Project description:In the present experiments, we used in vitro cultured bovine lymphocytes infected with T. annulata to purify the cycloartilaginous stage shizonts of T. annulata and the cycloartilaginous stage merozoites of T. annulata after using T. annulata-infected erythrocytes from live cows (with 30% staining rate), and then used the Proteomics and phosphorylated proteomics data of the two stages of T. annulata were measured on a Thermo Orbitrap Astral mass spectrometer for mass spectrometry uptake and resolution.
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.
Project description:Extracellular vesicles (EVs) are key mediators of intercellular communication, and often play critical roles in host-parasite interactions by facilitating parasite’s physiology and pathogenesis. Theileria annulata, an apicomplexan parasite, induces profound changes in host cells, leading to uncontrolled proliferation, apoptosis resistance, and increased invasiveness. In this study, we performed the comprehensive proteomic and small RNA analysis of EVs isolated from a T. annulata Kashi isolate-infected bovine lymphocyte cell line (TaXJS), B cell line (TaBC), dendritic cell line (TaDC), and from the sera of cattle before and after infection. Our label-free LC-MS/MS proteomics identified 2580 proteins, while small RNA sequencing revealed 6635 miRNAs associated with parasite development, host invasion, and immune evasion. Functional enrichment analyses recognized vesicular components involved in key pathways of the parasite-host such as ECM-receptor interaction, oxidative phosphorylation, and proton transport. These findings highlight the potential of Theileria-derived EVs in modulating host responses and their potential as therapeutic and vaccine targets.
Project description:Investigation of parasite (T. annulata) gene expression over the course of the life-cycle (sporozoite->macroschizont->merozoite->piroplasm). The study focused on the expression of known and putative transcription factors, in particular members of the ApiAP2 gene family. Up-stream motifs associated with stage-specifically expressed genes were identified during the course of the analysis. The experiment investigates Theileria annulata gene expression over a differentiation time-course - sporozoite through to piroplasm. 20 samples were analyzed - 4 x sporozoite (replicates), 3 x macroschizont (replicates), 3 x day 4, 3 x day 7, 3 x day 9 (replicates) and 4 x piroplasm (replicates)