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Genome Sequence of Theileria parva, a Bovine Pathogen That Transforms Lymphocytes

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Science  01 Jul 2005:
Vol. 309, Issue 5731, pp. 134-137
DOI: 10.1126/science.1110439

Abstract

We report the genome sequence of Theileria parva, an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum, and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.

Theileria parva is a tick-borne parasite that causes a fatal disease in cattle known as East Coast fever (ECF). This disease, which kills over 1 million cattle each year in sub-Saharan Africa, results in economic losses exceeding $200 million annually (1). Theileria organisms belong to the phylum Apicomplexa, which is predicted to have originated about 930 million years ago (2). Unlike other apicomplexans, penetration of host cells by T. parva is not orientation-specific. Rhoptries and microspheres discharge after invasion, coincident with dissolution of the surrounding host cell membrane, leaving the parasite free in the host cell cytoplasm. Morbidity and mortality due to ECF are attributed to the ability of the schizont stage to malignantly transform its host cell, the bovine lymphocyte. Parasitosis increases exponentially because the schizont divides in synchrony with the host cell and infected cells infiltrate all tissues; cattle die of this lymphoproliferative disease 3 to 4 weeks after infection. Little pathology is due to the tick infective piroplasm, the red blood cell stage (1).

We sequenced the genome of T. parva in order to facilitate research on parasite biology, assist the identification of schizont antigens for vaccine development (3), and extend comparative apicomplexan genomics, in particular with Plasmodium falciparum, which causes malaria. Comparison with T. annulata, which causes tropical bovine theileriosis and mainly transforms macrophages, is described in an accompanying report (4). (This whole-genome shotgun project has been deposited at DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank under the project accession AAGK00000000.)

The haploid T. parva nuclear genome is 8.3 × 106 base pairs (Mbp) in length and consists of four chromosomes (Table 1). We provide a complete sequence, except for a 1- to 2-kbp gap in chromosome 4 and a gap in chromosome 3 (Tpr locus) that contains a 41-kbp and a 13-kbp set of overlapping sequences (contig) (5). The parasite apicoplast and mitochondrial (6) genomes have also been sequenced. Like P. falciparum, T. parva chromosomes contain one extremely A+T-rich region (>97%) about 3 kbp in length that may be the centromere. The regions between the CCCTA3-4 telomeric repeats and the first protein-encoding gene are short, 2.9 kbp on average, and do not contain other repeats. Thus, the structure of the subtelomeric regions in T. parva is much less complex than that in P. falciparum, where arrays of repeats extend up to 30 kbp (7).

Table 1.

Comparison of T. parva nuclear genome coding characteristics with other sequenced apicomplexans. Gene length excludes introns; gene density calculated as genome size/number of protein-encoding genes. Source of data for P. falciparum was (7), and, for C. parvum, (20).

Features Apicomplexan organism
T. parvaP. falciparumC. parvum
Size (bp) 8,308,027 22,853,764 9,100,000
Number of chromosomes 4 14 8
Total G+C content (%) 34.1 19.4 30
Number of protein encoding genes 4035 5268 3807
Number of hypothetical proteins 2498 3208 925
Mean gene length (bp) 1407 2283 1795
Gene density (gene frequency in bp) 2057 4338 2382
Percent coding 68.4 52.6 75.3
Genes with introns (%) 73.6 53.9 5
Exons per gene (median) 4 2 1
Mean intergenic length (bp) 405 1694 566
G+C content intergenic regions (%) 26.1 13.6 23.9
Number of tRNA genes 47 43 45
Number of 5S rRNA genes 3 3 6
Number of rRNA units 2 7 5

The T. parva nuclear genome contains about 4035 protein-encoding genes, 20% fewer than P. falciparum, but exhibits higher gene density, a greater proportion of genes with introns, and shorter intergenic regions. There are two identical, unlinked 5.8S-18S-28S rRNA units, suggesting that unlike P. falciparum T. parva does not possess functionally distinct ribosomes (8). Putative functions were assigned to 38% of the predicted proteins (Table 1).

