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'The glideosome': A dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii
Abstract
Motion is an intrinsic property of all living organisms, and each cell displays a variety of shapes and modes of locomotion. How structural proteins support cellular movement and how cytoskeletal dynamics and motor proteins are harnessed to generate order and movement are among the fundamental and not fully resolved questions in biology today. Protozoan parasites belonging to the Apicomplexa are of enormous medical and veterinary significance, being responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coccidiosis and cryptosporidiosis. These obligate intracellular parasites exhibit a unique form of actin-based gliding motility, which is essential for host cell invasion and spreading of parasites throughout the infected hosts. A motor complex composed of a small myosin of class XIV associated with a myosin light chain and a plasma membrane-docking protein is present beneath the parasite's plasma membrane. According to the capping model, this complex is connected directly or indirectly to transmembrane adhesin complexes, which are delivered to the parasite surface upon microneme secretion. Together with F-actin and as yet unknown bridging molecules and proteases, these complexes are among the structural and functional components of the 'glideosome'.
... Les complexes d'adhésion entre le parasite et la membrane de la cellule hôte sont déplacés vers le pôle basal du parasite par l'action du moteur actino-myosine, puis clivés au niveau de leurs domaines transmembranaires par les rhomboïdes à activité protéase MPP1, Carruthers, Håkansson, et al., 2000;Corinna Opitz & Soldati, 2002;Rugarabamu et al., 2015;Zhou et al., 2004). Ceci constitue la fin du processus d'invasion, la dissociation entre la membrane du parasite et la PVM. ...
... L'ensemble des protéines impliquées dans cette motilité actine-myosine est appelé le glideosome (Frénal et al., 2010;Corinna Opitz & Soldati, 2002). Ce glideosome s'ancre entre la membrane plasmique du parasite et l'IMC (Keeley & Soldati, 2004). ...
L’invasion de la cellule hôte par les parasites Apicomplexes est médiée par la sécrétion séquentielle et coordonnée de deux types d’organites apicaux spécialisés : les micronèmes et les rhoptries. Les mécanismes de l’exocytose de ces organites restent peu connus. Les Apicomplexes font partie, avec les Ciliés et les Dinoflagellés, du phylum des Alveolés. Les Alveolés, bien qu’ayant de substantielles différences morphologiques, partagent notamment la présence d’organites sécrétoires. Chez Paramecium tetraurelia, un Cilié, de précédentes études ont permis d’observer que l’exocytose des trichocystes, leurs organites sécrétoires, pouvait être induite par diverses stimulations. En analysant des mutants déficients pour l’exocytose, nommés Nd pour « non-discharge », ces études ont identifié des composants essentiels du système de fusion membranaire.Notre équipe a démontré précédemment la conservation des gènes Nd6 et Nd9 chez Toxoplasma et Plasmodium et leur implication dans l’invasion et la sécrétion des rhoptries. Nd6 et Nd9 appartiennent à un complexe comprenant deux autres facteurs également conservés chez les Ciliés et que l’équipe a montré nécessaire à la sécrétion des rhoptries et des organelles de sécrétion d’un autre cilié Tetrahymena. Ces résultats supportent l’hypothèse d’un mécanisme d’exocytose des rhoptries conservé chez les Alveolés.Chez Tetrahymena, les gènes impliqués dans la biogenèse et la sécrétion des organites de défense sont exprimés au cours du cycle de façon synchronisée. Dans un criblage bio-informatique, nous avons identifié les gènes de Tetrahymena co-régulés avec le gène Nd6 conservés chez les Apicomplexes. Nous avons démontré que deux protéines de Toxoplasma gondii, appelées Nd11 et Nd12 et identifiées par ce criblage, sont impliquées dans l’invasion, et que l’incapacité à envahir en l’absence de ces protéines pourrait être liée à une perte de la capacité à secréter les rhoptries. Nd11 et Nd12 sont localisées dans le cytoplasme de manière ponctuée et possèdent de nombreux domaines transmembranaires ainsi que des domaines prédits d’interaction avec des protéines ou des sucres. Par Co-immunoprécipitation et spectrométrie de masse, nous avons démontré que Nd11 et Nd12 interagissaient entre elles et avec deux autres protéines inconnues, impliquées dans les mécanismes d’invasion et de sécrétion des rhoptries et possédant des domaines de liaison aux sucres. Notre étude supporte l’hypothèse de la conservation des mécanismes de sécrétion au sein des Alveolata et valide l’utilité des études comparatives entre espèces comme stratégie d’identification d’effecteurs de l’exocytose chez les Apicomplexes. En outre cette étude a permis l’identification d’un nouveau complexe protéique essentiel à la sécrétion des rhoptries chez T. gondii.
