Theileria annulata Subtelomere-Encoded Variable Secreted Protein-TA05575 Binds to Bovine RBMX2

Abstract

Tropical theileriosis is the disease caused by tick-transmitted apicomplexan parasite Theileria annulata, which has ability to transform bovine leukocytes, including B cells, macrophage cells, and dendritic cells. The T. annulata transformed cells are characterized as uncontrolled proliferation and shared some cancer-like phenotypes. The mechanism of the transformation by T. annulata is still not understood well. In previous reports, the subtelomere-encoded variable secreted proteins (SVSP) of T. parva were considered to contribute to phenotypic changes of the host cell, but the role of SVSP of T. annulata in host-pathogen relationship remains unknown. In the present study, a member of SVSP family, TA05575 of T. annulata was selected as the target molecule to analyze its expression profiles in different life cycle stages of T. annulata by qPCR and investigate its subcellular distribution of different passages of T. annulata transformed cells using confocal experiments. From the results, the transcription level of TA05575 at schizont stage was significantly higher than the other two life stages of T. annulata, and the protein of TA05575 was mainly distributed in nucleus of T. annulata infected cells. In addition, the potential proteins of host cells interacting with TA05575 were screened by Yeast-two hybrid system. The results of Co-IP experiment confirmed that TA05575 interacted with RBMX2-like protein that participated in transcription regulation of cells. In addition, a novel BiFC assay and flow cytometry were carried out, and the results further revealed that TA05575-RBMX2-like pair was directly interacted in cell context. Moreover, this interacting pair was found to distribute in intracellular compartments of HEK293T cells by using confocal microscopy. The results of the present study suggest that TA05575 may contribute for cells transformation due its distribution. According to the function of RBMX2, the interaction of TA05575 and RMMX2-like will provide a new information to further understand the mechanisms of cells transformation by T. annulata.

Keywords: BiFC; Co-IP; RBMX2-like; TA05575; Theileria annulata; Yeast-two hybrid.

Figure 1

Expression analysis of TA05575 at mRNA level in different life cycle stages of T. annulata. ****p < 0.0001.

Figure 2

The scheme of subcellular distribution of TA05575 in T. annulata-infected cell using CELLO v.2.5. ER represents the endoplasmic reticulum.

Figure 3

Subcellular localization of TA05575 in T. annulata-infected cells. Panels I to V displayed the subcellular localization of TA05575 in different culture passages (F10, F20, F55, F110, and F165) of T. annulata-infected cells. Panel VI was the negative control. The TA05575 (Panel I to V) were incubated with rabbit polyclonal antibody against TA05575 and then stained with an Alexa Fluor 488-conjugated goat anti-rabbit antibody. Hoechst 33342 was used to label cell nuclei, Alexa Fluor™ 594 phalloidin was used to label the cytoskeleton. Subcellular localization of TA05575 was visualized by confocal microscopy. Scale bar = 25 μm.

Figure 4

Screen of prey proteins interact with TA05575 by Y2H system. (A) Identification for autoactivation and toxicity of pGBKT7-TA05575 in Y2H Gold cells. (B) Confirmation for the transformation efficiency of pGBKT7-TA05575 recombinant bait plasmid. The colonies grown on SDO plates were randomly selected and performed to colony PCR using specific primers for TA05575. M: DL 2000 DNA marker; 1-8: the colonies picked from the SDO plate. (C) Screening and identification of the possible interacting proteins with TA05575. The co-transformants of pGBKT7-TA05575 and cDNA library of bovine B cells were cultured on DDO/X/A and then grown onto QDO/X/A plates. The co-transformants of pGADT7-T and pGBKT7-Lam plasmids, and pGADT7-T and pGBKT7-53 plasmids grown on DDO an DDO/X/A plates were served as negative and positive controls, respectively.

Figure 5

Expression and subcellular distribution analysis of TA05575and its potential interacting protein in HEK293T cells. Confocal microscopy analysis of HEK293T cells expressing TA05575 (4 µg) and its possible prey protein-RBMX2-like (4 µg) (A), and (TA05575 (2 µg)-RBMX2-like (2 µg) (B), respectively. Scale bar = 25 μm.

Figure 6

TA05575 interacts with RBMX2-like in HEK293T cells. Co-IP and immunoblotting (IB) detection of the cells expressing TA05575 (10 µg) and its potential pair RBMX2-like (10 µg). Whole HEK293T cells (WCL) represents IB analysis of the cell lysates for transfection without IP.

Figure 7

TA05575 directly binds to RBMX2-like in a cell context. TA05575-VN (4 μg), SVSP449-VC (4 μg), RBMX2-like-VN (4 μg), RBMX2-like-VC (4 μg), TA05575-VN (2 μg)-RBMX2-like-VC (2 μg), and TA05575-VC (2 μg)-RBMX2-like-VN (2 μg) were expressed individually or pairwise in HEK293T cells. The MFI of the green-fluorescent signals for BiFC assay was determined using flow cytometry, and the value of MFI is relative to fluorescent signal of un-transfected HEK293T cells, which were served as the control (A, B). HEK293T cells were co-transfected with TA05575-VN (2 μg)-RBMX2-like-VC (2 μg), TA05575-VC (2 μg)-RBMX2-like-VN (2 μg) pairs. The subcellular colocalization of the red of interacting pairs and green-fluorescent signals for BiFC were visualized by confocal microscopy (C). Scale bar =25 μ m. ****p < 0.0001.