Abstract
Host cells infected by Theileria annulata schizonts show the character of permanent proliferation in vitro, also named transformation. To explore the molecular mechanism a T. annulata Cyp1 (TaCyp1) protein potentially involved in regulating cell transformation was used as bait to screen for its interacting proteins by yeast-two-hybrid assay. Additional GST-pull down experiments confirmed that only MED21 specifically interacted with TaCyp1. Moreover, the distribution of TaCyp1 around T. annulata schizonts facilitated interaction with host cell MED21. As a component of mediator complex, MED21 is normally involved in regulating the transcription of nearly all RNA polymerase II-dependent genes. Therefore, to explore its influence on NF-κB signaling MED21 RNA interference and parasite killing with BW720c treatment were performed. Knock down of MED21 resulted in a significant decrease in NF-κB1/2 mRNA expressions, but no significant change in P105, P52 levels, nor detectable alteration in levels of phosphorylated IκBα/β. By contrast, BW720c treatment induced an obvious decrease in the phosphorylation status of P52 and IκBα/β, but no obvious change in that of P105. This suggests that BW720c-induced parasite death had a significant negative influence on NF-κB signaling, whereas knock down of MED21 had no obvious effect on NF-κB signaling. Characterization of TaCyp1 provides information on the function of parasite cyclophilins and leads to a better understanding of the interactions between T. annulata and its host leukocytes.
Highlights
Theileria annulata is a tick-borne protozoan parasite mainly distributed in North Africa, Southern Europe, India, the Middle East, and Asia (Bilgic et al, 2010)
Total proteins were extracted from Y2HGold cells transformed with pGBKT7-Theileria annulata Cyclophilin1 (TaCyp1) or pGBKT7-53 plasmid and detected with an anti-Myc tag mouse mAb
Only MED21 was confirmed to interact with TaCyp1 in GST-pull down experiments
Summary
Theileria annulata is a tick-borne protozoan parasite mainly distributed in North Africa, Southern Europe, India, the Middle East, and Asia (Bilgic et al, 2010). Cyclophilins (Cyps) known as immunophilins are a family of ubiquitous proteins present in the cytosol of all prokaryotic and eukaryotic cells. Due to their peptidyl-prolyl isomerase (PPIase) activity they catalyze the cis to trans conversion of proline-containing peptides that facilitates protein folding (Wang and Heitman, 2005; Naoumov, 2014). Among all family members CypA is most abundantly distributed in the cytosol (Dornan et al, 2003), where it’s involved in protein folding, trafficking, signal transduction and cell activation (Nigro et al, 2013). Ibrahim et al (2014) found Toxoplasma gondii cyclophilin regulated host cell migration and enhanced parasite dissemination in a CCR5-independent manner. For T. annulata cyclophilins, their role in cell transformation is almost unknown
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