Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes.

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.

Theileria highjacks JNK2 into a complex with the macroschizont GPI (GlycosylPhosphatidylInositol)-anchored surface protein p104.

Constitutive c-Jun N-terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI-(GlycosylPhosphatidylInositol)-anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase-A (PKA)-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages.

Identification and characterisation of a Theileria annulata proline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface.

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria-induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co-immunoprecipitation, we identified a CLASP1/CD2AP/EB1-containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14-3-3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta-p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain-interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1-binding SxIP motif, as well as functional SH3 domain-binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non-infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.

HIF-1α induction, proliferation and glycolysis of Theileria-infected leukocytes.

Within 2 h of infection by Theileria annulata sporozoites, bovine macrophages display a two- to fourfold increase in transcription of hypoxia inducible factor (HIF-1α). Twenty hours post-invasion sporozoites develop into multi-nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper-invasive and disease-causing leukaemia-like cell. Once immortalized Theileria-infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF-1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF-1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF-1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high Reactive Oxygen Species (ROS) environment. We propose a model where uninfected macrophages have low levels of H2 O2 output, whereas virulent-infected macrophages produce high amounts of H2 O2 . Further increase in H2 O2 output leads to dampening of infected macrophage virulence, a characteristic of disease-resistant macrophages. At the same time exposure to H2 O2 sustains HIF-1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF-1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukaemic-like cell, we propose that manipulating ROS levels, HIF-1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2 O2 drive virulent Theileria-infected macrophages towards attenuation it highlights how infection-induced pathology and redox balance are intimately linked.

Proteomic analysis of the Theileria annulata schizont.

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

Determination of adenosine deaminase activity in cattle naturally infected with Theileria annulata.

The purpose of this study was to determine serum ADA activity in cattle naturally infected with Theileria annulata. In this study, a total of 37 cross-bred cattle which 27 of it showing clinical signs of theileriosis constituted infected group and 10 healthy cattle as control group were used as animal materials. Infected group divided into three groups according to their PCV values. Cattle with PCV > or = 25 were put on group I (n = 9), those with PCV 13-24 were put on group II (n = 11) and those with PCV < or = 12 were put on group III (n = 7). Microscopical diagnosis of the disease was also made. Hematological parameters, serum enzyme activities (ADA, AST, ALT and ALP) were determined in all cattle. Hematological results revealed that significant progressive decreases in HGB, PLT, PBML counts and ratios from group I onwards to group III, whereas the WBC, PBPL counts and ratios showed an increase from group I onwards to group III. The serum ADA, AST, ALT and ALP activity increased significantly in all infected groups compared to control group. However, these parameters were also observed to decrease progressively from group I to group III. Furthermore, the highest increase in enzyme activities observed in the infected group I. But, these enzyme's activities started to decrease in infected group II and III in parallel with PBML and PLT counts. Eventhough, this decrease did not reach to the values obtained from control group. On the contrary, PBPL counts and ratios increased in infected group II and III in contrast to decrease in PCV. As a result, increased serum ADA activity in tropical theileriosis may reflect the involvement of the cellular immune responses.

Genome of the host-cell transforming parasite Theileria annulata compared with T. parva.

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

Molecular genetic characterization and subcellular localization of a putative Theileria annulata membrane protein.

A Theileria annulata protein (TaD) exhibiting an N-terminal signal sequence for endoplasmic reticulum membrane translocation and a conserved cysteine-rich region was isolated by screening the mRNA of a T. annulata-infected bovine lymphoblastoid cell line with degenerated primers directed against T. annulata-targeting sequences. The TaD-coding sequence was found to be most closely related to the genomic DNA sequence of T. parva (TIGR database, 72%) and the amino acid sequence of Plasmodium falciparum (41%), P. yoelii yoelii (38%) and Cryptosporidium parvum (36%). The TaD mRNA is expressed within the sporozoite, schizont and merozoite stages of the parasite, implying that it is constitutively transcribed throughout the parasite's life cycle. Allelic variants were found between isolates originating from different geographical regions, however not affecting conserved cysteines. The open reading frame encoded a protein of 19.5 kDa and non-reducing SDS-PAGE analysis demonstrated a homodimeric protein. Using confocal microscopy, the protein was found to be both located in the parasite cytoplasm and to colocalize with a transmembrane protein of the schizonts within infected cells.

