Project description: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.

Project description:The protozoan parasite Theileria parva infects and transforms bovine lymphocytes inducing uncontrolled proliferation. The transforming schizont resides free in the host cell cytoplasm and it is assumed that proteins released from the parasite contribute to host cell transformation and parasite persistence. The identification and characterisation of parasite genes encoding candidate secreted proteins constitutes a first step towards elucidating this complex process. In earlier work, it was shown that the genes encoding subtelomere-encoded variable secreted proteins (SVSPs) are located at the subtelomeres of all four T. parva chromosomes and, with 85 members, form the largest Theileria gene family. The majority of predicted proteins contain signal peptides, suggesting secretion into the host cell cytoplasm. We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Preliminary microarray followed by quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was neither influenced by the cell type transformed by T. parva, nor by the animal background. Instead, the pattern of SVSP mRNA expression was largely defined by the parasite genotype and found to be relatively stable when monitored in vitro over a period of two months. Experiments using antibodies raised against the SVSP TP03_0882 provided first evidence for protein expression. Interestingly, results indicate that SVSP expression in cell lines established from a cloned parasite is limited to only a small percentage of parasites, suggesting SVSP expression by individual parasites is restricted. Expression of epitope-tagged TP03_0882 in mammalian cells revealed nuclear translocation and localisation to different nuclear compartments, including nucleoli, nucleoplasm and other nuclear bodies. Nuclear translocation to the mammalian cell nucleus was shown to involve two different types of nuclear localisation signals present in the conserved C-terminal region of TP03_0882. This first characterisation, opens up possibilities for future studies on the regulation of gene expression and the biological role of these enigmatic proteins. Expression patterns of SVSP genes in different T. parva-infected cloned cell lines Keywords: Gene expression

Project description:The protozoan parasite Theileria parva infects and transforms bovine lymphocytes inducing uncontrolled proliferation. The transforming schizont resides free in the host cell cytoplasm and it is assumed that proteins released from the parasite contribute to host cell transformation and parasite persistence. The identification and characterisation of parasite genes encoding candidate secreted proteins constitutes a first step towards elucidating this complex process. In earlier work, it was shown that the genes encoding subtelomere-encoded variable secreted proteins (SVSPs) are located at the subtelomeres of all four T. parva chromosomes and, with 85 members, form the largest Theileria gene family. The majority of predicted proteins contain signal peptides, suggesting secretion into the host cell cytoplasm. We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Preliminary microarray followed by quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was neither influenced by the cell type transformed by T. parva, nor by the animal background. Instead, the pattern of SVSP mRNA expression was largely defined by the parasite genotype and found to be relatively stable when monitored in vitro over a period of two months. Experiments using antibodies raised against the SVSP TP03_0882 provided first evidence for protein expression. Interestingly, results indicate that SVSP expression in cell lines established from a cloned parasite is limited to only a small percentage of parasites, suggesting SVSP expression by individual parasites is restricted. Expression of epitope-tagged TP03_0882 in mammalian cells revealed nuclear translocation and localisation to different nuclear compartments, including nucleoli, nucleoplasm and other nuclear bodies. Nuclear translocation to the mammalian cell nucleus was shown to involve two different types of nuclear localisation signals present in the conserved C-terminal region of TP03_0882. This first characterisation, opens up possibilities for future studies on the regulation of gene expression and the biological role of these enigmatic proteins. Expression patterns of SVSP genes in different T. parva-infected cloned cell lines Keywords: Gene expression We performed a three-condition experiment comparing SVSP gene transcription in three T. parva infected cell lines: (1) 211T-A3 (consisting of CD4+ T cells), (2) 211B-A3 (consisting of B cells), and 951T-F44 (consisting of CD4+ T cells). We used direct two-color experiment design, comparing each cell line with the other, i.e. (1) 211T-A3 vs 211B-A3, (2) 211T-A3 vs 951T-F44 and (3) 211B-A3 vs 951T-F44. There were two biological replicates for each hybridization and dye-flips for each hybridization pair. Microarray consisted of four in-slide replicates peer gene as well as no-DNA spots as negative controls.

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Theileria annulata Subtelomere-Encoded Variable Secreted Protein-TA05575 Binds to Bovine RBMX2.

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