... Over the years, many labs have characterized key proteins involved in parasite motility [2,6,7,12,13,[39][40][41][42][43][44][45][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. ...
... How this dynamic interplay between imposed load and motor output occurs remains an unexplored area, as the outcome of manipulating the previously known motilityrelevant genes has been largely binary; i.e., motile vs. immotile [7,12,13,[41][42][43]46]. While the current model [47] provides a valuable framework connecting the functions of many proteins involved in motility (e.g. the IMC-anchored myosin motors for generating internal force, actin polymerization for providing tracks for the motor, actin-binding adapter proteins for linking the actomyosin complex to the transmembrane adhesins), it reflects the binary nature of the genetic manipulation experiments in that it describes a 2-state motility apparatus: the motor is always either "ON" or "OFF". ...
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.
... Cysts indeed proved to be a remarkable natural source of genomic DNA. Gliding is a characteristic apicomplexan movement that also happens to be essential for the invasion and egress of host cells, and thus for the intracellular parasitic lifestyle [20][21][22][23][24]. P. gigantea trophozoites are known to glide at rates of up to 60 μm/s [25], so are prime candidates in which to study the mechanism of gliding motility. ...
... Gliding motility is an essential feature of apicomplexans, and for some intracellular parasites among them, glideosome proteins have been shown to be crucial for host cell invasion and egress [22,23,26,63,74]. However, our sequence analysis of the glideosome components shows that the currently known mechanistic model based on T. gondii and P. falciparum does not fully account for gliding in all apicomplexans, as anticipated [26,63,67]. ...
Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~ 9 Mb) and coding capacity (~ 5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16–38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sisters to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions such as gliding, well known to allow access to the intracellular parasitic lifestyle in Apicomplexa.
... The IMC is known to carry out three major functions in infection of host cells and intracellular replication. First, it hosts the glideosome, an actin-myosin motor that interacts with adhesins secreted onto the parasite's surface for motility and invasion (8). Second, it serves as a scaffold for the formation of daughter cells via the internal budding process known as endodyogeny (6). ...
... The IMC is able to carry out its diverse functions by partitioning the organelle into distinct subcompartments, each containing its own cargo of proteins (6,7). The glideosome components that power motility are localized to the membrane vesicles of the IMC body and apical cap, with the motor facing the plasma membrane to tether to secreted micronemal adhesins (8). The apical cap portion of the organelle hosts the AC9/AC10/ERK7 complex, which regulates the stability of the conoid, which in turn is essential for release of the micronemes and rhoptries for attachment and penetration, respectively (9,10). ...
The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identified here a novel protein that localizes to discrete puncta that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis showed that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then used deletion analyses to identify a domain in the N terminus of the protein that is critical for both localization and function. Finally, we used the protein as bait for in vivo biotinylation, which identified several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, these data reveal an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggest that the suture puncta are likely a portal for trafficking cargo across the IMC. IMPORTANCE The inner membrane complex (IMC) is a peripheral membrane and cytoskeletal system that is organized into intriguing rectangular plates at the periphery of the parasite. The IMC plates are delimited by an array of IMC suture proteins that are tethered to both the membrane and the cytoskeleton and are thought to provide structure to the organelle. Here, we identified a protein that forms discrete puncta that are embedded in the IMC sutures, and we show that it is important for the proper sorting of a group of IMC suture proteins as well as maintaining parasite shape and IMC cytoskeletal integrity. Intriguingly, proximity labeling experiments identified several proteins that are involved in membrane trafficking or endocytosis, suggesting that the IMC puncta provide a gateway for transporting molecules across the structure.
