Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata

Apicomplexan parasites are major pathogens of humans and domesticated animals. Infection of the mammalian host requires establishment within a range of different host cell types that can vary in a species-specific manner. While the molecular mechanisms these parasites use to manipulate the phenotype of the infected cell are beginning to be understood [1], little is known about how different parasite species within a genus have evolved to establish infection in cells of different lineages or novel host species. The genus Theileria encompasses species of tick-transmitted parasitic protozoa that infect domestic livestock and other mammals. Of the five species that cause clinical disease in cattle, the two most important are T. annulata, the agent of tropical theileriosis, which is widespread in North Africa, Southern Europe and Asia and T. parva, the agent of East Coast fever a highly fatal disease of cattle in East and Central Africa. Following inoculation by the tick vector, the parasite invades and transforms host leukocytes, which divide in synchrony with the intracellular multi-nucleate macroschizont stage. For T. annulata, parasite infection results in establishment of transformed myeloid cells but never T lymphocytes. For T. parva, T cells are the preferred host cell type and while macrophages can be infected they are not transformed [2]. Establishment of the transformed leukocyte is characterised by activation of a number of host cell transcription factors and the production of inflammatory cytokines, the profile of which varies considerably between T. annulata and T. parva infected cells [3]. In T. annulata, recovery from primary challenge results in non-sterile immunity, involving T cell recognition of class I presented parasite peptides, followed by the development of the persistent carrier state [4]. This state is highly important for transmission of the parasite and in T. annulata causes significant economic losses due to sub-clinical infection [5]. The mechanisms by which the parasite avoids clearance from the host are obscure although in part, it is believed that low-grade infection is maintained by macroschizont-infected leukocytes residing in immunologically privileged sites [4]. It is also likely that the macroschizont-infected cell actively evades and subverts the bovine immune response [2, 3, 6] but the molecular mechanisms involved have not been elucidated.

A comparative analysis of the genomes of T. annulata and T. parva has shown that genes encoding polypeptides predicted to be either on the surface of the merozoite (the extra-cellular, bloodstream stage) or secreted by the macroschizont exhibit relatively high inter-species ratios of non-synonymous to synonymous nucleotide substitutions (d_Nd_S) [7]. For the macroschizont stage, this indicates that genes encoding secreted proteins are more likely to be under positive selection at an inter-species level than genes encoding non-secreted products. In many other pathogens, elevated d_Nd_S ratios have been observed for antigen-encoding genes. In an early study, d_Nd_S ratios of a large number of homologous sequences lodged in Genbank were calculated, identifying 17 groups of genes across a range of species as being under the influence of positive selection, nine of which encoded surface antigens of parasites and viruses [8]. It has been shown that significant allelic diversity of antigen genes with positive d_Nd_S ratios occurs within pathogen species [9] and in the case of T. annulata merozoite surface antigens, positive selection for allelic diversity has been attributed to host immune selection [10]. However, it is unlikely that full-length Theileria proteins expressed by the macroschizont are directly exposed to the host immune response since no parasite-encoded products have been identified on the surface of the infected leukocyte, despite intensive investigation [11]. Thus the reasons for elevated d_Nd_S and positive selection in macroschizont-expressed genes encoding secreted products remain to be determined.

A number of genus-specific gene families have been identified in the genomes of T. annulata and T. parva, several of which are predicted to encode products secreted by the macroschizont into the host cell compartment [7]. Together with a number of single-copy genes, these families encode the predicted Theileria secretome that is likely to represent a critical interface between host and parasite systems. The present study was designed to investigate parasite gene families encoding products that may play a role at this interface and are evolving under positive, diversifying selection. In this study we have conducted an analysis of allelic diversity of T. annulata secretome gene families in order to provide insight into their evolution and putative function. For example, genes that have evolved to allow adaptation to a particular biological niche, such as a cell lineage or host species, would be predicted to be conserved within a parasite species but show divergence between species that exploit different host backgrounds or cell types. Consequently, gene families involved in host adaptation would be predicted to stabilise within each species and this would be reflected in evidence of purifying selection operating at the allelic level. In contrast, a gene family representing multiple paralogous antigens may be predicted to show evidence of diversification at both the inter-species and intra-species level. To investigate this hypothesis, the published Theileria genomes were compared in order to identify two positively selected gene families with distinctive and contrasting bioinformatic signatures. This would allow representative genes to be identified and the broad pattern of diversity exhibited by each family to be characterised and interpreted.

