A single exon-encoded Theileria parva strain Muguga cysteine protease (ThpCP): Molecular modelling and characterisation.

The tick-transmitted apicomplexan Theileria parva causes East Coast fever, a bovine disease of great economic and veterinary importance in Africa. Papain-like cysteine proteases play important roles in protozoan parasite host cell entry and egress, nutrition and host immune evasion. This study reports the identification and characterisation of a T. parva strain Muguga cathepsin L-like (C1A subfamily) cysteine protease (ThpCP). Molecular modelling confirmed the papain-like fold of ThpCP, hydrophobic character of the S2 substrate binding pocket and non-covalent interaction between the pro- and catalytic domains preceding low pH autoactivation. ThpCP was recombinantly expressed in a protease deficient E. coli (Rosetta (DE3)pLysS strain) expression host as a 46 kDa proenzyme. Following Ni-chelate affinity chromatography and acidification, the 27 kDa mature ThpCP was purified by cation-exchange chromatography. Purified ThpCP hydrolysed typical cathepsin L substrates N-α-benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methyl-coumarin (AMC) (kcat/Km = 4.49 × 105 s-1M-1) and Z-Leu-Arg-AMC (kcat/Km = 4.20 × 105 s-1M-1), but showed no activity against the cathepsin B-selective substrate Z-Arg-Arg-AMC. Recombinant ThpCP was active over a broad pH range from pH 4.5 to 7.5, thereby showing potential activity in the acidic parasite food vacuole and close to neutral pH of the host lymphocyte cytoplasm. Recombinant ThpCP was inhibited by the cysteine protease inhibitors E64, iodoacetate, leupeptin, chymostatin, Z-Phe-Ala-diazomethylketone (DMK) and Z-Phe-Phe-DMK and hydrolysed bovine proteins: haemoglobin, immunoglobulin G, serum albumin and fibrinogen as well as goat IgG at pH 6 and 7. Functional expression and characterisation of Theileria cysteine proteases should enable high throughput screening of cysteine protease inhibitor libraries against these proteases.

Re-annotation of the Theileria parva genome refines 53% of the proteome and uncovers essential components of N-glycosylation, a conserved pathway in many organisms.

BACKGROUND: The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome. RESULTS: The re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized. CONCLUSIONS: The process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translational modification in apicomplexan parasites.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata.

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Highly syntenic and yet divergent: a tale of two Theilerias.

The published genomic sequences of the two major host-transforming Theileria species of cattle represent a rich resource of information that has allowed novel bioinformatic and experimental studies into these important apicomplexan parasites. Since their publication in 2005, the genomes of T. annulata and T. parva have been utilised for a diverse range of applications, ranging from candidate antigen discovery to the identification of genetic markers for population analysis. This has led to advancements in the quest for a sub-unit vaccine, while providing a greater understanding of variation among parasite populations in the field. The unique ability of these Theileria species to induce host cell transformation is the subject of considerable scientific interest and the availability of full genomic sequences has provided new insights into this area of research. This article reviews the data underlying published comparative analyses, focussing on the general features of gene expression, the major Tpr/Tar multi-copy gene family and a re-examination of the predicted macroschizont secretome. Codon usage between the Theileria species is reviewed in detail, as this underpins ongoing comparative studies investigating selection at the intra- and inter-species level. The TashAT/TpshAT family of genes, conserved between T. annulata and T. parva, encodes products targeted to the host nucleus and has been implicated in contributing to the transformed bovine phenotype. Species-specific expansion and diversification at this critical locus is discussed with reference to the availability, in the near future, of genomic datasets which are based on non-transforming Theileria species.

Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa.

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage.

Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1,095,000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4-52,256 transcripts per million (t.p.m.). Rare transcripts (<50 t.p.m.) were detected from 36% of genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.

Comparative studies of the efficacy of parvaquone and parvaquone-plus-frusemide in the treatment of Theileria parva infection (East Coast fever) in cattle.