... This actin-myosin motor is known as the glideosome which is conserved in the Apicomplexan phylum and empowers the parasites motility, invasion and egress (Frénal et al., 2017;Jacot et al., 2016;Keeley & Soldati, 2004;Santos et al., 2009). The system is fixed on the inner membrane complex (Opitz & Soldati, 2002) and connected to actin filaments that pull adhesin molecules (MIC), located on the plasma membrane of the parasite but fixed to host cell (for invasion) or substrate (for gliding), leading to their anterior-toposterior translocation to drive parasite motion ( Figure 6A). Thus, it is thought that the actinmyosin motor of parasite powers invasion ( Figure 6A) and the MJ anchors it to the host cell ( Figure 6B) (detailed in sections 1.8.1.1 and 1.8.2.1). ...
... The glideosome is located between the inner membrane complex (IMC) and the plasma membrane (PM) at the pellicle (Opitz & Soldati, 2002). A class XIV myosin A (TgMyoA), a myosin light chain (TgMLC1) and three additional gliding-associated integral membrane glycoprotein TgGAP45, TgGAP50 and TgGAP40 form the glideosomal structure (Frénal et al., 2010;Gaskins et al., 2004). ...
Toxoplasmosis is classified as a neglected parasitic infection necessitating public health control. It is due to Toxoplasma gondii, an obligatory intracellular parasite. One route of infection occurs following the oral ingestion of bradyzoite tissue cysts. Thereupon, bradyzoites invade enterocytes and convert rapidly to tachyzoites, leading to the acute infection. Under the tight control of host immune response, tachyzoites transform back into bradyzoites which sustain a lifelong chronic disease. If left untreated, chronic toxoplasmosis may reactivate and become fatal in immunocompromised patients. Invasion of Toxoplasma involves the formation of a tight connection between the parasite and the host cell membranes, referred to a structure called Moving Junction (MJ). In tachyzoites, the MJ is shaped by the assembly of a microneme protein, AMA1, and a rhoptry neck protein, RON2, as part of a complex involving additional RONs. Whilst the MJ process is well characterized in tachyzoites, invasion in bradyzoites remains underexplored. Here, we demonstrated that tachyzoite MJ proteins are also expressed at the MJ of invading bradyzoites, showing for the first time that bradyzoites do form a MJ during their invasion. We also characterized more paralogs of MJ proteins, which are barely expressed in tachyzoites, and demonstrated that AMA2, AMA4 and RON2L1, are highly expressed in bradyzoites. Remarkably, we found AMA4 and AMA2 at the MJ of invading bradyzoites, highlighting that bradyzoite MJ exhibits a different protein composition than that of invading tachyzoites. Moreover, we generated AMA4 knockout parasites and unraveled the importance of AMA4 for invasion in tachyzoites in some extend, but its predominant role for bradyzoite invasion and for establishing the chronic phase of infection.In light of the absence of an approved vaccine against human toxoplasmosis and the promises of a vaccine strategy based on immunization with AMA1-RON2 complex against Plasmodium, we built on the importance of MJ in invasion of both tachyzoite and bradyzoite stages, and interrogated the potential protection against toxoplasmosis by targeting MJ complexes. Immunization with recombinant AMA1 protein in complex with a RON2 peptide, revealed a high protective efficacy both after oral challenge of mice with bradyzoites (≈80% reduction of cysts burden) or intraperitoneal challenge with a lethal dose of tachyzoites (80% increased mice survival). Yet, AMA4 in complex with its binding RON2L1 peptide did not protect against acute infection, a consistent result with its low expression and function in tachyzoites. In contrast, supporting its role in the invasion of bradyzoites, AMA4-RON2L1 complex mainly protected against chronic infection. In both cases, immunization with AMA-RON2 complexes is more efficient than immunization with AMA alone. Finally, IgG against either complexes inhibited MJ formation and invasion in vitro, suggesting that the inhibition of invasion may be one plausible mechanism for in vivo protection. Importantly, antibodies against AMA1-RON2 are more efficient to inhibit the invasion of tachyzoites while antibodies against AMA4-RON2L1 target preferentially the invasion of bradyzoites; both supporting a predominant role of AMA1-RON2 in tachyzoites and AMA4-RON2L1 in bradyzoites. Altogether, these results validate different MJ complexes as potential vaccine candidates to protect against toxoplasmosis.
