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.

Polymorphic open reading frames encoding secretory proteins are located less than 3 kilobases from Theileria parva telomeres.

Polymorphic, multicopy gene families are frequently located in subtelomeric regions of the genomes of parasitic protozoa. Theileria parva telomere-associated (TA) DNA from two chromosomes contained long open reading frames (ORFs) 54% identical at the N-termini, whose 3' ends were 2670 and 2680 bp from the telomeric repeats. Probes derived from these ORFs revealed related sequences close to additional telomeres. The 3' end of an unrelated ORF was approximately 2720 bp from a third telomere. These are among the closest ORFs to telomeres in any organism. Reverse transcription PCR detected transcripts originating within the telomeric multicopy gene family. Additional ORFs, with complex sequence similarities, were located centromeric to the telomere-adjacent ORFs. Transcripts from the schizont stage of T. parva, containing domains with significant amino acid similarity to a 3529 codon ORF located 6900 bp upstream of the telomeric repeats, were mapped to a subtelomeric locus at a fourth telomere. Five telomeric ORFs contained predicted N-terminal signal peptides and one of these signal peptides was functional in a heterologous system. Hybridisation data suggested extensive strain polymorphism between ORFs. Two of the telomere-adjacent ORFs were absent from the genome of a cloned T. parva parasite which can, nonetheless, be passaged through ticks and cattle. T. parva is unusual, among organisms so far studied, in the high density of potential coding sequences located directly adjacent to telomeres and the apparent absence of extensive tracts of repeated sequences within the TA DNA.

Microsequence heterogeneity and expression of the LSU rRNA genes within the two single copy ribosomal transcription units of Theileria parva.

The nucleotide sequences of the large subunit ribosomal RNA coding genes within the two single copy ribosomal DNA transcription units of a cloned Theileria parva isolate from a buffalo were determined. The two LSU rRNA coding units differed by 11 nucleotide substitutions and two deletions of 1 and 6 bp, all located in the 5' end of the LSU coding region. We also observed microsequence heterogeneity between the two buffalo parasite LSU sequences and the previously determined LSU rRNA gene of a T. parva parasite isolated from cattle. At all positions which were variable between the two LSU rRNA coding sequences of the buffalo-derived parasite, either unit 1 or unit 2 matched the LSU rRNA coding sequence of the cattle-derived T. parva parasite in a mosaic pattern. Synthetic oligonucleotides specific for LSU units 1 and 2 of the buffalo T. parva were developed, and used to assay expression of the two units. Both units were expressed in the intra-lymphocytic schizont stage of T. parva. A 2.5-10-fold excess of rRNA derived from LSU unit 1, compared with unit 2, was observed in the schizont stage, the difference being attributable to variation in the level of expression of unit 2. Theileria represents the third genus of Sporozoan protozoa, in addition to Plasmodium and Babesia, exhibiting rRNA coding genes, which are divergent in sequence between different transcription units.

Linear peptide specificity of bovine antibody responses to p67 of Theileria parva and sequence diversity of sporozoite-neutralizing epitopes: implications for a vaccine.

A stage-specific surface antigen of Theileria parva, p67, is the basis for the development of an anti-sporozoite vaccine for the control of East Coast fever (ECF) in cattle. By Pepscan analysis with a series of overlapping synthetic p67 peptides, the antigen was shown to contain five distinct linear peptide sequences recognized by sporozoite-neutralizing murine monoclonal antibodies. Three epitopes were located between amino acid positions 105 to 229 and two were located between positions 617 to 639 on p67. Bovine antibodies to a synthetic peptide containing one of these epitopes neutralized sporozoites, validating this approach for defining immune responses that are likely to contribute to immunity. Comparison of the peptide specificity of antibodies from cattle inoculated with recombinant p67 that were immune or susceptible to ECF did not reveal statistically significant differences between the two groups. In general, antipeptide antibody levels in the susceptible animals were lower than in the immune group and neither group developed high responses to all sporozoite-neutralizing epitopes. The bovine antibody response to recombinant p67 was restricted to the N- and C-terminal regions of p67, and there was no activity against the central portion between positions 313 and 583. So far, p67 sequence polymorphisms have been identified only in buffalo-derived T. parva parasites, but the consequence of these for vaccine development remains to be defined. The data indicate that optimizations of the current vaccination protocol against ECF should include boosting of relevant antibody responses to neutralizing epitopes on p67.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM).

To identify the genes encoding novel immunodominant antigens of Theileria parva a lambda gt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host-sporozoite interaction.

Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva.

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich i minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T2-5ACACA (6-17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10-17bp (3-7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites. 1998 Elsevier Science B.V.

Concerted evolution at a multicopy locus in the protozoan parasite Theileria parva: extreme divergence of potential protein-coding sequences.