Comparative studies of the efficacy of parvaquone (Parvexon) and parvaquone-plus-frusemide (Fruvexon) Bimeda Chemicals, Ireland, were done on 60 naturally infected cases of East Coast fever (ECF; Theileria parva infection in cattle). Small-scale dairy farmers in the peri-urban of Dar Es Salaam city reported ECF-suspected cases from March to mid-October 2001 and were treated with the two drugs alternately, as were diagnosed positive for ECF. Four sub-groups of 15 cattle each (early stage, 15; advanced stage, 15) were treated with parvaquone and parvaquone-plus-frusemide. Twenty-eight out of 30 (93.3%) cattle treated with parvaquone-plus-frusemide were cured, so do 24 out of 30 (80.0%) cattle treated with parvaquone without frusemide. Early diagnosis and prompt management of pulmonary signs, which accounted for 30.0% of total ECF cases is advised in order to improve cure rates. Unlike parvaquone without frusemide (Parvexon), parvaquone-plus-frusemide (Fruvexon) proved useful in the management of pulmonary signs, hence, a drug of choice in the treatment of ECF cases that are accompanied by or are likely to manifest pulmonary signs.

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

Characterisation of single domain ATP-binding cassette protien homologues of Theileria parva.

Two distinct genes encoding single domain, ATP-binding cassette transport protein homologues of Theileria parva were cloned and sequenced. Neither of the genes is tandemly duplicated. One gene, TpABC1, encodes a predicted protein of 593 amino acids with an N-terminal hydrophobic domain containing six potential membrane-spanning segments. A single discontinuous ATP-binding element was located in the C-terminal region of TpABC1. The second gene, TpABC2, also contains a single C-terminal ATP-binding motif. Copies of TpABC2 were present at four loci in the T. parva genome on three different chromosomes. TpABC1 exhibited allelic polymorphism between stocks of the parasite. Comparison of cDNA and genomic sequences revealed that TpABC1 contained seven short introns, between 29 and 84 bp in length. The full-length TpABC1 protein was expressed in insect cells using the baculovirus system. Application of antibodies raised against the recombinant antigen to western blots of T. parva piroplasm lysates detected an 85 kDa protein in this life-cycle stage.

Trapping parasite secretory proteins in baker's yeast.

Because the function of signal sequences has been conserved during evolution it has been possible to develop both bioinformatics resources to identify them and techniques to clone genes that encode secretory proteins. The latter entail insertion of heterologous signals upstream of signal peptide deleted reporter genes. We discuss the advantages of using Saccharomyces cerevisiae for signal sequence trap technology. The yeast protein-translocation system appears to be less discriminating than that of higher eukaryotes - for example, a Theileria parva cysteine protease gene containing a recessed, nonclassical signal allows access to the secretory pathway--and yeast technology could have general application in studying elements of parasite protein trafficking.

A Rab1 homologue with a novel isoprenylation signal provides insight into the secretory pathway of Theileria parva.

As a first step in developing compartment-specific markers for protein trafficking within Theileria parva, we have isolated cDNAs encoding homologues of the small GTP binding proteins Rab1 and Rab4. The T. parva homologue of Rab1 (TpRab1), a protein which regulates vesicular transport between the endoplasmic reticulum and cis golgi in other organisms, was unusual in that it contained a unique 17 amino acid C-terminal extension. The C-terminal motif sequence KCT (XCX) contrasted with the CXC or XCC motifs which act as as signals for isoprenylation by geranylgeranyl in most Rab proteins, including all known Rab1 homologues, in containing only a single cysteine. [C14]mevalonic acid lactone and [H3]geranylgeranyl pyrophosphate were specifically incorporated into recombinant TpRab1 in vitro, demonstrating that the novel motif was functional for isoprenylation. Recombinant TpRab1 bound radiolabeled GTP, and this binding was inhibited by excess unlabeled GTP and GDP and also partially by ATP. The TpRab1 gene contained four short (34-67 bp) introns with a distinct pattern of occurrence within the protein sequence as compared to the introns of other lower eukaryote Rab1 genes. Immunofluorescence microscopy using antiserum specific for the novel C-terminal peptide in combination with labelling of cells using the nucleic acid-staining dye DAPI, indicated that TpRab1 was located in the vicinity of the schizont nucleus within the infected lymphocyte.

Theileria parva 104 kDa microneme--rhoptry protein is membrane-anchored by a non-cleaved amino-terminal signal sequence for entry into the endoplasmic reticulum.