... They include proteins with perforin-like properties, adhesins, and serine proteases (subtilisins) [39,55]. Some of the micronemal proteins are involved in the assembly (together with rhoptry proteins) of the moving junction [41,56,57]. Morphological analysis has shown that the number of micronemes is higher in sporozoites, lower in bradyzoites, and intermediate in tachyzoites [3,42]. ...
... Intermediate hosts can be infected through several pathways (Fig. 1). Both tissue cyst and oocysts walls are removed by digestive enzymes, liberating, respectively, bradyzoites or sporozoites that inside the new host, move by a unique mechanism of gliding [24,56]. Micronemal proteins are the first to be secreted and are essential for protozoan motility by gliding and initial adhesion to the host cell surface. ...
Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies.
Graphical Abstract
... In addition, one of the important roles of the IMC is to act as an anchor for the actin-myosin 376 motor complex which is necessary for both parasite invasion and egress (58,59). GAP40 and 377 GAP50 are glideosome-associated proteins that stably anchor the motor complex to the IMC (52, 378 60, 61), which is significantly downregulated in TgAP2XII-9 deficient parasites and is directly 379 controlled by TgAP2XII-9. ...
Toxoplasma gondii is an intracellular parasitic protozoan that poses a significant risk to pregnant women and immunocompromised individuals. T. gondii tachyzoites duplicate rapidly in host cells during acute infection through endodyogeny. This highly regulated division process is accompanied by complex gene regulation networks. TgAP2XII-9 is a cyclical transcription factor, but its specific role in the parasite cell cycle is not fully understood. Here, we demonstrate that TgAP2XII-9 is identified as a nuclear transcription factor and is dominantly expressed during the S/M phase of the tachyzoite cell cycle. CUT&Tag results indicate that TgAP2XII-9 targets key genes for the moving junction machinery (RON2, 4, 8) and daughter cell inner membrane complex (IMC). TgAP2XII-9 deficiency resulted in a significant downregulation of rhoptry proteins and rhoptry neck proteins, leading to a severe defect in the invasion and egress efficiency of tachyzoites. Additionally, the loss of TgAP2XII-9 correlated with a substantial downregulation of multiple IMC and apicoplast proteins, leading to disorders of daughter bud formation and apicoplast inheritance, and further contributing to the inability of cell division and intracellular proliferation. Our study reveals that TgAP2XII-9 acts as a critical S/M-phase regulator that orchestrates the endodyogeny and apicoplast division in T. gondii tachyzoite. This study contributes to a broader understanding of the complexity of the parasite's cell cycle and its key regulators.
... Parasites lacking GAP40 or 315 GAP50 start replication but fail to complete it, implicating a structural role in maintaining the 316 stability of the developing IMC during replication(Harding et al, 2016). It was shown that 317 IMC is critical for the anchorage and stabilization of the glideosome(Opitz & Soldati, 2002) 318 and is required during the invasion of the host cell (Bargieri et al, 2013; Egarter et al, 2014; 319 Togbe et al, 2008; Meissner et al, 2013) ...
Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.
... Chemical inhibition, as well as genetic manipulations, have shown that actin and a series of apical and cortical myosin motors provide the underlying force for the parasite motility (Dobrowolski et al., 1997;Gaskins et al., 2004;Graindorge et al., 2016;Heaslip et al., 2011;Meissner et al., 2002). The current model posits that the internal force is transmitted to the parasite surface through a coupling between the actomyosin complex and the secreted transmembrane adhesins (Opitz and Soldati, 2002). The characterization of these factors has significantly advanced the understanding of parasite motility (Dobrowolski et al., 1997;Frenal et al., 2010;Gaskins et al., 2004;Graindorge et al., 2016;Heaslip et al., 2011;Huynh and Carruthers, 2006;Huynh et al., 2003;Jacot et al., 2016;Meissner et al., 2002;Tosetti et al., 2019). ...
Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3-D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here we address the role of the cortical microtubules in parasite motility, invasion, and egress by utilizing a previously generated mutant (dubbed "TKO") in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in∼80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization is further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication is not affected. In a 3-D Matrigel matrix, the TKO mutant moves directionally over long distances, but along trajectories significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory does not impact either movement speed in the matrix or the speed and behavior of the parasite's entry into and egress from the host cell.
... This process is driven by the actin-myosin system, which is located underneath the plasma membrane. The actin-myosin system termed glideosome is formed by the interaction of the apical complex, the actin cytoskeleton, myosin motor proteins, and the associated proteins [20][21][22] . The gliding motility comprises three movement types: circular gliding, upright twirling, and helical rotation 23,24 . ...
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in human and warm-blood organisms. Cyclic nucleotide signaling is crucial for the successful intracellular survival and replication of the parasites. Here, we dissected the physiological and biochemical importance of the essential phosphodiesterases (PDEs) in Toxoplasma gondii tachyzoite. By C-terminal tagging of 18 PDEs, we detected the expression of 11 PDEs. Immunogold staining revealed that TgPDE1, TgPDE2 and TgPDE9 are distributed throughout the parasite body, including the inner membrane complex, the apical pole, the plasma membrane, the cytosol, dense granules, and rhoptry, suggesting the spatial control of signaling within tachyzoites. Subsequently, we identified that most enzymes are notorious dual-specific phosphodiesterases, and TgPDE2 is cAMP specific differently, whilst T.gondii lacks of cGMP specific phosphodiesterase. Our enzyme kinetic data demonstrated that the highest affinity to its substrate belongs to TgPDE2, while the dual PDEs (TgPDE1, TgPDE7 and TgPDE9) have higher affinity with cGMP than cAMP. Inhibition screening of commonly-used PDE inhibitors on TgPDEs, signifying TgPDE1 as the target of BIPPO and zaprinast. Furthermore, the biological significance revealed TgPDE1 and TgPDE2 are individually necessary for parasite growth, and their loss associatively results in parasite death, implying their functional redundancy. In addition, we identified kinases and phosphatases within the TgPDE1 and TgPDE2 interactomes, which may operate the enzymatic activity via protein-protein interactions or posttranslational modifications. Collectively, our findings on subcellular localization, catalytic function, drug inhibition, and physiological relevance of major phosphodiesterases highlight the unforeseeable plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii. The data set of cAMP-binding interactors, which we disclosed in another aspect of this study, will provide valuable insight into the pervasive nature of cAMP-mediated signaling in T. gondii tachyzoites.
... During the invasion, micronemes (MICs) release their contents that are required for motility, host cell attachment and egress (Dubois and Soldati-Favre, 2019). MIC proteins (adhesins and escorters) form complexes that link host cell receptors to the glideosome (Opitz and Soldati, 2002). These complexes required for invasion are translocated backwards, allowing the parasite to propel itself into the host cell by membrane invagination. ...
Rhoptries and micronemes are essential for host cell invasion and survival of all apicomplexan parasites, which are composed of numerous obligate intracellular protozoan pathogens including Plasmodium falciparum (malaria) and Toxoplasma gondii (toxoplasmosis) that infect humans and animals causing severe diseases. We identified Toxoplasma gondii Tg SORT as an essential cargo receptor, which drives the transport of rhoptry (ROP) and microneme (MIC) proteins to ensure the biogenesis of these secretory organelles. The luminal domain of 752 amino acid long situated at the N-terminus end of TgSORT has been described to bind to MIC and ROP proteins. Here, we present an optimized protocol for expression of the entire luminal N-terminus of TgSORT (Tg-NSORT) in the yeast Pichia pastoris . Optimization of its coding sequence, cloning and transformation of the yeast P. pastoris allowed the secretion of Tg-NSORT. The protein was purified and further analyzed by negative staining electron microscopy. In addition, molecular modeling using AlphaFold identified key differences between the human and the T gondii sortilin. The structural features that are only present in T. gondii and other apicomplexan parasites were highlighted. Elucidating the roles of these specific structural features may be useful for designing new therapeutic agents against apicomplexan parasites
... Thus far, the IMC has three known functions. First, the IMC is an anchor for the glideosome, an actomyosin motor complex that powers gliding motility, which is necessary for both parasite invasion and egress (16,17). Second, the apical cap of the IMC plays critical roles in stabilizing the apical complex to govern motility, invasion, and egress (9,10,18). ...
