Infection with the Intracellular Protozoan Parasite Theileria parva Induces Constitutively High Levels of NF-κ B in Bovine T Lymphocytes.

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-κ B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-κ B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-κ B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-κ B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-κ B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-κ B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-κ B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-κ B. We propose that high levels of activated NF-κ B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite

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

Background The 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 Findings 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. 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. Conclusions Our 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

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused by Theileria parva infection

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva -infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast, T. parva mediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored following T. parva killing, indicating that viable parasites are required for IκBβ degradation. Treatment of T. parva -infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidant N- acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function

Theileria parva isolate of low virulence infects a subpopulation of lymphocytes

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parvaChitongo (TpC) is an isolate of lower virulence than T.parvaMuguga (TpM). Lower virulence appeared correlated with a delayed onset of thelogarithmic growth phase of TpC-tranformedperipheral blood mononuclear cells after in vitro infection. In the current study, infection experiments of WC1(+)-gammadelta-T cellsrevealed thatonly TpMcould infect these cells andthat no transformed cells could be obtained withTpC sporozoites. Subsequent analysis of susceptibility of different cell lines and purified populations of lymphocytes for infection and transformation by both isolates showed that TpMsporozoites could attach and infectCD4(+), CD8(+) and WC1(+) T lymphocytes, but that TpCsporozoites were only observed to bind to the CD8(+) T cell population. Flow cytometry analysis of established,transformed clones confirmed this bias in target cells. TpM-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+)or WC1(+)T cells. In contrast, all in vitro and in vivoTpC-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the TpC-transformed target cells were exclusivelyinfected CD8(+) lymphocytes.So a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100 % identical between the two strains, a second, high affinity adhesin that determines target cell specificity appears to exist

List of phosphorylated proteins with a predicted transmembrane-domain and/or a signal peptide.

<p>Phospho-epitopes indicated in bold were those found more abundantly in samples from M- or S-phase (p<0.05).</p

Synchronisation of TaC12 cells in S- and M-phase.

<p>TaC12 were treated with thymidine for 24 hours or nocodazole for 16 hours to synchronise cells in S-phase or mitosis. Synchronised cells were fixed with 4% PFA and analysed with anti-p-Thr, anti-p-Thr-Pro and anti-p-Ser antibodies. The parasite was detected with anti-p104 or TaSP antibodies and DNA is visualised with DAPI. Merge: phospho-epitopes (green), schizont (red), DAPI (blue). A: Thymidine synchronised TaC12 cells in S-phase. B: Nocodazole synchronised TaC12 cells in mitosis. Scale bar represents 10 µm.</p