Li et al. Parasites Vectors (2021) 14:319 https://doi.org/10.1186/s13071-021-04820-4 Parasites & Vectors RESEARCH Open Access Screening and identifcation of Theileria annulata subtelomere-encoded variable secreted protein-950454 (SVSP454) interacting proteins from bovine B cells Zhi Li1, Junlong Liu1*, Quanying Ma1, Aihong Liu1, Youquan Li1, Guiquan Guan1, Jianxun Luo1 and Hong Yin1,2* Abstract Background: Theileria annulata is a protozoan parasite that can infect and transform bovine B cells, macrophages, and dendritic cells. The mechanism of the transformation is still not well understood, and some parasite molecules have been identifed, which contribute to cell proliferation by regulating host signaling pathways. Subtelomeric vari- able secreted proteins (SVSPs) of Theileria might afect the host cell phenotype, but its function is still not clear. There- fore, in the present study, we explored the interactions of SVSP454 with host cell proteins to investigate the molecular mechanism of T. annulata interaction with host cells. Methods: The transcription level of an SVSP protein from T. annulata, SVSP454, was analyzed between diferent life stages and transformed cell passages using qRT-PCR. Then, SVSP454 was used as a bait to screen its interacting proteins from the bovine B cell cDNA library using a yeast two-hybrid (Y2H) system. The potential interacting proteins of host cells with SVSP454 were further identifed by using a coimmunoprecipitation (Co-IP) and bimolecular fuores- cence complementation (BiFC) assays. Results: SVSP454 was transcribed in all three life stages of T. annulata but had the highest transcription during the schizont stage. However, the transcription level of SVSP454 continuously decreased as the cultures passaged. Two proteins, Bos Taurus coiled-coil domain 181 (CCDC181) and Bos Taurus mitochondrial ribosomal protein L30 (MRPL30), were screened. The proteins CCDC181 and MRPL30 of the host were further identifed to directly interact with SVSP454. Conclusion: In the present study, SVSP454 was used as a bait plasmid, and its prey proteins CCDC181 and MRPL30 were screened out by using a Y2H system. Then, we demonstrated that SVSP454 directly interacted with both CCDC181 and MRPL30 by Co-IP and BiFC assays. Therefore, we speculate that SVSP454-CCDC181/SVSP454MRPL30 is an interacting axis that regulates the microtubule network and translation process of the host by some vital signaling molecules. Identifcation of the interaction of SVSP454 with CCDC181 and MRPL30 will help illustrate the transforma- tion mechanisms induced by T. annulata. Keywords: Theileria annulata, SVSP454, Yeast two-hybrid, Co-IP, BiFC, CCDC181, MRPL30 *Correspondence: [email protected]; [email protected] 1 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. Parasites Vectors (2021) 14:319 Page 2 of 13 Background Terefore, in the present study, we explored the inter- Teileria annulata is an intracellular apicomplexan actions of SVSP proteins and host cell proteins. First, parasite transmitted by Hyalomma tick species, and is the mRNA level of SVSP454 was compared between T. the pathogen responsible for tropical theileriosis. Tropi- annulata at diferent life stages and transformed cells at cal theileriosis causes more than 1.1 million cattle to diferent passages with qPCR. Ten, we used SVSP454 as die and approximately hundreds of millions of dollars the bait plasmid to screen the interacting proteins of the of losses per year in tropical and subtropical areas [1]. host by a yeast two-hybrid system. We demonstrated that Teileria annulata together with T. parva are referred SVSP454 interacts with both CCDC181 and MRPL30 to as “transforming species” for their ability to induce using a coimmunoprecipitation (Co-IP) assay. With a the uncontrolled proliferation of infected cells [2]. Te bimolecular fuorescence complementation (BiFC) assay, target cells of these two T. species are diferent, where T. the interaction of SVSP454 with CCDC181 and MRPL30 annulata mainly infects B cells, macrophages and den- was confrmed in the cell context. Te subcellular colo- dritic cells, while T. parva invades T and B lymphocytes. calization of the interacting proteins indicated that their However, infection of both parasites could cause acute interaction occurs in the intracellular compartments. In lymphoproliferative disease, which shares some clini- addition, fow cytometry was used to observe the median