Project description:The experiment investigates bovine gene expression in response to BW720c treatment in uninfected and Theileria annulata-infected cell cultures Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. 50% of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of expression and chromatin modification. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and reversible manner. Six experimental conditions with three replicates per condition. Total RNA prepared from cell cultures. BL20 (uninfected bovine lymphosarcoma cell line), BL20 24 hours post-BW720c treatment, BL20 48 hours post-BW720c, TBL (T. annulata infected bovine cell line), TBL 24 hours post-BW720c, TBL 48 hours post-BW720c. Each hydridisation represents bovine and parasite gene expression on a single channel and 2 technical replicates of each probeset are represented on the chip.

Project description:Plasmodium falciparum merozoite surface protein MSPDBL2 is a highly polymorphic target of naturally acquired immune responses encoded by a single copy gene under strong balancing selection in natural populations. The MSPDBL2 protein is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene expression in culture is increased in response to overexpression of the gametocyte development inducer GDV1 in an engineered parasite clone, so there is a need to characterise its natural expression variation. In this study, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 102 clinical isolates from four endemic populations in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as eleven isolates had more than 3% schizonts positive and one had 73% positive. The expression frequencies were similar across the four endemic populations, and there was no significant difference between the clinical isolates overall and frequencies of positive schizonts previously seen in cultured P. falciparum laboratory lines. To explore whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBP2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of many genes previously implicated as likely to be involved in gametocytogenesis, but not including other genes including the gametocytogenesis master regulator ap2g. Although MSPDBL2 expression occurs in a highly variable proportion of schizonts in clinical isolates, and correlation with expression of some gametocytogenesis-related genes is consistent with regulation by GDV1, it is not apparently a direct marker of sexual commitment and its function in the parasite remains to be determined.

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:Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by depletion of H3K4me3 in their promoter regions. These genes were upregulated in merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic merozoites, suggesting roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial approaches that may have activity against both the liver and blood stages.

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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	DX121213_S2.raw	Raw
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	DX121213_W1.raw	Raw
	DX121213_W2.raw	Raw

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Cell cycle dependent phosphorylation of transmembrane proteins on the Theileria annulata schizont surface

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Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

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