The complexity of the T. parva life cycle is not matched by a large number of recognizable cell cycle regulators. Thus, the parasite is more akin to yeasts than higher eukaryotes, lacking discernable components of both the p53-MDM2-p14ARF-p21 and the Ink4-retinoblastoma-E2F pathways (9). There are four predicted cyclins and five cyclin-dependent kinases (cdks), most of which have close homologs in P. falciparum. However, T. parva lacks one cyclin and two cdks found in P. falciparum. These parasite cyclins are poorly conserved (∼25% identity), making cross-species comparisons difficult. The reduced recognizable T. parva cell cycle machinery suggests that a number of novel regulatory features remain to be discovered.

A unique aspect of T. parva biology is that infection of T and B lymphocytes results in a reversible transformed phenotype with uncontrolled proliferation of host cells that remain persistently infected. Parasite proteins that may modulate host cell phenotype are described in an accompanying report (4). Host cell microtubules that decorate the surface of schizonts are captured by the host cell spindle during mitosis, favoring infection of both daughter cells (1). T. parva encodes putative secreted forms of EMAP115- and Tau-like proteins, which are absent from P. falciparum; in higher eukaryotes, these proteins interact with microtubules (10). In addition, T. parva may modulate host cell mitosis by influencing disassembly of the host cell spindle via a secreted cdc48-like AAA–adenosine triphosphatase (ATPase associated with diverse cellular activities) (11). A likely P. falciparum homolog of this protein contains an N-terminal signal anchor sequence, whereas the T. parva protein contains a signal peptide and lacks a recognizable endoplasmic reticulum retention signal.

We used the Tribe-MCL algorithm (5) to identify 384 protein families containing 1063 proteins in the T. parva proteome (table S1). The largest family, containing 85 proteins, exists primarily in tandem arrays in the subtelomeric regions of all chromosomes. Many members of the family have a similar architecture, consisting of a secretion signal at the N terminus and a low-complexity glutamine- and proline-rich central domain that may be difficult for vertebrate immune systems to recognize (12). These genes are polymorphic between parasite isolates, and specific genes are absent from certain isolates (13). Each telomere has a conserved ∼140-bp sequence immediately adjacent to the telomeric repeat (14), and several subtelomeric regions exhibit 70 to 100% sequence similarity (fig. S1). As in other eukaryotic pathogens, these features may facilitate interchromosomal recombination and the generation of antigenic diversity.

Proteins in the most rapidly evolving T. parva protein family, the Tpr (T. parva repeat) family, contain complex domain structures reminiscent of a system that has evolved to generate diversity (15). Unlike the majority of hypervariable gene families in parasitic protozoa (16), Tpr sequences are not telomere-associated. This family comprises a tandem array of highly conserved open reading frames (ORFs) on chromosome 3, located ∼570 kbp from a telomere. The locus, estimated to span 100 kbp, contains at least 28 ORFs, of which 18, ranging in length from 192 to 674 amino acids, lack methionine codons in the first 50 amino acids (fig. S2). Eleven additional dispersed copies of Tpr, also of varying length, contain a 268–amino acid membrane-associated helical domain typical of the Tpr family. Massively parallel signature sequencing (17) and expressed sequence tags suggest that some genes in the locus are only transcribed in the piroplasm stage, whereas at least two of the dispersed genes are transcribed in the schizont stage. In common with the var genes of P. falciparum (18), domains within the Tpr genes are isolate-specific (19), and the 3′ end of Tpr has been used for genotyping of T. parva isolates. Tpr proteins have not yet been detected in piroplasms, and the function of these proteins remains unknown.

