 24 hr post-transfection; stable transgenic lines expressing either 55.m05046 (RH-ROP21) or 49.m03275 (RH-ROP38) were isolated by chloranphenicol selection and cloned by limiting dilution.

For subcellular localization, 4×105 parasites were inoculated into confluent host cell monolayers on 22 mm glass coverslips, incubated 24 hr at 37°C, fixed in 3.7% paraformaldehyde, permeabilized with 0.25% Triton X-100, and stained with (i) mouse monoclonal anti-HA conjugated to Alexa 488 (Roche; 1:1,000), (ii) mouse monoclonal anti-TgROP2/3/4 (1:50,000, kindly provided by Jean François Dubremetz) followed by Alexa 594-conjugated goat anti-mouse (Molecular Probes; 1:5,000), and (iii) 2.8 μM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen). This antibody recognizes proteins in the rhoptries, with PVM localization observed only immediately after infection, presumably due to inaccessibility of the epitope (Jean François Dubremetz, personal communication). Samples were visualized on a Leica DM IRBE inverted microscope equipped with a motorized filter wheel, 100W Hg-vapor lamp and Orca-ER digital camera (Hamamatsu). Openlab software (Improvision) was used for all image acquisition and manipulation.

For immunoblot analysis of phosphorylated ERK, T. gondii-infected HFF host cells were passed through a 26 ga needle to ensure efficient egress, filtered through a 3 μm filter, and resuspended in CO2 equilibrated reduced serum medium (Opti-MEM; GIBCO) prior to inoculation into serum-starved confluent HFF cells in 25 cm2 T-flasks (MOI 5:1). At various times post-infection, samples were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Sigma #P5726; Roche complete mini protease inhibitor tablets) and frozen. Lysates were resuspended in Invitrogen NuPage LDS buffer containing 5 mM β-mercaptoethanol, incubated 5 min at 85oC, and 4 μg protein (determined by Bradford assay) loaded on a NuPage 10% BT SDS gel, followed by semi-dry transfer to nitrocellulose membrane, and sequential 1 hr incubations in 10% non-fat milk in PBS, monoclonal anti-diphosphorylated ERK 1&2 (Sigma, 1:4000) and monoclonal anti-α-tubulin (Sigma, 1:7500), and HRP conjugated goat-anti-mouse (BIO-RAD, 1:3000), proteins were visualized by 5 min incubation with Immobilon Western Chemiluminescent HRP substrate (Millipore) and 2 min exposure to BioMaxMR Film (Kodak).

Toxoplasma gondii expression profiling

T. gondii parasites were cultivated in vitro in HFF cells (MOI 10:1) using standard methods (Roos et al 1994), and RNA harvested from purified parasites (RNeasy; Qiagen); quality was ascertained using a spectrophotometer (NanoDrop), and confirmed with an Agilent Bioanalyzer. The Affymetrix Expression 3′ One-Cycle amplification kit was used to prepare labeled cRNA, which was hybridized to a custom T. gondii Affymetrix microarray (ToxoDB.org), and the fluorescent signal collected by excitation at 570 nm and confocal scanning at 3 μm resolution; low affinity probes and sequences redundant in the parasite genome were excluded (not a concern for ROP38, which harbors few SNPs). T. gondii microarray data has been deposited with GEO (GSE22315, GSE22258), and is also available for querying and downloading at ToxoDB.org.

Two sequential scans were averaged for each microarray feature, and Robust Multiarray Analysis (RMA) normalization used to calculate relative RNA abundance for each gene (log2 values). Three replicates of each experiment were used to assign a p-value and average log fold change between strains or time points during differentiation using the limma R package (Bioconductor). Tachyzoite expression was determined for strains RH, GT1, Prugniaud, ME49, VEG, RH-ROP38 and RH-ROP21, and bradyzoite differentiation data was obtained at various time points after alkaline induction of the Prugniaud strain (Dzierszinski et al 2004). The following known bradyzoite markers were used as controls: 9.m03411, 72.m00004, 80.m00010, 55.m00009, 641.m01562, 72.m00003, 59.m03410, 44.m00006, 59.m00008, 44.m00009, 641.m01563. A power analysis was carried out for all pairwise strain comparisons to establish statistical significance of ≥2-fold differential expression, based on a calculated false discovery rate of 8% at p = 0.05 (1% at p = 0.01). Maximum log fold change between strains was defined as the maximum difference between any two strains.

Host-cell expression profiling and cluster analysis of functional enrichment

Freshly-harvested VEG, RH, RH-ROP21 and RH-ROP38 parasites were inoculated and incubated 24 hr before harvest and RNA isolation as described above (MOI 20:1). At least three experimental replicates were conducted for each assay. Only samples with comparable ratios of parasite:host RNA (determined by Agilent Bioanalyzer) were used for hybridization. Illumina HumanRef8_V2 and V3 microarrays were hybridized according to the manufacturer’s instructions, scanned on a BeadScan unit, analyzed using the gene expression module of GenomeStudio software (Illumina), and deposited in the GEO database (GSE22402). Due to incompatibilities between V2 and V3 probes, only 15,554 human genes were analyzed in comparisons across all samples. Genes were defined as expressed if they displayed a detection p-value <0.05 in at least one sample. Comparison with uninfected or RH-infected HFF cells was used to determine differential expression based on an Illumina differential expression score of >|30| (nominally equivalent to p < 0.001.)

Datasets for functional annotation were defined as the set of genes exhibiting statistically significant up- or down-regulation >2X. Enrichment of functional annotation (GO Biological process, GO Molecular Function, KEGG pathways, Biocarta pathways, InterPro and PFAM domains, SwissProt and Protein Information Resource keywords) was assessed using the DAVID package (Dennis et al 2003; Huang et al 2009). Enrichment relative to the 15,554 Illumina probes common to V2 and V3 arrays, was defined as a p <0.05 with at least 3 genes per term per dataset. Fuzzy heuristical clustering was performed using kappa similarity >0.3–0.35, and requiring an enrichment p-value geometric mean > 0.05.

Highlights

  • The 159 gene T. gondii kinome includes a family of 44 secreted ‘ROPK’ kinases.
  • ROPKs show evidence of evolutionary selection and are differentially expressed.
  • Integrative genomic analyses highlight ROP38 as likely to be functionally important.
  • ROP38 expression alters the abundance of ~1200 host cell transcripts in infected cells.

Supplementary Material

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Acknowledgments

We thank the Roos laboratory and T. gondii and ISCB research communities for helpful discussions. Neospora sequence data were produced by the Wellcome Trust Sanger Institute, funded by the BBSRC(UK) and are available from http://www.sanger.ac.uk/Projects/N_caninum/. This work was supported by research grants AI28724, AI077268, AI075846 and RR016469 from the NIH.

Footnotes

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