Concerted evolution of multicopy gene families in vertebrates is recognized as an important force in the generation of biological novelty but has not been documented for the multicopy genes of protozoa. A multicopy locus, Tpr, which consists of tandemly arrayed open reading frames (ORFs) containing several repeated elements has been described for Theileria parva. Herein we show that probes derived from the 5'/N-terminal ends of ORFs in the genomic DNAs of T. parva Uganda (1,108 codons) and Boleni (699 codons) hybridized with multicopy sequences in homologous DNA but did not detect similar sequences in the DNA of 14 heterologous T. parva stocks and clones. The probe sequences were, however, protein coding according to predictive algorithms and codon usage. The 3'/C-terminal ends of the Uganda and Boleni ORFs exhibited 75% similarity and identity, respectively, to the previously identified Tpr1 and Tpr2 repetitive elements of T. parva Muguga. Tpr1-homologous sequences were detected in two additional species of Theileria. Eight different Tpr1-homologous transcripts were present in piroplasm mRNA from a single T. parva Muguga-infected animal. The Tpr1 and Tpr2 amino acid sequences contained six predicted membrane-associated segments. The ratio of synonymous to nonsynonymous substitutions indicates that Tpr1 evolves like protein-encoding DNA. The previously determined nucleotide sequence of the gene encoding the p67 antigen is completely identical in T. parva Muguga, Boleni, and Uganda, including the third base in codons. The data suggest that concerted evolution can lead to the radical divergence of coding sequences and that this can be a mechanism for the generation of novel genes.

The nucleotide sequence of the bovine interleukin-5-encoding cDNA.

The nucleotide (nt) sequence encoding the bovine homologue of interleukin-5 (boIL-5) was determined. Total cDNA generated from stimulated bovine peripheral blood lymphocytes was used to amplify the boIL-5 gene using primers based on the 5' and 3' untranslated regions of the ovine IL-5 cDNA sequence. The boIL-5 coding sequence is 405 bp long, coding for a 15.2 kDa precursor protein of 134 amino acids (aa). Cleavage of a putative signal peptide of 19 aa yields a mature form of 13.1 kDa. Comparisons at the nt level revealed 96%, 81%, 74% and 73% identity with the ovine, human, mouse and rat IL-5 sequences, respectively, and 97%, 66%, 59% and 58% aa identity, respectively.

Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different cross-immunity profiles.

Immunity to Theileria parva infection in cattle is often parasite stock specific. The antigenic diversity which is expressed at the schizont stage of the parasite together with a wild reservoir of the organism in buffalo has complicated the development of effective disease control by immunization. We have previously shown that about 70% of cattle inoculated with recombinant forms of p67, a sporozoite stage-specific surface antigen from the cattle-derived Muguga stock of the parasite, are immune to a homologous challenge. Thus, immune responses to p67 can play a role in immunity. The genes encoding this protein in five other parasite stocks have been sequenced. Here, we report that the p67 molecule encoded by four cattle-derived parasite stocks (Boleni, Uganda, Mariakani, and Marikebuni) that fall into different cross-immunity groups is identical in sequence to Muguga p67. The protein encoded by a buffalo-derived parasite exhibits 95% sequence identity with Muguga p67, the major difference being the presence of a 43-residue peptide insert. As predicted by these data, cattle inoculated with recombinant p67 can resist a heterologous cattle-derived parasite challenge. Seven of 12 cattle receiving a homologous Muguga challenge and 6 of 11 cattle receiving a heterologous Marikebuni challenge were immune to East Coast fever. These results extend earlier data suggesting that p67 is a conserved molecule and confirm its potential as a broad-spectrum vaccine antigen for the control of T. parva infection.

Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

Structure and sequence variation of the genes encoding the polymorphic, immunodominant molecule (PIM), an antigen of Theileria parva recognized by inhibitory monoclonal antibodies.

The polymorphic, immunodominant molecule (PIM) of Theileria parva is the predominant antigen recognized by sera from infected cattle and by monoclonal antibodies (mAb) used to differentiate parasite strains. As such, the antigen is under consideration as a diagnostic antigen, and since the mAbs can neutralize sporozoite infectivity in vitro, in immunization experiments. Initial comparison of two PIM cDNA sequences suggested that the PIM genes consist of conserved 5' and 3' termini flanking a central variable region. We present further evidence, based on sequence analysis, supporting this general structure for the PIM genes. Evidence is also presented for a single copy of the PIM gene per haploid genome, implying that the different versions of PIM are encoded by distinct alleles. The central variable region of the PIM allele from the T. parva (Marikebuni) stock was found to contain 13 copies of the tetrapeptide repeat Gln-Pro-Glu-Pro. We also detected point mutations in the 5' and 3' termini of the PIM alleles, including regions recognized by the neutralizing and typing mAb. This contrasted with the high sequence conservation of the two introns of the genes, suggesting that the protein is undergoing rapid evolution. Sequence comparison of PIM genes from buffalo- and cattle-derived parasites supported earlier results that the parasites infecting buffaloes constitute a more heterogeneous population than those from cattle.

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.

Theileria: improved species discrimination using oligonucleotides derived from large subunit ribosomal RNA sequences.