The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δimc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.
... ROMs have been demonstrated to be crucial for the invasion of some apicomplexan parasites due to their ability to cleave adhesins from the surface of the parasites, allowing them entry into the host cell completely (32)(33)(34)(35)(36). For instance, it has been confirmed that TgMIC2, TgMIC6, TgMIC12, and TgAMA1 were hydrolyzed by ROMs during the invasion of T. gondii, and PfEBA-175, PfAMA1, PfRh1, PfRh4, and PfTRAP was hydrolyzed by ROMs during the invasion of Plasmodium falciparum (32). ...
Introduction
Avian coccidiosis, caused by apicomplexan protozoa belonging to the Eimeria genus, is considered one of the most important diseases in the intensive poultry industry worldwide. Due to the shortcomings of live anticoccidial vaccines and drugs, the development of novel anticoccidial vaccines is increasingly urgent.
Methods
Eimeria maxima rhomboid-like protein 1 (EmROM1), an invasion-related molecule, was selected as a candidate antigen to evaluate its protective efficacy against E. maxima in chickens. Firstly, the prokaryotic recombinant plasmid pET-32a-EmROM1 was constructed to prepare EmROM1 recombinant protein (rEmROM1), which was used as a subunit vaccine. The eukaryotic recombinant plasmid pVAX1.0-EmROM1 (pEmROM1) was constructed as a DNA vaccine. Subsequently, 2-week-old chicks were separately vaccinated with the rEmROM1 and pEmROM1 twice every 7 days. One week post the booster vaccination, induced cellular immune responses were determined by evaluating the mRNA level of cytokines including IL-2, IFN-γ, IL-4, IL-10, TGF-β, IL-17, and TNFSF15, as well as the percentages of CD4⁺ and CD8⁺ T cells from spleens of vaccinated chickens. Specific serum antibody level in the vaccinated chickens was determined to assess induced humoral immune responses. Finally, the protective efficacy of EmROM1 was evaluated by a vaccination-challenge trial.
Results
EmROM1 vaccination significantly upregulated the cytokine transcription levels and CD4⁺/CD8⁺ T cell percentages in vaccinated chickens compared with control groups, and also significantly increased the levels of serum-specific antibodies in vaccinated chickens. The animal trial showed that EmROM1 vaccination significantly reduced oocyst shedding, enteric lesions, and weight loss of infected birds compared with the controls. The anticoccidial index (ACI) from the rEmROM-vaccination group and pEmROM1-vaccination group were 174.11 and 163.37, respectively, showing moderate protection against E. maxima infection.
Discussion
EmROM1 is an effective candidate antigen for developing DNA or subunit vaccines against avian coccidiosis.
... The machinery involved in the gliding movement is termed the glideosome and is responsible for powering the parasite's motility, migration, invasion, and egress from the host cell. It is situated between the plasma membrane from one side and the IMC from the other and is conserved among members of the Apicomplexa (Opitz & Soldati, 2002). The glideosome consists of Myosin A (TgMyoA), the associated myosin light chain1 (TgMLC1) (Herm-Gotz, 2002), the essential light chain (TgELC1) (Bookwalter et al., 2014;Williams et al., 2015) and three glidingassociated proteins GAP45, GAP50 (Gaskins et al., 2004), and GAP40 (Frénal et al., 2010). ...