The genome sequence provides a global view of the metabolic potential of T. parva and allows a comparative analysis with P. falciparum metabolism. We predict a reduced functional role for the T. parva apicoplast and a greater dependence on the host for many substrates (fig. S3). T. parva lacks many enzymes in the shikimic acid, porphyrin, polyamine, and type II fatty acid biosynthetic pathways, but it retains the ability to produce isoprenoids via a methyl erythritol phosphate pathway in the apicoplast. T. parva cannot salvage purines, its ability to interconvert amino acids is very limited, and it lacks enzymes that permit the alternative nonoxidative production of pentoses and tetroses via the pentose phosphate pathway. Analysis of predicted transporters revealed fewer transporters of organic nutrients and inorganic cations than are present in P. falciparum. However, T. parva has more adenosine 5′-triphosphate-binding cassette (ABC) transporters of unknown substrate specificity. Another difference is that T. parva encodes an amino acid–cation symporter that is not present in P. falciparum (7) or C. parvum (20). In contrast to P. falciparum, T. parva encodes trehalose-6-phosphate synthase and trehalose phosphatase. Trehalose is a disaccharide that plays a role in desiccation and stress tolerance. It may protect the parasite during its long developmental cycle in the tick.

T. parva genes encode all of the enzymes necessary for glycolysis, glycerol catabolism, and the tricarboxylic acid (TCA) cycle. Unlike P. falciparum, T. parva does not encode malate dehydrogenase, but this could be functionally replaced by malate-quinone oxidoreductase, an activity also predicted to be present in P. falciparum. The origin of mitochondrial acetyl-coenzyme A (CoA) in both parasites presents a problem, because P. falciparum encodes a single pyruvate dehydrogenase that is targeted to the apicoplast (21) and T. parva does not encode all the subunits of this enzyme. Both parasites are predicted to contain cytoplasmic acetyl-CoA synthetase and a plasma membrane acetyl-CoA–CoA anti-porter, but how mitochondrial oxidation of carbon chains is fueled in these two pathogens remains enigmatic because glycolysis and the tricarboxylic acid cycle do not appear to be linked by a classical route (22). Thus, it is not clear whether the complete TCA cycle is functional. Nitrogen metabolism differs from P. falciparum because T. parva lacks glutamate-ammonia ligase and only contains a nicotinamide adenine dinucleotide (NAD+)–dependent glutamate dehydrogenase, which is usually associated with glutamate catabolism. This suggests that imported glutamate could play a role in supplementing intermediates in the TCA cycle.

The ionophores valinomycin and gramicidin D kill T. parva, suggesting that a mitochondrial electrochemical gradient is essential for parasite survival (23), but it is not known whether this is coupled to ATP synthesis. All subunits of the F1 catalytic domain of ATP synthase and subunit c of the F0 domain are present, but genes coding for subunits a and b of F0 were not found. The T. parva respiratory complexes are similar to those described in P. falciparum. Buparvaquone, a hydroxynapthaquinone drug used in the chemotherapy of ECF, probably inhibits electron transport through complex III (23).

The apicoplast is found in most apicomplexans and plays an essential role in parasite metabolism (24). An A+T-rich, ∼35-kbp apicoplast genome encoding 30 proteins, rRNAs, and tRNAs is present in Plasmodium, Toxoplasma, and Eimeria, but not in Cryptosporidium (20); the latter lacks an apicoplast. The 39.5-kbp T. parva apicoplast genome differs from that of P. falciparum in that all of its genes are transcribed in the same direction. In addition, it has one rather than two copies of the rRNA genes, clpC is duplicated, the rpoC2 gene encoding the β″ subunit of RNA polymerase is split into two parts, and it lacks the sufB gene (Fig. 1). Twenty-six of the 44 T. parva apicoplast genome protein-coding genes share sequence similarity (27 to 61%) with proteins encoded by the P. falciparum apicoplast genome.

Fig. 1.

Comparison of the apicoplast genomes of T. parva (A) and P. falciparum (B). A circular contig of the T. parva apicoplast genome was obtained after assembly of shotgun sequences, but the in vivo conformation has not been determined. The P. falciparum apicoplast genome is circular in vivo (30). The genomes are displayed in linear format beginning with the small subunit rRNA genes. Abbreviations and color coding: light orange, small (SSU) and large (LSU) subunit rRNAs; magenta, tRNAs [single-letter amino acid code (31)]; pink, ribosomal proteins (s and l for small and large subunit ribosomal proteins, respectively) and elongation factor Tu (tufA); blue, protein import; stippled gray, hypothetical proteins; purple, transcription; brown, SufB subunit of the SufABCDE Fe-S cluster assembly complex. The black and red bars indicate a region containing repeats and short ORFs and another region containing repeats and potential selenocysteine tRNAs, respectively (5). Scale bar equals 1 kbp.