Large multi-gene families located at the sub-telomeres of protozoan parasite genomes often encode antigens that display sequence hyper-variability to allow escape from a protective immune response, while genes encoding proteins that engender adaptation to particular host environments are more likely to be conserved within-species. In this study we have analysed allelic diversity of members of two distinct families of proteins of T. annulata that are predicted to be secreted into the host compartment and have been considered as candidate parasite molecules that enable evasion of a protective immune response (SVSP s [20]) or function to control host cell phenotype (TashAT s [14, 22]). SVSP genes are arranged in sub-telomeric arrays while all the TashAT s are located in an internal cluster on chromosome one.

Allelic sequencing identified a high level of diversity at the nucleotide and amino acid level across the length of all four SVSP genes analysed, with hyper-variability identified at the site of the predicted PEST degradation motifs (see Figure 5). However, the combined analyses performed in this study indicate that the SVSP genes are evolving in a neutral manner. The McDonald-Kreitman test, specifically designed for detecting intra-specific selection, also indicated these genes have not been subject to a strong positive selective pressure (see Table 2) with Tajima's D and Fu and Li's D and F tests failing to provide any evidence of a deviation from neutrality. Thus, with a large proportion of neutral mutations, SVSP genes appear to be evolving in the absence of strong purifying selection within T. annulata populations. In contrast to the findings for SVSP s, the level of allelic diversity of the four TashAT family genes that were analysed was, in general, less extensive with evidence for strong conservation of the predicted AT-hook functional motifs. Moreover, analysis of the TashHN sequences provided significant data for purifying selection to conserve amino acid sequence at the intra-specific level, while the SuAT1 alleles showed evidence for positive selection, with a greater number of non-synonymous mutations compared to synonymous mutations resulting in a higher within-species ratio and a neutrality index greater than one. We conclude that it is likely that the differences in the pattern of allelic diversity in extant populations displayed by the representative loci of these two major secretome gene families are a consequence of different evolutionary pressures that reflect contrasting functional properties of the proteins they encode.

Motif prediction analysis suggests the majority of SVSP proteins are secreted into the leukocyte cytoplasm [7] and it can be postulated that they play a role in the interaction between the parasite and the infected host cell. In addition, many SVSP s in both T. annulata and T. parva encode predicted nuclear localisation NLS motifs, and the vast majority possess the 'frequently associated in Theileria' FAINT domain, characteristic of proteins that constitute the macroschizont secretome. The conservation of the NLS in the allelic sequences of SVSP 3 and 4 identified in this study indicate this motif is functionally conserved, supporting the recent demonstration in T. parva that a typical SVSP protein can locate to the nucleus of transfected mammalian cells [20]. In addition, the allelic sequence data highlighted the L-PETIPVEIGSDED motif notable for its level of conservation in the midst of a region that was shown to be divergent. Related motifs can be identified in a number of predicted proteins of the T. parva secretome (data not shown) and nearly all TashAT family members [1]. Despite the identification of conserved motifs, a biological function for SVSPs has yet to be proposed. Although a T. parva-encoded SVSP (TP03_0882) was shown to locate to the nucleolus in transfected U2OS cells, a host nuclear/nucleolar location for SVSPs in Theileria infected cells was not demonstrated [20]. Indeed, detection of macroschizont reactivity by an anti-SVSP serum was limited to a small number of cells and endogenous polypeptide was not detected by immunoblotting [20]. This is in stark contrast to members of the TashAT cluster analysed in this study, which have been shown to be present at significant levels in the nucleus of the majority of T. annulata macroschizont-infected leukocytes [13, 14].

One explanation for the difficulty in detecting SVSPs is that these proteins are likely to be rapidly degraded within the host compartment, and the possession of multiple PEST motifs that are known to target eukaryotic proteins for proteolytic degradation supports this. The presence of such motifs and signal peptide on SVSPs is compatible with the hypothesis that these proteins are secreted into the host compartment, degraded and subsequently presented as peptides on MHC Class I molecules [25]. Recognition of class I presented peptides by cytotoxic T cells (CTL) has been shown to play an important role in protective immunity against T. parva[26] and an SVSP family member has been identified among a panel of T cell antigens in this species (I. Morrison, personal communication). The recognition of pathogen peptides by CTL is known to exert an immune selection pressure that results in selection of amino acid substitutions in critical residues of the epitope [27], and evidence for diversifying selection of a predominant (non-SVSP) T cell epitope of T. annulata has been obtained (Weir and Morrison, unpublished data). However, despite showing a significant level of allelic diversity, none of the T. annulata SVSP gene sequences analysed in this study provide evidence to support the hypothesis that divergent allelic forms of the SVSP proteins studied have evolved to escape recognition by CTL. Nevertheless, it has been argued that Theileria CTL antigens may be subject to weak selection as a result of bovine MHC Class I polymorphism [28] and therefore this possibility cannot be completely discounted.