cal characteristics with human leukemias [3]. In addi- fuorescence intensity (MFI) emitted during the interac- tion to uncontrolled proliferation, the cells transformed tions between bait and prey proteins. by T. annulata also have other cancer hallmarks, such as increased metastasis and invasiveness and deregulated Methods cellular energetics [3–6]. Te transformation induced by Cell culture T. annulata is reversible, once the cells are treated with T. annulata schizont-infected cell lines were provided a theilericidal drug, they lose cancer-like characteristics by the Vector and Vector-borne Disease (VVBD) labo- and undergo the programmed apoptosis [7]. ratory, Lanzhou Veterinary Research Institute (LVRI), To drive host cell transformation, some molecules of T. China. Cells were cultured in RPMI 1640 medium (Bio- annulata have been confrmed to highjack host signal- logical Industries, Kibbutz Beit Haemek, Israel) plus 10% ing pathways that contribute to the immortality of cells, fetal bovine serum (Biological Industries, Kibbutz Beit such as EB1 and TaPin1 [8–10]. However, the detailed Haemek, Israel) and 100 mg/ml penicillin/streptomycin, mechanisms by which T. annulata manipulates the host and maintained at 37 °C in an incubator with 5% CO 2. cell and by which parasitic molecules are responsible HEK293T cells were obtained from the China Center for for cancer-like phenotypes remain largely unknown. Type Culture Collection, Shanghai, China, and cultured To understand the mechanisms of the transformation in Dulbecco’s Modifed Eagle Medium (DMEM) (Gibco, induced by Teileria, the genome of T. annulata was New York, USA) containing 10% fetal bovine serum sequenced, and 3792 putative proteins were predicted (Gibco, New York, USA). [11]. Te proteins expressed at the macroschizont stage and released into the cell cytoplasm or expressed on the Quantitative real‑time polymerase chain reaction surface of parasites are possibly involved in the process (qRT‑PCR) of transformation [11]. Teileria annulata schizont AT- Total RNA of T. annulata-infected cells at diferent pas- hook protein (TashAT) family proteins, which matched sages (F10, F20, F55, F110, and F165), and the RNA for these criteria, were confrmed to localize in the host cell suspensions of sporozoite and merozoite for T. annu- nucleus and afected the host cell phenotype and prolif- lata infection were extracted using a RNeasy Mini kit eration [3, 12] (QIAGEN, Dusseldorf, Germany) according to the user Te SVSP multigene family, another large gene family, manual. cDNA was then synthesized via a PrimeScript™ is mainly expressed at the schizont stage of both T. parva RT Reagent Kit with gDNA Eraser (Perfect Real Time) and T. annulata, and has been suspected to be involved (Takara, Dalian, China). Te specifc primers of SVSP454 in host cell transformation, invasion, and immune eva- (Accession No: XM950454.1) were as follows: SVSP454-F sion [3, 13, 14]. Most SVSP genes are distributed across (5′-TAA AGG GAA ATG GGT GTC TA-3′) and SVSP454-R the subtelomeres, and have unique features, including (5′-CAC TTG TGC TAA GGC TAA CG-3′). qPCR reactions diversity in length, atypical codon usage and extensive were carried out in a fnal volume of 20 μL containing nucleotide variability. Moreover, the majority of SVSPs 10 μL TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) have signal peptides and are localized in the nuclear and (2 ×) (TaKaRa, Dalian, China), 10 μM of each primer, cytoplasmic regions of host cells [11, 15], which makes it 0.4 μL ROX Reference Dye II (50 ×) (TaKaRa, Dalian, more likely that SVSP molecules interfere with host cell China), 2 μL of cDNA template and 10 μL Easy Dilu- signaling “hubs” to regulate host cell functions. tion for Real Time PCR System (TaKaRa, Dalian, China). Li et al. Parasites Vectors (2021) 14:319 Page 3 of 13 And the cDNA from PBMC for bovine uninfected T. and then fxed with 4% paraformaldehyde at room tem- annulata was used as the negative control in the qPCR perature for 30 min. After three washes with PBS, the system. Te qPCR protocol composed of initial dena- cells were permeabilized with 0.5% Triton X-100 for turation at 95 °C for 30 s followed by 40 cycles of 2 step 10 min at room temperature. Following three washes, PCR (95 °C for 5 s, 60 °C for 34 s) and dissociation stage the cells were blocked with 3% bovine serum albumin (95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s).