An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii

Abstract

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

A circular zone of attachment to the extracellular matrix provides directionality to the motility ofToxoplasma gondiiin 3D

“…Munera Lopez et al [36] also recently noted that tachyzoites show constrictions as they move through Matrigel. These authors ascribed the constrictions to the parasite having to squeeze through small pores in the matrix.…”

The cortical microtubules ofToxoplasma gondiiunderlie the helicity of parasite movement

“…Host cell and T. gondii tachyzoite parasite cultures were maintained as described previously [15,22,34,39]. The TKO (∆tlap2∆spm1∆tlap3) mutant was generated in [22] and the ∆dcx parasite was generated in [33].…”

“…The 3-D motility assay for Toxoplasma, originally developed in [3], was carried out as described in [34]. For cold-treated parasites, cultures with intracellular parasites were placed at ~ 7ºC for 2 hr, before harvesting as described in [34]. Parasites were harvested and imaged immediately at 37ºC.…”

“…Image stacks were acquired from at least ~10 µm above the coverslip. To represent the 3-D DIC images in 2-D format (Fig 2 and Video S1&3), we performed focus-stacking using Fiji by applying the Extended Depth of Field Sobel Projection plugin from [40,41] DIC image stacks processing and cell tracking DIC images were processed using our custom scripts extended from [34]. High contrast images were generated by subjecting the DIC images to 1) a 3-D median filter (footprint nx = ny = nz = 2 px) to reduce noise, 2) a 2-D Laplacian filter to attenuate out-of-focus portions of the cells, and 3) a 3-D gradient filter of Sobel type for edge detection.…”

“…For cold-treated TKO, intracellular parasite cultures were placed at 7 degree for two hours before harvesting and mixed with Matrigel. For the other samples/conditions, the parasites were harvested as described previously in [34]. All motility assays were carried out at 37°C.…”