The SVSP family of genes are located at the sub-telomeric regions of the chromosomes and show evidence of expansion and diversification in T. annulata and T. parva. A telomeric location of gene families can promote ectopic recombination and the presence of a large number of indels across T. annulata SVSP allelic sequences (see Figure 5) suggests that generation of sequence diversity via recombination could be a relatively frequent event, at least in the family members studied. These characteristics bear similarity to families of sub-telomerically located genes in other protozoan parasites that perform an essential biological function but, due to exposure to a protective immune response, have evolved into multiple divergent antigenic forms. Variant expression of different antigenic types allows escape from protective immunity, and the genes act as a contingency against parasite killing. The two most extensively studied systems are the vsg[29] and var[30] gene families of Trypanosome and Plasmodium parasites, respectively. The manner in which these contingency gene families have evolved has been difficult to assess and consequently there is no experimental data for or against diversifying selection or neutrality. Moreover, since it is possible that selection against contingency genes operates, in principle, against the whole antigenic repertoire, the finding of neutrality for allelic diversity of SVSP s does not exclude a role for SVSP s as a contingency gene family. Indeed, the observation that SVSP sub-telomeric gene location bears similarity to vsg and var gene families has been made and it was proposed that restricted expression of individual SVSP s could be responsible for the small number of parasites expressing detectable SVSP antigens [20].

For SVSP s to act as a family of classical contingency genes, like the vsg and var genes, it can be expected that SVSP mRNA expression patterns would show evidence of variant expression between distinct parasite genotypes and over time. However, transcription profiling of SVSP genes in both T. annulata and T. parva infected cell lines does not fit the classical pattern of variant expression, as the majority of SVSP genes are co-expressed at the RNA level [7, 20]. In addition, comparison of different infected cell lines showed that the SVSP expression profile was largely comparable and did not vary over time. While the possibility of differential translational control or protein stability cannot be discounted, an alternative model that merits consideration is that in all macroschizont infected cells, the majority of SVSP proteins are continually generated and rapidly degraded. If such a situation occurred then each infected cell would simultaneously generate a plethora of random variant peptides with the potential to be presented at the host cell surface with either class I or class II MHC antigens, assuming the SVSPs contain the relevant motifs for presentation. The consequence of this large peptide repertoire on subversion of the host immune response to these proteins is unknown, but it would be reasonable to predict that a wide range of peptide specificities would be generated with each peptide present at a relatively low frequency. Like the contingency theory, selection for a particular amino acid variant may not operate, as it is the generation of the divergent pool itself that confers a selectable advantage. Investigation of SVSP gene expression at the level of the single infected cell and evidence for stimulation of cells of the immune response by SVSP peptides will be required to provide supporting evidence for the postulation that the SVSP family has evolved to subvert the bovine immune response.

In direct contrast to the results obtained from analysis of T. annulata SVSP genes, TashHN was found to have very limited allelic diversity within the species. Indeed, several codons were found to be under the influence of negative selection whereas none showed evidence of positive selection. The lack of diversity within T. annulata is not unexpected as there is a high level of sequence identity with the T. parva orthologue and predicted functional motifs are located in identical positions within the polypeptide [23]. Based on this, it is proposed that an ancestral gene with significant identity to TashHN was present in the common ancestor of T. annulata and T. parva. TashHN has no identified DNA binding domains but is known to locate to the leukocyte nucleus [31]. Evidence for phosphorylation of the protein has been generated and its expression levels are significantly elevated in infected cell lines that show an attenuated phenotype [31]. In conclusion, the results obtained in this study strongly support the hypothesis that the function of the TashHN protein as a modulator of host cell phenotype is conserved across transforming Theileria species and predicts that sequence diversity would also be limited in T. parva.