TashHN, a Theileria annulata encoded protein transported to the host nucleus displays an association with attenuation of parasite differentiation.

The intracellular apicomplexan parasite, Theileria annulata, manipulates its bovine host cell by over-riding the cells natural apoptotic response and inducing proliferation of the infected leukocyte. We have recently identified a T. annulata encoded family of polypeptides (TashATs) with characteristics that indicate that they are involved in control of host cell gene expression. Here we present data on another member of this family, TashHN, showing that it is located to the parasite and host cell nucleus. Immunoblot analysis demonstrated that, unlike TashAT2 and 3, TashHN displays three forms, the largest of which is enriched in the host nuclear fraction and appears to be phosphorylated. Northern and 5 prime race analyses identified multiple TashHN RNA species in infected cells that have retained the ability to differentiate. These transcripts showed subtly different kinetics, but all decreased during differentiation to the merozite, and two showed reduced levels prior to down-regulation of the other TashATs. In addition, analyses of multiple cell lines that have become severely attenuated in their potential to differentiate, indicated a substantial increase in TashHN expression, with host nuclear reactivity particularly enhanced.

Initiation of translation and cellular localization of Theileria annulata casein kinase IIalpha: implication for its role in host cell transformation.

Theileria annulata and T. parva are protozoa that infect bovine leukocytes which leads to subsequent transformation and uncontrolled proliferation of these cells. It has been proposed that the CKIIalpha subunit of T. parva induces mitogenic pathways of host leukocytes by being exported into the host cell. The evidence for this is the existence of a predicted N-terminal secretion signal-like peptide. We tested this hypothesis by analyzing gene structure, translation, and protein localization of the T. annulata CKIIalpha (TaCKIIalpha). The determined TaCKIIalpha-ORF potentially codes for a 50 kDa protein with an N-terminal extension including a possible signal sequence not present in CKIIalpha proteins of non-Theileria species. However, antisera raised against TaCKIIalpha recognized a protein of a molecular weight of about 40 kDa and, therefore, inconsistent with this predicted molecular weight. We demonstrate by in vitro transcription/translation that this discrepancy is due to translation from a downstream initiation site omitting the putative N-terminal signal sequence and thus excluding the notion that the protein product is secreted via the classical secretory pathway. In corroboration immunofluorescence investigations suggest that the TaCKIIalpha subunit is confined to the parasite schizonts within the host cell. On the basis of the above findings it seems highly unlikely that export via the classical pathway of the parasite CKIIalpha is the way in which this protein possibly contributes to host cell transformation.

Identification of a 40S Ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata.

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileria annulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed.

TaCRK3 encodes a novel Theileria annulata protein kinase with motifs characteristic of the family of eukaryotic cyclin dependent kinases: a comparative analysis of its expression with TaCRK2 during the parasite life cycle.

The TaCRK3 gene from the bovine apicomplexan parasite Theileria annulata, encodes a 46 kDa polypeptide with strong homology to the eukaryotic family of cyclin-dependent kinases. TaCRK3 does not show significant alignment with any particular CDK group, other than the Pfmrk kinases from the related apicomplexans Plasmodium falciparum and Plasmodium yoelii. It has a putative bipartite nuclear localization signal and is located to parasite nuclei by IFAT. Protein levels are constitutive throughout differentiation of the intra-lymphocytic macroschizont. This contrasts with the expression pattern of TaCRK2 (Kinnaird et al., 1996, Mol. Microbiol., 22, 293-302) which is closely related to the eukaryotic CDK1 /2 families involved in regulation of cell cycle progression. TaCRK2 is also located to the parasite nuclei but has no nuclear localization signal and exhibits transient up-regulation in protein levels during mid-merogony. However compared to TaCRK3, it shows down-regulation near the end of merogony. We predict that TaCRK3 may have a role in regulation of gene transcription while TaCRK2 is more likely to be involved in control of parasite nuclear division.

Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70.

A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T. annulata-infected bovine lymphoblastoid cell line with an antibody raised against T. annulata schizonts. The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens). The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle. The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins. The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C. By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells. Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites. However, it was also found in small amounts in the cytoplasm of the parasite and host cell. This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm.

