Abstract

Toxoplasma gondii is a single-celled eukaryotic parasite with prolific invasion capability. The parasite uses an apical complex comprised of proteinaceous structures and secretory organelles to efficiently enter host cells. As a result, the apical complex remains a vital structure of interest, with many studies dedicated to understanding its protein organization. One such protein is the motor Myosin H (MyoH), which is indispensable for parasite motility and host cell invasion. Given the small size of the complex, roughly a diffraction-limited volume in the visible, high-resolution techniques are required to make precise determinations of protein organization. In this work, we use 3D single-molecule localization microscopy in both traditionally fixed and gel-expanded parasites to localize the indispensable motor MyoH within the apical complex. Labeling of the N- and C-termini of MyoH in fixed parasites resolved the orientation of the motor protein in the apical complex, showing the motor head radially exterior to the tail. Two-color imaging of MyoH with tubulin in fixed parasites allowed for localization of the MyoH termini relative to the conoid, a barrel of tubulin-based fibrils in the apical complex, and showed the MyoH tail toward the interior face of the conoid and the head at the conoid exterior. Gel expansion showed improved labeling density for both tubulin and MyoH but altered MyoH localization, highlighting the nuanced effects of gel expansion on protein organization.

View details for DOI 10.1016/j.bpj.2026.09.012

View details for PubMedID 42723283

Abstract

As a master of host-cell reprogramming, Toxoplasma gondii (T. gondii) tachyzoites manipulate diverse signaling networks to establish a niche permissive for long-term infection. While the parasite's subversion of canonical NF-κB signaling (p65/p50) is well established, how infection impacts the non-canonical NF-κB pathway has been largely unexplored. Here, we report that T. gondii infection induces nuclear accumulation of the non-canonical NF-κB subunits RelB and p52 in both human and murine fibroblasts. This response is conserved across both type I and type II parasite genetic backgrounds. We demonstrate that this reprogramming is dependent on the MYR1-mediated export of dense granule effectors. Mechanistically, T. gondii infection drives the depletion of the negative regulator TRAF3, leading to the stabilization of NF-κB-inducing kinase (NIK), phosphorylation of p100, and its subsequent processing into p52. Utilizing a panel of combinatorial knockout parasites, we reveal that no single effector is responsible for this phenotype. Instead, a suite of eight MYR1-dependent effectors, IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, and GRA84, functions through a collaborative, additive network to drive most of the non-canonical response. These findings highlight a distributed regulatory strategy used by the parasite to overcome host transcriptional robustness and shape host signaling.Toxoplasma gondii infects nearly one-third of the global population and establishes infection by extensively rewiring host immune signaling. While decades of work have focused on how the parasite modulates canonical NF-κB activity, whether it also engages the alternative, non-canonical arm of this pathway has remained unclear. Here, we show that T. gondii tachyzoites activate non-canonical NF-κB signaling, driving nuclear accumulation of RelB/p52 through MYR1-dependent effector export. Unexpectedly, no single effector is responsible. Instead, eight secreted proteins act cooperatively to enable NIK stabilization and engage the non-canonical NF-κB cascade, revealing a networked mode of immune control. This discovery highlights a regulatory logic evolved by the parasite to overcome host transcriptional robustness. Together, these findings identify non-canonical NF-κB activation as a new axis of host-parasite interaction and expand our understanding of how T. gondii reprograms central immune signaling circuits through multi-effector networks.

View details for DOI 10.1128/mbio.01652-26

View details for PubMedID 42663465

Abstract

Toxoplasma gondii is remarkable for its intermediate host range, which includes most warm-blooded animals so far tested. Being such a generalist poses challenges for how the parasite can be optimized for growth in cell types that might be radically different in their metabolism and other aspects, including host defenses and microenvironments. To explore how the parasite might adapt to finding itself in a different host, we started with a Type II line of T. gondii (ME49) that had been grown for at least the past 20 years exclusively in vivo. This line was then used to repeatedly infect two cell lines in vitro, human foreskin fibroblasts and Madin-Darby bovine kidney cells. After at least 70 such passages in one or other host cell type, the resulting lines were compared with respect to growth differences, secretion of an important adhesin (MIC2), and transcriptome. The results showed that passage on these two host cell lines results in profound and reproducible differences in parasite phenotype, including attachment/invasion, MIC2 secretion, and gene expression. The transcriptomic differences were especially pronounced for parasite surface antigen genes. The implications of these results for how T. gondii deals with its breadth of possible intermediate hosts are discussed.

View details for DOI 10.1093/femsmc/xtag006

View details for Web of Science ID 001690839000001

View details for PubMedID 41704854

View details for PubMedCentralID PMC12908928

View details for DOI 10.1038/s41567-024-02652-4

View details for Web of Science ID 001331252700003

Abstract

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

View details for DOI 10.1128/mbio.02864-23

View details for PubMedID 38456679

Abstract

Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.

View details for DOI 10.1093/pnasnexus/pgac183

View details for PubMedID 36329726

View details for PubMedCentralID PMC9615128

Abstract

Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

View details for DOI 10.1073/pnas.2111661119

View details for PubMedID 35121661

Abstract

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCE Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

View details for DOI 10.1128/mBio.00260-21

View details for PubMedID 34749525

Abstract

Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins.IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

View details for DOI 10.1128/mSphere.00858-19

View details for PubMedID 32075880

Abstract

Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.

View details for DOI 10.7554/eLife.54129

View details for PubMedID 32065584