(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii [1] ['Jonathan Munera Lopez', 'Biodesign Center For Mechanisms Of Evolution School Of Life Sciences', 'Arizona State University', 'Tempe', 'Arizona', 'United States Of America', 'Isadonna F. Tengganu', 'Jun Liu', 'Department Of Biology', 'Indiana University'] Date: 2022-11 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. The thousands of species of apicomplexan parasites are responsible for many devastating human and animal diseases, including malaria and toxoplasmosis. Cell movement, needed to disseminate among tissues and invade into a host cell, is essential for the apicomplexan parasites to maintain their intracellular parasitic life style. Here we investigate the movement of a very successful apicomplexan, Toxoplasma gondii. We discovered that Preconoidal region protein 2 (Pcr2), a new component of the parasite apical complex, is important for the persistence of Toxoplasma movement. The movement of the Pcr2 knockout parasite is spasmodic, which compromises egress and invasion, two vital steps in the parasite’s lytic cycle. As a result, Pcr2 knockout parasites cause much less host tissue destruction. Furthermore, the ability of the Pcr2 knockout parasite to assemble a ring-like structure (the moving junction) at the entry point of invasion, is impaired. The discovery of Pcr2 and the analysis of its function open new opportunities to determine how mechanical interactions with its environment impact parasite motility, and how the regulation or maintenance of persistence in parasite movement is functionally connected with and imposed onto the motility apparatus and other structures involved in invasion and egress. Funding: This study was supported by funding from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01-AI132463) awarded to K.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2022 Munera Lopez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pcr2 is a novel protein located in the apical complex of the parasite. Knockout of Pcr2 results in stuttering parasite movement, a phenotype that has not been observed before. Calcium stimulated basal accumulation of mEmeraldFP tagged actin chromobody (actin-Cb-mE) is not blocked in the Pcr2 knockout parasite. This suggests that Pcr2 does not regulate motility through actin polymerization or actomyosin activity, as both have been shown to be necessary for the basal accumulation of actin-Cb-mE to occur [ 13 , 48 ]. The abnormal motility of the Pcr2 knockout parasite is associated with significantly reduced efficiency of parasite egress, invasion, and destruction of host cells, indicating the importance of Pcr2 for the lytic cycle. Infectious apicomplexan parasites move by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. Over the years, many labs have characterized key proteins involved in parasite motility [ 2 , 6 , 7 , 12 , 13 , 39 – 46 ]. The functions of these proteins can be largely explained in the context of a working model [ 47 ], in which the internal motor activity powers parasite gliding via the coupling of the actomyosin machinery with transmembrane adhesin complexes (more in discussion). Here we report the discovery of a new apical complex protein, Preconoidal region protein 2 (Pcr2), which is required for persistent movement. The loss of Pcr2 results in an unusual motility phenotype. During invasion, the parasite first makes contact with the host cell "head-on" with its apical complex, which features an elaborate cytoskeletal apparatus and associated structures that contain both structural and signaling proteins important for invasion [ 7 – 13 ]. As the parasite enters the host cell, it assembles a ring-like structure (the moving junction) at the entry point from proteins secreted from the rhoptries and the micronemes, which is critical for the invasion process [ 14 – 22 ]. As it invades, the parasite also extensively modifies the host cell’s plasma membrane at the entry point, and uses that modified membrane to form the parasitophorous vacuole that eventually completely envelops the intracellular parasite [ 16 , 23 – 35 ]. The integrity of the host plasma membrane and viability of the host cell are preserved during invasion, thus allowing for subsequent replication cycles during which the parasites exploit host resources for proliferation. Parasite exit (i.e. egress) on the other hand, is lethal for the host cell [ 3 , 7 , 36 – 38 ]. During Toxoplasma egress, the combination of pore formation by lytic proteins secreted from the parasite and mechanical disruption due to parasite moving through the host cell membrane results in destruction of the host cell, and allows parasite dissemination to initiate the next round of the lytic cycle. Successive cycles of parasite invasion, replication, and egress lead to extensive tissue damage such as seen in toxoplasmic encephalitis and congenital toxoplasmosis. Therefore, parasite