Partial nucleotide sequences of large subunit ribosomal RNA (LSU rRNA) coding genes of Theileria parva and T. taurotragi were determined. Alignment of the T. parva and T. taurotragi sequences revealed species-specific regions within the LSU rRNA genes. A major species-variable region, located between the first and second conserved secondary structure domains of LSU rRNA, was identified. The sequence of the major species-variable region was determined for T. annulata, T. buffeli, and an unclassified Theileria species isolated from buffalo. Species-specific oligonucleotides complementary to LSU rRNA sequences were designed for five species of Theileria. The oligonucleotide probes discriminated between Theileria species when hybridized to amplified parasite LSU rRNA genes or directly to parasite rRNA. Three species-specific oligonucleotides derived from nonoverlapping sequences were developed for each of T. parva and T. taurotragi. A simplified procedure was developed in which T. parva- and T. taurotragi-specific oligonucleotides were used to discriminate the two species under nonstringent hybridization and washing conditions. Use of the nonstringent conditions also increased the sensitivity of target detection. Comparison of Theileria species-specific oligonucleotides derived from large subunit and small subunit rRNA genes demonstrated that, for closely related species, probes derived from LSU rRNA sequences had improved specificity.

Evidence for two single copy units in Theileria parva ribosomal RNA genes.

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.

Bovine T cells specific for Trypanosoma brucei brucei variant surface glycoprotein recognize nonconserved areas of the molecule.

The specificity of bovine CD4+ T-cell responses to Trypanosoma brucei variant surface glycoprotein (VSG) has been examined by using a panel of seven T-cell clones and nested deletions of the ILTat 1.3 VSG gene expressed in Escherichia coli. All clones recognized the polymorphic N-terminal domain of the antigen, and the recognition sites of five of the clones were resolved to three areas with lengths of 14, 18, and 21 amino acids. Comparison of these regions with corresponding areas of other VSG molecules, including those derived from the same trypanosomal serodeme, has shown that the sites are not conserved. In the light of recent observations that VSG-specific T-cell responses are induced in mice infected with T. brucei, these results confirm with the belief that immune pressure from T cells may contribute to the generation of antigenic diversity on the surface of African trypanosomes.

A 7.1 kb linear DNA molecule of Theileria parva has scrambled rDNA sequences and open reading frames for mitochondrially encoded proteins.

Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.

Early expression of a Trypanosoma brucei VSG gene duplicated from an incomplete basic copy.

Intrachromosomal variant surface glycoprotein (VSG) genes in Trypanosoma brucei are expressed by a mechanism involving gene conversion. The 3' boundary of gene conversion is usually within the last 130 bp of the VSG gene, a region of partially conserved sequences. We report here the loss of the predominant telomeric A VSG gene in the cloned variant antigenic type (VAT) 5A3, leaving only an intrachromosomal A VSG gene (the A-B gene). The nucleotide sequence of the A-B VSG gene reveals that it lacks the normal VSG 3' sequence. Surprisingly, we find cells expressing this A-B VSG gene in relapse populations arising from VAT 5A3. Since the A VSG mRNAs from these cells have a normal 3' sequence, the incomplete A-B VSG gene must be expressed via a partial gene conversion that supplies the functional 3' end. Although the A-B VSG gene is no longer predominant like the telomeric A VSG gene, it is still expressed more frequently than other intrachromosomal VSG genes, suggesting that factors other than a telomeric location determine whether a VSG gene is expressed early in a serodeme.

Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites.

We have cloned and characterised the gene encoding the 67-kilodalton stage-specific surface antigen, p67, of Theileria parva (Muguga) sporozoites. The gene which is present in a single copy, is divided into 2 exons by an intron 29 bp long and is transcribed into mRNA of about 2500 nucleotides. The gene is present in all stocks of T. parva and there is a related gene in Theileria annulata. The deduced amino acid sequence of 709 residues predicts that p67 is a membrane protein and that it lacks tandemly repeated sequences. Recombinant p67 has been expressed in Escherichia coli as a fusion protein with Sj-26, a glutathione-S-transferase of Schistosoma japonicum. Antibodies to purified recombinant proteins containing residues 9-316 or 397-709 of p67 bind to p67 in immunoblots and neutralise sporozoite infectivity in vitro. Recombinant p67 is, therefore, a candidate antigen for development of an anti-sporozoite vaccine for East Coast fever in cattle.

A single exon codes for the enzyme domain of a protozoan cysteine protease.

Theileria parva is an intracellular protozoan parasite of cattle. We have determined the nucleotide sequence of a gene and cDNA coding for a cysteine protease of T. parva. The gene is divided into two exons. The first exon codes for a signal sequence and for part of the "pro" region of the zymogen. The second exon codes for the remainder of the pro region, including residues thought to be involved in zymogen processing, and for the entire enzyme domain. Part of this exon cross-hybridizes, at high stringency, with DNA isolated from Plasmodium falciparum. It is likely that the T. parva gene is functionally active. Parasite extracts contain hydrolytic activity against benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin. This activity is optimal at pH 6.0 and is inhibited by the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64). This is, to our knowledge, the first description of a gene coding for a eukaryotic cysteine protease which lacks an intron in DNA coding for the enzyme domain.