The 104 kDa microneme-rhoptry protein (p104) is the only known apical complex organelle-specific protein of Theileria parva. p104 exhibits striking structural similarities to circumsporozoite protein and sporozoite surface protein 2 of Plasmodium yoelii. Their primary sequences contain two hydrophobic segments, located at the amino-and the carboxy-terminus. The p104 amino-terminal hydrophobic region was suggested to be a signal peptide for entry into the endoplasmic reticulum and the extreme carboxy-terminal region to function as a membrane anchor. We have studied the biogenesis of p104 in a cell-free expression system and found that p104 is co-translationally transported into membranes derived from endoplasmic reticulum. The amino-terminal signal peptide is not cleaved off and anchors the protein in the membrane with the carboxy-terminal portion translocated into the lumen. We suggest that in vivo p104 is co-translationally integrated into the membrane of the endoplasmic reticulum, from where it is further transported to the microneme-rhoptry complex. Thus, p104 appears to be a suitable marker to study the development of micronemes and rhoptries in T. parva.

Proteolytic cleavage of surface proteins enhances susceptibility of lymphocytes to invasion by Theileria parva sporozoites.

A flow cytometric method using anti-parasite antibodies was developed to measure binding of Theileria parva sporozoites to the target bovine lymphocyte membrane. Parasite-host cell interactions could be inhibited by monoclonal antibodies to bovine MHC class I and partially by one of two antibodies to BoCD45R. Proteolysis of the lymphocyte surface removed CD45R but not MHC class I determinants, and enhanced sporozoite binding. These observations support the hypothesis that CD45R and CD45R antibodies may nonspecifically prevent close approximation between sporozoites and lymphocytes. Interestingly, under normal conditions, sporozoites of T. parva did not attach to lymphocytes from goats, but did so when the cells were treated with the protease, suggesting that receptor(s) for T. parva sporozoites might be present on caprine cells but are not easily accessible. These and other results indicate that proteases may be involved in binding and entry of T. parva sporozoites. Electron microscopy revealed that the process of binding and entry of sporozoites into protease-treated goat lymphocytes was very similar to that of the bovine cells. However, schizonts did not develop and lymphocyte proliferation was not induced, indicating that cell entry by sporozoites and cellular transformation are separate processes.

Characterization of a secretory type Theileria parva glutaredoxin homologue identified by novel screening procedure.

The schizont stage of the protozoan parasite Theileria parva induces features characteristic of tumor cells in infected bovine T-cell lines. Most strikingly T. parva-infected cell lines acquire unlimited growth potential in vitro. Their proliferative state is entirely dependent on the presence of a viable parasite within the host cell cytoplasm. It has been postulated that parasite proteins either secreted into the host cell or expressed on the parasite surface membrane are involved in the parasite-host cell interaction. We used an in vitro transcription-translation-membrane translocation system to identify T. parva-derived cDNA clones encoding secretory or membrane proteins. Within 600 clones we found one encoding a 17-kDa protein which is processed by microsomal membranes to a 14-kDa protein (11E), presumably by signal peptidase. The processed form is expressed in the T-cell line TpM803 harboring viable parasites. By immunolocalization we show that the 11E protein mostly resides within the parasite, often in close vicinity to membranous structures, but in addition it appears at the surface membrane. Amino acid sequence comparison suggests that 11E belongs to the glutaredoxin family, but is unique so far in containing a signal sequence for endoplasmic reticulum membrane translocation.

Sequence and expression of a 90-kilodalton heat-shock protein family member of Theileria parva.

A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.

Characterisation of a glutamine- and proline-rich protein (QP protein) from Theileria parva.

We have isolated a clone from a Theileria parva infected lymphocyte cDNA library which has the potential to encode a protein of 480 amino acids. This protein is particularly rich in glutamine and proline and has some short repeated amino acid motifs based on the sequences QPXP and QPXQ. We have called it the 'QP protein'. Southern blotting suggests that the QP protein gene is present as a single copy in the T. parva Muguga genome. Northern blotting revealed that the gene is transcribed in both schizonts and piroplasms. We have expressed part of the QP protein as a fusion with glutathione S-transferase in Escherichia coli and used this product to raise an anti-QP protein serum. Western blots of T. parva lysates using this serum showed a major polypeptide of approximately 100 kDa and two further polypeptides of approximately 67 and 72 kDa. Indirect immunofluorescence assays using the anti-QP protein serum on infected cells showed that the protein is associated with the schizont. The pattern of staining in the indirect immunofluorescence assays and the structure of the protein suggest that it is a component of the schizont membrane.