Unlike TashHN, T. annulata TashAT family genes encoding polypeptides possessing AT-hook motifs (TashAT1-3 and SuAT1-3) show significant divergence from their T. parva orthologues, which completely lack AT-hooks. This finding could be interpreted as indication that the AT-hook motifs do not perform a critical biological function. However, the results of this study clearly imply that species-specific functional divergence of TashAT family genes has occurred following the T. annulata/T. parva split, as strong conservation of the AT-hook motifs was observed in all TashAT and SuAT1 sequences analysed in this study. Further studies are required to demonstrate whether motifs specific to the T. parva orthologues are conserved across alleles. Interestingly, the TashAT3 allele cluster does not segregate at the first branch of the tree displayed by Figure 7, suggesting that the ancestral TashAT3 may have been more closely related to particular alleles of TashAT2, and that complete sequence divergence has not occurred at these loci. For the majority of SuAT1 alleles, strong conservation of the sequence defining AT-hook 1 and 2 was found, together with conservation of the spacing between the motifs. This suggests that binding to the target AT-rich sequence is an essential function of this protein. In mammalian systems, specificity of promoter recognition by AT-hook proteins has been attributed to differences in core motif sequence and spacing between motifs: the HMGA1 protein, for example, has three AT-hooks and specifically binds to tandem repeats of AT-rich sequence in the INF-β gene using the central AT-hook in combination with either the first or third hook [32]. The arrangement of AT-hook motifs in TashAT sequences (Figures 6 and 7) shows similarity to HMGA proteins and the AT-hook region of TashAT2 has been shown to bind tandem repeats of AT-rich DNA [14]. We conclude that the results of this study support the postulation that members of the TashAT gene family have evolved to modulate the activation outcome of the host cell lineage preferentially infected by T. annulata.

Despite displaying strong conservation of functional motifs, variation was identified across TashAT/SuAT allelic sequences. Firstly, TashAT2 and 3 alleles display extensive variability over the AT-hook region with allelic variants having between one and five hook motifs. There is also variation in the distance between pairs of hook motifs, fixed around three gap sizes, and different alleles show different combination of AT-hook gaps. Variation in number of and spacing between AT-hook motifs has been documented for a number of proteins in different organisms and is thought to be associated with distinct target recognition [32]. AT-hooks have been described as evolutionary mobile modules dispersed by translocation of complete and contiguous units of the motif [24] and our results support this model. Why T. annulata exhibits this level of diversity in DNA binding proteins implicated in modulation of host cell gene expression is not clear. In this context, further allelic sequence analysis of other members of the gene family will determine whether these conclusions apply to the family as a whole. It is of interest to note that different T. annulata and T. parva infected cell lines show significant variability in the profile of cytokine genes they express [33, 34], despite activation of the same host cell transcription factors [35].

Geographical sub-structuring of TashHN and SuAT1 allelic sequences (Figures 4 and 6) was identified in this study. The simplest explanation for this divergence is isolation and genetic drift, whereby mutations in the gene arising in separate areas eventually resulted in different allelic types. However, to date clear sub-structuring of divergent allelic sequences appears to be restricted to TashAT family genes, as this property is limited or absent for all other loci analysed to date. In addition, the evidence of positive selection of SuAT1 sequences within the species is most likely to be due to the evolution of two distinct groups of SuAT1 alleles that show significant differences in amino acid sequence, but maintain the predicted basic functional motifs. A more radical hypothesis is that evolution of TashAT family proteins has been driven by recognition of differences in chromatin targets and that these have evolved to be subtly different in cattle or buffalo indigenous to geographically distinct regions endemic for T. annulata.

The results of this study indicate that contrasting selection pressures have shaped the evolution of two gene families encoding secretome proteins of T. annulata and that these differences are likely to relate to the function of the encoded proteins. SVSP genes have evolved to be divergent (or perform a function that has limited requirement for amino acid conservation) and a logical prediction is that SVSP proteins interact at an undefined level with the bovine immune system. In contrast, TashHN shows a high level of constraint and may function to alter a property of the host leukocyte that is fundamental to infection by both T. annulata and T. parva. Finally, TashAT genes are constrained within, but not between, species and show clear evidence of divergence in the arrangement of conserved functional motifs. These proteins are most likely to perform a species-specific function that allows adaptation to infection and activation of the preferred host cell for each species and, more controversially, host genetic backgrounds.