Project description:Tropical theileriosis in a cattle disease of global economic importance, caused by the tick-borne protozoan parasite Theileria annulata. Conventional control strategies are failing to contain the disease and an attractive alternative is the use of pre-existing genetic resistance or tolerance. However, tropical theileriosis tolerant cattle are less productive than some susceptible breeds. To combine resistance and production traits requires an understanding of the mechanisms involved in resistance. Therefore, we have compared the response of monocytes derived from tolerant (Sahiwals, Bos indicus) and susceptible (Holstein-Friesians, B. taurus) cattle to in vitro infection with T. annulata. Over 150 genes exhibited breed-specific differential expression during the course of infection and nearly one third were differentially expressed in resting cells, implying that there are inherent differences between monocytes from the breeds. Fifty sequences currently only match ESTs or are unique to the library used to generate the microarray. The differential expression of a selection of genes was validated by quantitative RT-PCR, e.g. CD9, prion protein and signal-regulatory protein alpha. A large proportion of the differentially expressed genes encode proteins expressed on the plasma membrane or in the extracellular space and cell adhesion was one of the major Gene Ontology biological processes identified. We therefore hypothesise that the breed-specific tolerance of Sahiwal cattle compared to Holstein-Friesians is due to the interaction of infected cells with other immune cells, which influences the immune response generated against T. annulata infection. The BoMP microarray is available from the ARK-Genomics facility (www.ark-genomics.org).
Project description:A bovine lymphosarcoma cell line infected with Theileria annulata exhibits an irreversible reconfiguration of host cell gene expression
Project description:To understand the immune response of cows to the apicomplexan parasite Theileria annulata, we used ex vivo isolate cells derived from two infected calve : Holstein 12886 (Bos taurus), which is known to be susceptible to the disease, and Sahiwal 82H (Bos indicus), which is known to be resistant. The infected bovine macrophages of the two species with Theileria were collected and performed multiome 10X Chromium genomics scRNA-Seq.
Project description:The experiment investigates bovine gene expression in response to LPS in uninfected and Theileria annulata-infected cell cultures A subset of genes are identified which are activated in response to LPS stimulation with further modulation due to parasite infection.
Project description:The experiment investigates bovine gene expression in response to LPS in uninfected and Theileria annulata-infected cell cultures A subset of genes are identified which are activated in response to LPS stimulation with further modulation due to parasite infection. Six experimental conditions with three replicates per condition. Total RNA prepared from cell cultures. BL20 (uninfected bovine lymphosarcoma cell line), BL20 4 hours post-LPS stimulation, BL20 18 hours post-LPS, TBL (T. annulata infected bovine cell line), TBL 4 hours post-LPS, TBL 18 hours post-LPS. 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:The tick-borne protozoan parasites, Theileria annulata and T. parva are unique amongst intracellular eukaryotic pathogens because of their ability to induce a phenotype akin to transformation of the host cell. Infection with T. annulata, the causative agent of tropical theileriosis, is frequently fatal, via a cycle of neoplastic growth and tissue dissemination. Infected leukocytes become highly metastatic, forming foci of infected cells in multiple organs with subsequent destruction and disorganisation of the lymphoid system. Studies on extracellular vesicles (EV) have revealed that inter-cellular communication is critical for metastatic progression in cancer, and that the release of exosomes, a sub-set of EV, from tumour cells into the micro-environment induces pre-metastatic niche formation. Here we compared EV from a bovine lymphosarcoma cell line (BL20) and the same line infected in vitro with T. annulata (TBL20) by comparative mass spectrometry and miRNA profiling. Ingenuity Pathway Analysis revealed that many infection-associated proteins essential to migration and extracellular matrix digestion were up-regulated in EV from TBL20 cells compared with those from BL20 controls. Analysis of EV-associated miRNA revealed an altered repertoire of six host miRNA, each with a known role in tumour and/or infection biology. Focusing on bta-miR-181a and bta-miR-181b, we identified relevant mRNA targets and confirmed the interaction of bta-miR181a with intracellular adhesion molecule 1 (ICAM1). These data support a role for EV and their miRNA cargo in the pathobiology of Theileria infection.
Project description:Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumors that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria-induced transformation and highlighted a small set of genes associated with leukocyte dissemination. CRISPR/Cas9-mediated knock-down of GZMA and RASGRP1 in macrophages attenuated for dissemination led to a regain in their dissemination in Rag2/gC mice confirming their suppressor roles in vivo. Comparing the transcriptomes of 934 human cancer cell lines to that of Theileria-transformed bovine B cells again highlighted GZMA and RASGRP1 and CRISPR-mediated overexpression of GZMA and RASGRP1 dampened the dissemination potential of human B-lymphomas. The ensemble provide evidence for a novel suppressor function in the dissemination of both T. annulata-transformed bovine leukocytes and human B-lymphomas.
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:Theileria annulata is an apicomplexan parasite that infects and transforms bovine macrophages that disseminate throughout the animal causing a leukaemia-like disease called tropical theileriosis. Using deep RNAseq of T. annulata-infected B cells and macrophages we identify a set of microRNAs induced by infection, whose expression diminishes upon loss of the hyper-disseminating phenotype of virulent transformed macrophages. We describe how infection-induced upregulation of miR-126-5p ablates JIP-2 expression to release cytosolic JNK to translocate to the nucleus and trans-activate AP-1-driven transcription of mmp9 to promote tumour dissemination. In non-disseminating attenuated macrophages miR-126-5p levels drop, JIP-2 levels increase, JNK1 is retained in the cytosol leading to decreased c-Jun phosphorylation and dampened AP-1-driven mmp9 transcription. We show that variation in miR-126-5p levels depends on the tyrosine phosphorylation status of AGO2 that is regulated by Grb2-recruitment of PTP1B. In attenuated macrophages Grb2 levels drop resulting in less PTP1B recruitment, greater AGO2 phosphorylation, less miR-126-5p associated with AGO2 and a consequent rise in JIP-2 levels. Changes in miR-126-5p levels therefore, underpin both the virulent hyper-dissemination and the attenuated dissemination of T. annulata-infected macrophages.