During its complex life cycle, the T. gondii parasite differentiates into distinct developmental stages. The transition between these life stages is associated with changes in the parasite's transcriptome. In Toxoplasma, it has been demonstrated that specific transcription factors of the ApiAP2 family play a role in regulating gene expression during these developmental transitions. ApiAP2 transcription factors are characterized by the presence of an AP2 domain. These AP2 TFs possess an Apetala2/ERF integrase DNA binding domain similar to those of plants. While certain ApiAP2 TFs have been demonstrated to play a role in controlling the tachyzoite to bradyzoite developmental transition, several remain unstudied. Thus, for the first part of this thesis, the role of two constitutively expressed ApiAP2 TFs, TgAP2X-10 and TgAP2III-1 was studied. The effect of TgAP2X-10 and TgAP2III-1 on bradyzoite differentiation was determined using two in vitro models. We also study the effect of TgAP2X-10 and TgAP2III-1 loss in vivo in mice and demonstrate that these proteins might be potential regulators of bradyzoite differentiation. In an attempt to continue with the characterization of ApiAP2 TFs, a third ApiAP2 TF, TgAP2IX-5 was studied. The study of TgAP2IX-5 represents the second part of this PhD project. TgAP2IX-5 was demonstrated to have a role in asexual cell cycle division of the T. gondii tachyzoite. The conditional depletion of TgAP2IX-5 blocks the progression of the cell cycle at a precise time-point when the plastid is elongated before its segregation. By using RNA-seq, we determined that TgAP2IX-5 differentially regulates hundreds of genes, a majority of which are targeted to the Inner Membrane Complex (IMC) and apical complex. ChIP-seq allowed to identify the promoters of hundreds of genes targeted by TgAP2IX-5 which are necessary for the progression of the budding cycle. In addition, TgAP2IX-5 was demonstrated to activate known bradyzoite repressors. Strikingly, we demonstrate that the re-expression of TgAP2IX-5 re-initiates cell cycle division, yet the parasite switches its mode of division from endodyogeny to endopolygeny. Further studies regarding the elucidation of the different regions of the TgAP2IX-5 protein were carried out and a novel domain was identified and shown to be essential for the function of TgAP2IX-5. In the third and final part of this PhD study, we demonstrated the crucial role of the TgPP1 phosphatase in the cell cycle. The production of the daughter cell IMC and the nuclear cycle is affected in absence of this protein. Phospho-proteomics analysis revealed that the depletion of TgPP1 results in the differential phosphorylation of several IMC proteins. Overall, this study suggests that TgPP1 is important for controlling phosphorylation events within the tachyzoite's cell cycle.
... Simultaneously, they demonstrated that TgMyoA is critical for parasite motility and host cell invasion (Meissner et al., 2002b). Finally, the name "glideosome" was proposed by Dominique's lab to describe this new and unique actomyosin system (Opitz and Soldati, 2002). Together with other labs, the team then identified and functionally characterized the glideosome components (Gaskins et al., 2004;Freńal et al., 2010;Nebl et al., 2011;Williams et al., 2015;Jacot et al., 2016), as well as regulators of actin dynamics (Plattner et al., 2008;Mehta and Sibley, 2010;Daher et al., 2010;Yadav et al., 2011;Salamun et al., 2014;Jacot et al., 2016). ...
... For intracellular parasites, efficient entry of host cells is essential to parasitize a wide range of host cells. Toxoplasma can parasitize almost any warm-blooded animal because of its unique gift to attach to (38,44), and the glideosome-centered motility system provides a strong impetus for entering the host cell (45). Recent studies have recognized that Toxoplasma attachment to host cells appears to be regulated by more than just microneme secretion. ...
Infection with Toxoplasma gondii can lead to severe and even life-threatening diseases in people with compromised or suppressed immune systems. Unfortunately, drugs to combat the parasite are limited, highly toxic, and ineffective against the chronic stage of the parasite.
... This is done through a type of cellular movement without shape deformation known as gliding motility, which includes three movement types: circular gliding, upright twirling, and helical rotation [119,120]. Gliding is achieved from the interaction of the apical complex, the actin cytoskeleton, myosin motor proteins, and associated proteins which together form the glideosome [121][122][123][124][125]. Endocytosis is involved in retrograde motility [126]. ...
