Screening and identification of host proteins interacting with Theileria annulata cysteine proteinase (TaCP) by yeast-two-hybrid system

Abstract

Background Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.MethodsThe cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.ResultsTwo host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.ConclusionsThis study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.

Establishment and Expression of Cytokines in a Theileria annulata-Infected Bovine B Cell Line

“…Flow cytometry is a powerful technique for studying fluorescence-labeled surfaces as well as intracellular molecules [27]. CD21 + bovine mature B cells [28] were isolated by magnetic separation according to a previously described method [29,30] with minor modifications to obtain a highly pure population (>95%). Briefly, whole blood (from infected and normal cattle separately) was collected into 9 mL tubes containing K 3 EDTA by venipuncture of the jugular vein.…”

Theileria annulata Cyclophilin1 (TaCyp1) Interacts With Host Cell MED21

“…Separation of B cell from bovine peripheral blood mononuclear cells (PBMCs) and construction of a yeast two-hybrid cDNA library have been previously described (Zhao et al, 2017). Briefly, 2 × 10 7 B cells with purity of 95.3% were obtained and used for cDNA library construction and library titer was 2 × 10 6 cfu with the size of inserted fragments varying from 750 to 3500 bp.…”

“…Following the protocols of the Yeastmaker TM Yeast Transformation System 2 kit (Clontech, United States) both pGBKT7-TaCyp1 and pGBKT7-53 (positive control) were transformed into the yeast strain Y2HGold. Total proteins were extracted from yeast cells and detected using mouse anti-Myc tag McAb (1/1000, Proteintech, United States) to detect TaCyp1 expression (Zhao et al, 2017).…”

“…The protocol has been described previously (Zhao et al, 2017) and according to white colonies on SD/-Trp and SD/-Trp/X plates, and absence of colony growth on SD/-Trp/X/A plates, the bait plasmid was confirmed to lack auto-activation. Moreover, similar colonies size between bait and pGBKT7 plasmids indicated no toxicity indicating that expression of the bait plasmid had no auto-activation and toxicity and could be used in the yeast-two-hybrid screen.…”

“…To screen for host interacting proteins TaCyp1 bait and prey plasmids were co-transfected into Y2HGold, and screened as previously described (Zhao et al, 2017). The blue colonies on QDO/X/A agar plates were confirmed as positive hits.…”