motility not only is required to initiate an infection, but also directly contributes to the pathogenesis of the disease. Cell movement allows cells to explore their environment, initiate physical interaction with other cells, and respond accordingly. Movement forms the basis of numerous processes such as embryonic development and the inflammatory response in animals. For the thousands of species of apicomplexan parasites that are responsible for many devastating diseases (including malaria and acute toxoplasmosis), movement is integral to their parasitic lifestyle. For instance, upon injection into the human host by a mosquito, parasite motility enables the Plasmodium sporozoites to migrate through the skin tissue, and cross a blood vessel wall into the bloodstream to be carried to the liver, where the sporozoites move out of the blood vessel and invade into liver cells [ 1 ]. Similarly, Toxoplasma, an extremely successful parasite that permanently resides in ~ 20% of the people on Earth and can infect any nucleated cell and any warm-blooded animal, relies on motility to shove its way into a host cell, as well as to rapidly escape from a resource-exhausted host cell, disseminate, and reinvade into a fresh host [ 2 – 6 ]. Results Loss of Pcr2 results in significant defects in parasite invasion To assess parasite invasion, we used an assay in which invaded (i.e., intracellular) and non-invaded (i.e., extracellular) parasites are distinguished based on accessibility of the parasite surface, before and after permeabilizing cells, to an antibody against a major surface antigen (SAG1) of the parasite [22,62]. Consistent with the smaller number of plaques formed in the plaque assay, the invasion efficiency of Δpcr2 parasites is significantly lower than the parental lines (Table 2). The invasion efficiency was restored in the complemented line. PPT PowerPoint slide PNG larger image TIFF original image Download: Table 2. Quantification of invasion for the four T. gondii strains. The number of intracellular parasites per field was counted in ten fields per strain, in each of three independent biological replicates. s.e.: Standard error of the mean. P-values from unpaired Student’s t-tests are indicated on the right. https://doi.org/10.1371/journal.ppat.1010776.t002 Loss of Pcr2 results in significant defects in parasite egress In contrast to the rapid dispersal of wild-type parasites after egress, in the Δpcr2 cultures, clusters of parasites are often observed in the vicinity of a lysed vacuole, suggesting that these parasites do not egress efficiently. To examine parasite behavior during egress, we carried out time-lapse microscopy to monitor egress induced by calcium ionophore (A23187) (Fig 5B–5C, S2 Video). Unlike wild-type parasites, Δpcr2 parasites in most vacuoles (37 out of 49 vacuoles) failed to disperse even 10 min after A23187 treatment (Fig 5B). For the parental, knock-in and complemented lines, it is common to observe reinvasion immediately after egress (Fig 5C, cyan arrows). In contrast, reinvasion of egressed Δpcr2 parasites was never observed in a substantially longer recording period (> 10 min), consistent with the low invasion efficiency demonstrated by the immunofluorescence-based assay (Table 2). Δpcr2 parasites can secrete effectors to lyse the host cell during calcium-induced egress and the localization and secretion of the major adhesin, MIC2, are normal Lysis of the host cell in response to elevated calcium [38] occurs normally in Δpcr2 parasites. Shortly after the Δpcr2 infected culture was exposed to A23187, nuclear labeling by a cell-impermeant DNA binding dye (DAPI) included in the culture medium was detected within the now-permeable host cell. DAPI in the culture medium and in the cytoplasm is not seen because its fluorescence is low until it binds to DNA. DAPI fluorescence first appears starting at the rim of the host cell nucleus, then spreads inwards (Fig 6A, S3 Video). The host cell also showed other symptoms of lysing, including the roundup of mitochondria, blebbing and contraction (S2 and S3 Videos). Note that the dynamics of the DAPI binding captures the local nature of the initiation of host cell lysing, as the fluorescence always appears first on the side of the nucleus closer to the parasitophorous vacuole. The nuclei of neighboring uninfected host cells are not labeled by DAPI (Fig 6A), further confirming that the host cell lysing is a parasite driven process. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 6. Calcium ionophore-induced micronemal secretion is not significantly affected in Δpcr2 parasites. A. Images selected from time-lapse experiments of intracellular RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (mE-Pcr2 KI), and knockout (Δpcr2) treated with 5 μM A23187 (also see S3 Video). The cell-impermeant DNA-binding dye, DAPI, was added to the medium to monitor the permeabilization of the host cell. Δpcr2 parasites are able to secrete effectors that lyse the host cell upon A23187 treatment, indicated by DAPI entering the host cell nucleus and binding to DNA, as well as by the dramatic change in the morphology of the host cell (see S2 and S3 Videos). Insets are DAPI images of the nuclear region of the host cell shown at 0.5X. Brackets in the mE-Pcr2 KI panels indicate the host cell nucleus included in the insets. Contrast was adjusted so that the DAPI labeling at the rim of the nucleus is easily visible. The nuclei of uninfected fibroblasts (marked by dashed circles) remained unlabeled by DAPI ~19 min after A23187 treatment as shown in the larger field of view images in the right-hand column. B. Projections of deconvolved wide-field fluorescence images of intracellular WT, mE-Pcr2 KI, Δpcr2, and complemented (Comp) parasites labeled with a mouse anti-MIC2 (red), a rat anti-GAP45 (cyan) and corresponding secondary antibodies. C. Western blots of the secreted (supernatant, S) and unsecreted (pellet, P) fractions of WT, mE-Pcr2 KI, Δpcr2, and complemented (Comp) parasites after A23187 or BAPTA-AM (a calcium chelator; negative control) treatment. The blots were probed by antibodies against MIC2 and GRA8. M: molecular weight markers, the masses of which are indicated in kDa by the numbers on the left. D. Levels of MIC2 in the secreted fractions relative to that from the wild-type in 3 independent biological replicates. For each sample, the MIC2 secretion upon A23187 stimulation is normalized against GRA8 in the pellet from the same sample. Error bars: standard error. https://doi.org/10.1371/journal.ppat.1010776.g006 Efficient host cell lysing suggests functional secretion of micronemal lytic proteins by the parasite. To further confirm this hypothesis, we investigated the impact of Pcr2 knockout on microneme distribution and secretion directly by examining the intracellular distribution and A23187-induced secretion of Micronemal Protein 2 (MIC2), a major transmembrane adhesin important for parasite motility [44,45,63]. Immunofluorescence analysis showed no significant differences in MIC2 distribution between intracellular wild-type and Δpcr2 parasites (Fig 6B). A23187-induced MIC2 secretion from the Δpcr2 parasites is also not significantly different from the wild-type, knock-in and the complemented lines (Fig 6C and 6D). This indicates that the major egress defect seen in the Δpcr2 parasite is not due to the loss of adhesin secretion. For the secretion assays, the dense granule protein GRA8 was used as a control, the secretion of which is negatively regulated by calcium as previously reported for other dense granule proteins [64]. Loss of Pcr2 does not block actomyosin-based motion nor AKMT dynamics Toxoplasma motility is generally thought to be driven by actin polymerization and associated myosin motors [2,6,7,12,39–43]. To investigate the dynamics of actin-containing structures, several groups have used an actin-chromobody tagged with a fluorescent protein (actin-Cb-mE or actin-Cb-GFPTy) and found that the distribution of the tagged actin-Cb is sensitive to changes in intra-parasite calcium concentration, induced by BIPPO (a cGMP phosphodiesterase inhibitor) or the calcium ionophore, A23187 [13,48,66,67]. The redistribution of actin-Cb in response to elevated calcium appears to be dependent on actin polymerization and myosin activity [13,48]. Indeed, in live egress experiments, we observed that A23187 treatment induced an actin-Cb-mE accumulation at the basal end in 100% of the wild-type parasites (n = 127) (Fig 8B). In contrast, the actin-Cb-mE accumulation was not observed in ~ 58% of the Δakmt parasites (n = 125) when treated with A23187. In the ~42% of A23187 treated Δakmt parasites that showed some basal concentration of actin-Cb-mE, the accumulation typically was not nearly as pronounced as observed in wild-type parasites. This is largely in agreement with a prior observation that BIPPO for the most part failed to induce actin-Cb-GFPTy basal accumulation when AKMT was knocked down [13]. We previously discovered that jasplakinolide, which stabilizes actin filaments, compensates the defect in magnitude of the cortical force generated by Δakmt parasites in laser trap measurements [68]. Thus, both lines of evidence suggest that AKMT might regulate parasite motility by controlling actin polymerization. While the Δpcr2 parasites also have a pronounced motility defect, the basal accumulation of actin-Cb-mE occurred in 95% of these parasites (n = 159) when stimulated by A23187. This raises the possibility that Pcr2 acts downstream of or in parallel with the actomyosin machinery and AKMT. Consistent with this idea, we found that the loss of Pcr2 does not affect the apical localization of AKMT, and that the calcium-triggered dispersal of AKMT from the parasite apex [7] still occurs in the Δpcr2 parasites (Fig 8C). 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