Recruitment of Host Nuclear Pore Components to the Vicinity of Theileria Schizonts

Abstract

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface.IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

Keywords: Theileria; annulate lamellae; apicomplexan; importin; nuclear pore complex.

FIG 1

TEM analysis of T. annulata-infected macrophages reveals the presence of porous membranes in close proximity to the schizont surface. Glutaraldehyde-fixed and Epon-embedded TaC12 cells were analyzed using TEM. Parasite associated annulate lamellae are labeled with white arrows, the schizont surface is labeled with black arrows, and nuclear pores are labeled with white arrowheads. (A) T. annulata schizont surrounded by a porous membrane. Note that pores are visualized as spheres or cross-sections, depending on the plane of view. Bar, 380 nm. (B) T. annulata schizont surrounded by a porous membrane. The white box indicates a potential cytostome. Bar, 500 nm.

FIG 2

Cell cycle-dependent association of Nups with the schizont surface. (A) Deconvolution images of T. annulata-infected macrophages were taken at different stages of the cell cycle. Cells were fixed and analyzed by indirect immunofluorescence analysis with anti-nucleoporin (MAb414 [green]) antibodies. The parasite was labeled with anti-TaSP (red), microtubules ware labeled with anti-alpha tubulin (cyan) antibodies, and DNA was labeled with DAPI (blue). Representative cells are shown in interphase, prometaphase, metaphase, anaphase, and telophase/cytokinesis. Bar, 10 μm. (B) A T. annulata-infected macrophage in the late stages of cytokinesis/G₁ phase was imaged using 3D-SIM microscopy (DeltaVision OMX Blaze). RanGAP1 is labeled with green, and the parasite surface was labeled with anti-p104 (1C12) antibodies (red). One z-stack is shown. Bar, 5 μm.

FIG 3

TEM and immunogold labeling with MAb414. T. annulata-infected macrophages were fixed with formaldehyde and processed according to the Tokuyasu technique prior to labeling with rabbit anti-TaSP (10-nm-diameter gold particles; white arrows) and mouse MAb414 (15-nm-diameter gold particles; black arrows). The white box in the inset indicates the magnified area. Bar, 220 nm.

FIG 4

Localization of Nups in T. annulata-infected and T. parva-infected B and T cells. (A) T. annulata-infected bovine B cells (TBL20) and their noninfected counterparts (BL20) were processed for immunofluorescence analysis with MAb414 (green). The parasite was labeled with anti-TaSP (red), and DNA was labeled with DAPI (blue). (B) T. parva-infected T cells (Tpm803) were labeled with anti-RanGAP1 (green). The parasite was labeled with anti-T. parva PIM (red), and DNA was labeled with DAPI (blue). Bar, 10 μm.

FIG 5

Localization of Nups in Toxoplasma gondii and Plasmodium berghei-infected cells. (A and B) Human foreskin fibroblast (HFF) cells (A) or TaC12 cells (B) were infected with T. gondii and processed for immunofluorescence analysis. Nups are labeled with MAb414 (green). Toxoplasma was detected with rabbit anti-Toxoplasma serum (red), and Theileria was visualized with anti-CLASP1 antibodies (cyan). A merge is shown of MAb414 with Toxoplasma (A [green and red]) and MAb414 with Toxoplasma and Theileria (B [green, red, and cyan]). (C) HeLa cells were infected with mCherry expressing-P. berghei sporozoites and were processed for microscopy 24 h postinfection. Nups were labeled with MAb414 (green), the parasite is indicated in red, and the parasitophorous vacuole (PVM) was detected with anti-UIS4 antibodies (cyan). A merge is shown with MAb414 and Plasmodium (green and red) and additionally with the PVM (green, red, and cyan). Bar, 10 μm.

FIG 6

Components of the host cell nuclear transport machinery are recruited to the schizont surface. (A and B) T. annulata-infected macrophages were fixed and analyzed by indirect immunofluorescence analysis with anti-RanGAP1 (A) or anti-importin beta 1 (B) antibodies (green). The parasite was labeled with anti-TaSP (red), and DNA was labeled with DAPI (blue). Bar, 10 μm. (C) TaC12 cells were transfected with GFP-Ran and processed for indirect immunofluorescence analysis. Nucleoporins were labeled with MAb414, the parasite surface was labeled with anti-TaSP (red), and DNA was labeled with DAPI. A merge of GFP-Ran and the schizont (green and red) is shown. Bar, 10 μm.

FIG 7

Secreted Theileria proteins SuAT1 and TaMISHIP did not colocalize with Nups or importin beta 1 at the schizont surface. T. annulata-infected macrophages were fixed and subjected to indirect immunofluorescence analysis with anti-SuAT1 (A and B) or anti-TaMISHIP (C [red]) together with MAb414 (to label Nups) (A) or anti-importin beta 1 antibodies (green) (B and C). DNA was labeled with DAPI (blue). Bar, 10 μm.