Most apicoplast proteins are encoded by nuclear genes and imported into the organelle by means of a bipartite targeting presequence (24). Comparison of the 345 T. parva (5) and 551 P. falciparum (7) predicted apicoplast-targeted (AT) proteins revealed similarities and differences in apicoplast function. The apicoplasts of Plasmodium and Toxoplasma participate in heme biosynthesis and are the sites of type II fatty acid and isoprenoid biosynthesis. Apicoplast-derived fatty acids in these parasites might contribute to the establishment and modification of the parasitophorous vacuole membrane (25). It may be notable that both T. parva and T. annulata, which have only retained isoprenoid biosynthesis, do not exist within a parasitophorous vacuole. About 100 AT proteins were found in both species, but 40% of these were hypothetical proteins, indicating that many core apicoplast functions have yet to be defined.

Fe-S clusters are required in mitochondria and plastids for the maturation of apoproteins. Fe-S cluster formation in the T. parva mitochondrion appears to be similar to that in yeast and Plasmodium (26) (table S3). However, of the sufABCDES genes involved in the assembly of Fe-S clusters in Arabidopsis thaliana (27) and P. falciparum plastids (26), only sufS was identified in T. parva. SufS is a cysteine desulfurase that requires SufE for catalytic activity. The parasite T. para genome encodes a plastid-targeted tRNA thiolation enzyme (MnmA) that has an additional domain similar both in sequence and predicted structure to the sulfur-binding domain of SufE. Thus, a previously unknown complex of SufS/MnmA may catalyze thiolation of tRNA in the T. parva apicoplast. The T. parva nuclear genome also encodes an AT protein with homology to NFU1, a scaffold protein for Fe-S cluster assembly in A. thaliana plastids (28), suggesting that assembly of Fe-S clusters occurs in the T. parva apicoplast despite the absence of most Suf proteins.

T. parva and T. annulata exhibit near-complete synteny across all chromosomes (4). To examine the extent of conservation of gene synteny between the evolutionarily distant P. falciparum and T. parva, we applied an iterative syntenic block algorithm and Jaccard-filtered COGs to whole-genome data from P. falciparum clone 3D7 (7), P. y. yoelii (29), C. parvum (20), and T. parva. Extensive synteny was found between P. falciparum and P. y. yoelii but not between P. falciparum and C. parvum or between T. parva and C. parvum. A total of 435 microsyntenic regions containing 1279 orthologs were observed between P. falciparum and T. parva, consisting of groups of 2 to 11 orthologs conserved in position between the two genomes (Fig. 2). This may be an underestimate of the degree of microsynteny as it is possible that, due to its long-term in vitro culture, clone 3D7 may represent an atypical genome. Syntenic clusters were distributed uniformly along each chromosome except for the subtelomeric regions, which contain species-specific gene families.

Fig. 2.

Regions of microsynteny between T. parva and P. falciparum. Schematic of a representative P. falciparum chromosome showing synteny with three other apicomplexan species. Top row, P. falciparum chromosome 14 proteins. Second row, P. y. yoelii orthologs from P. y. yoelii chromosomes 6, 10, and 13. Third row, T. parva orthologs from T. parva chromosomes 1, 2, 3, and 4. Fourth row, C. parvum orthologs from C. parvum chromosomes 6 and 8.

The genome sequence of T. parva shows remarkable differences from the other apicomplexan genomes sequenced to date. It provides significant improvements in our understanding of the metabolic capabilities of T. parva and a foundation for studying parasite-induced host cell transformation and constitutes a critical knowledge base for a pathogen of significance to agriculture in Africa. Mining of sequence data has already proved useful in the search for candidate vaccine antigens (3).

Supporting Online Material

www.sciencemag.org/cgi/content/full/309/5731/134/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S3

References and Notes

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