Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Abstract

BackgroundThe intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm.Methodology/Principal FindingsWe analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals.ConclusionsOur analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.

Extensive Polymorphism and Evidence of Immune Selection in a Highly Dominant Antigen Recognized by Bovine CD8 T Cells Specific for Theileria annulata

“…DNA and protein database searches did not reveal any orthologues of Ta9 in parasite genera other than Theileria or in any mammalian species. The general features of the central region of the Ta9 protein, namely, its PQ-rich nature and variation in sequence and length due to multiple insertions and deletions, are found in other Theileria proteins, including the polymorphic immunodominant antigen in T. parva (PIM), the Tasp protein of T. annulata, and the subtelomere-encoded variable secreted protein (SVSP) family in T. parva and T. annulata (49,50,57). The presence of a predicted signal peptide indicates that Ta9 is likely to be secreted by the parasite and hence be available for cytosolic antigen processing.…”

“…Parasite-specific CD8 T cell lines derived from three of these A10 ϩ animals were used to investigate responses to the Ta9 antigen. In order to determine the proportion of the response directed against Ta9, panels of 90 CD8 ϩ T cell clones from each animal were tested for cytotoxicity on autologous and MHC haplotypematched T. annulata-infected target cells and on autologous T. parva-infected target cells pulsed with peptide representing Ta9 [40][41][42][43][44][45][46][47][48][49] . A summary of the results is shown in Table 2.…”

“…In contrast, only a minority of the clones (5/51) that showed specific cytotoxicity from the third animal (219) were restricted by the A10 haplotype. All of the A10-restricted T cell clones from the three animals demonstrated specific killing of Ta9 [40][41][42][43][44][45][46][47][48][49] peptide-pulsed targets but no killing of unpulsed targets. The levels of killing of pulsed targets were substantially higher (69 to 100% for all clones) than those obtained with T. annulata-infected target cells (5 to 80%) and two additional T cell clones that did not kill the parasitized cells demonstrated significant killing of peptide pulsed targets (Table 2).…”

“…5), while in the slightly longer C terminus none were identified (P Ͻ 0.1). Two of these sites are within the A10 T-cell epitope (Ta9 [40][41][42][43][44][45][46][47][48][49] ) identified for the T. annulata antigen, and statistical analysis confirmed that, compared to the rest of the gene, the epitope region was enriched for positively selected codons (P ϭ 0.016, Fisher exact test). In addition, three of the sites have amino acid substitutions in the two T. parva alleles.…”

“…The results confirm that amino acid substitutions in the epitope are associated with differential recognition by Ta9 40-49 -specific T cell clones. They also demonstrate that CD8 T cell clones differ in their tolerance of amino acid substitutions in the Ta9 [40][41][42][43][44][45][46][47][48][49] epitope, allowing recognition of some alleles (with up to 3 amino acid substitutions) by a component of the CD8 T cell response. The data shown in Table 4 suggested that the representation of CD8 T cell clones capable of recognizing the TaG.3 and TaG.5 parasites differed between the three animals, but the numbers of clones examined were not sufficient to draw clear conclusions.…”