An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata.

Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5' upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5'-TTTGTAGGG-3'). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.

Macrophages behaving badly: infected cells and subversion of immune responses to Theileria annulata.

The protozoan parasite Theileria annulata is the causative agent of the tick-borne disease tropical theileriosis, responsible for morbidity and mortality of cattle in many developing countries. Here, John Campbell and Roger Spooner discuss how the parasite might evade immune destruction during an acute primary infection. Theileria annulata macroschizont-infected macrophages act as over-efficient antigen-presenting cells within the infected draining lymph node. Infected cells activate CD4+ and CD8+ T cells abnormally, giving rise to a cascade of cytokine production. This altered immune response does not reject the parasitized cells, and might actively participate in the growth of the developing parasite.

Directing differentiation in Theileria annulata: old methods and new possibilities for control of apicomplexan parasites.

Apicomplexan parasites are major pathogens of humans and domesticated animals. The ability of these organisms to evade the host immune response and the emergence of drug-resistant parasites indicates a need for the identification of novel control strategies. Ideally, selected targets should be shared by a range of apicomplexans and fundamental to parasite biology. One process of apicomplexan biology which may provide this type of target is the molecular regulation of stage differentiation. This paper has reviewed studies carried out on differentiation of Theileria annulata and has highlighted general similarities with other apicomplexan differentiation steps. Similarities include asynchrony of differentiation, the loss (attenuation) of differentiation potential and an association between reduced proliferation and differentiation. In addition, novel data are presented assessing a possible role for a signal transduction mechanism or a direct involvement of classical heat-shock polypeptides in regulating differentiation of T. annulata in vitro. These studies, and previously published data, have led to the postulation that progression to the next stage of the life-cycle can be predetermined and involves the attainment of a quantitative threshold by regulators of gene expression. A modification of this model takes into account that for certain in-vitro systems, or differentiation steps in vivo, the process has to be initiated by alteration of the extracellular environment. Work which has shown that the time taken to achieve differentiation can be increased or decreased is also outlined. The ability to change the timing of differentiation suggests that the associated regulatory mechanism could be manipulated directly to significantly influence the outcome of an apicomplexan infection. The observation that a number of existing drugs and control strategies may exert their protective effect by altering differentiation potential supports this possibility.

A stage-specific, parasite-induced, "window" of in vivo interferon-gamma production is associated with pathogenesis in Theileria annulata infection.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe leukoproliferative disease of cattle, which naive susceptible animals fail to control. The parasite infects and transforms macrophages, developing in the local draining lymph node. IFN gamma has been shown to block parasite development in newly infected cells, and inhibits the growth of fully differentiated macroschizont stage-infected cells in vitro. However, the parasite has been found to specifically induce IFN gamma production by T cells and appears to flourish in the face of this T cell-derived response in vivo. Here we show that the production of IFN gamma in vivo is tightly controlled by the parasite. Induction of cytokine production by T cells is not initiated until the parasite has developed beyond the IFN gamma sensitive trophozoite stage. Cytokine production is kept high as infected macrophages develop, and IFN gamma appears to play an active role in maintaining the growth of these cells. Once the infection is fully established, IFN gamma is down regulated, avoiding potential inhibitory effects. Thus by controlling T cell IFN gamma production, the parasite induces a "window" of cytokine expression which promotes its own growth, but avoids potential inhibitory effects of the cytokine.

Metastasis of Theileria annulata macroschizont-infected cells in scid mice is mediated by matrix metalloproteinases.

Theileria annulata (Ta)-infected leucocytes are able to disseminate in scid mice. The dose of virulent parasites of the Ta-Ode line required to achieve quantifiable dissemination was found to be 2 x 10(6) cells given i.p. Dissemination was higher on day 11 post-inoculation than on day 18. The attenuated Ta-Ode cells were found to disseminate very poorly compared to their virulent progenitors, which correlates with a marked reduction in matrix metalloproteinase (MMP) expression. A daily i.p. injection of mice with BB94, a synthetic inhibitor of MMPs, almost completely ablated dissemination compared to controls. This provides strong evidence that metastasis of Theileria annulata macroschizont-infected host cells is mediated by host MMPs induced by the parasite. This has important implications for explaining a number of pathological features of tropical theileriosis in cattle.

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.