Toxoplasma gondii is a ubiquitous zoonotic parasite with an obligatory intracellular lifestyle. It relies on a specialized set of cytoskeletal and secretory organelles for host cell invasion. When infecting its felid definitive host, T. gondii undergoes sexual reproduction in the intestinal epithelium, producing oocysts that are excreted with the feces and sporulate in the environment. In other hosts and/or tissues, T. gondii multiplies by asexual reproduction. Rapidly dividing tachyzoites expand through multiple tissues, particularly nervous and muscular tissues, and eventually convert to slowly dividing bradyzoites which produce tissue cysts, structures that evade the immune system and remain infective within the host. Infection normally occurs through ingestion of sporulated oocysts or tissue cysts. While T. gondii is able to infect virtually all warm-blooded animals, most infections in humans are asymptomatic, with clinical disease occurring most often in immunocompromised hosts or fetuses carried by seronegative mothers that are infected during pregnancy.
... In addition, the parasite has a cortical actin and myosin cytoskeleton, called "glideosome" located between the plasma membrane and the IMC, which promotes parasitic motility (Opitz and Soldati, 2002). It consists of actin, myosin A, and IMC-anchoring proteins (GAPs). ...
Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into the host cell. These factors are contained in specific secretory organelles, the rhoptries (ROP), micronemes (MIC) and dense granules (DG) that release their content upon host adhesion and active invasion. DG proteins (GRA) are also secreted in a so called « constitutive manner » during parasite replication and play a crucial role in modulating host responses, ensuring parasite survival and dissemination. While the molecular mechanisms regulating ROP and MIC protein release during parasite invasion have been well studied, constitutive secretion of DG remains a fully unexplored aspect of T. gondii vesicular trafficking. During this thesis, we first investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. We demonstrated that during parasite replication, TgRab11A regulates actin-dependent DG motion and stimulates the final step of their exocytosis at the parasite plasma membrane and therefore GRA protein release in the vacuolar space and host cytosol. Moreover, we demonstrated a novel function for TgRab11A in the early steps of parasite adhesion to host cells and parasite motility, and thus host cell invasion. In agreement with these findings, the secretion of the MIC2 adhesin was severely perturbed in extracellular TgRab11A-defective parasites. Strikingly, extracellular adhering and invading parasites exhibited an apically polarized and focalized accumulation of TgRab11A-positive vesicles, suggesting a role for TgRab11A in early secretory events triggered during parasite invasion. Collectively, our data revealed TgRab11A as a crucial regulator of the constitutive secretory pathway in T. gondii. In a second part of this thesis, we functionally characterized a novel TgRab11A-binding partner, containing a unique HOOK-domain, that we called TgHOOK. We found that this protein forms a stable complex with a homologue of the Fused Toes (FTS) protein and a newly identified HOOK Interacting Protein (HIP) specific to coccidian parasites. HOOK and FTS are two conserved endosomal trafficking regulators known to promote vesicle trafficking and/or fusion in other eukaryotes. In T. gondii, we found that the TgHOOK-TgFTS-HIP complex accumulates at the apical tip of parasites and promotes microneme secretion, thereby contributing to parasite invasion.
... sliding movement generated by the action of an actin-myosin motor) necessary for the motility of the zoite and its invasion and egress of/from the host cell and has been mostly described for species displaying intracellular life modes(Opitz and Soldati, 2002; Keeley and Soldati, 2004). Recently several articles have brought together the latest results of studies about the different components of this machinery and have updated the different understandings of this molecular architecture(Boucher and Bosch, 2015;Tardieux and Baum, 2016; Frénal et al., 2017). ...
Apicomplexan are unicellular eukaryotic microorganisms that have evolved towards strict parasitic lifestyle. Some apicomplexan groups include species that cause serious pathologies such as malaria (Plasmodium ssp.), toxoplasmosis (Toxoplasma gondii) and cryptosporidiosis (Cryptosporidium spp.). While the genomes of these highly pathogenic agents are now well documented, this is not the case for other apicomplexan lineages such as gregarines, which are considered basal within the Apicomplexa, have low pathogenicity and above all are non-cultivable. Their molecular study currently represents a major bottleneck, whereas a precise knowledge of their genomes would be essential to better understand the evolutionary history of apicomplexan parasites and the diversity of their adaptive paths to parasitic lifestyle. During this thesis the genome caracterisation of 2 marine gregarines, Porospora gigantea, parasite of the European lobster Homarus gammarus and Diplauxis hatti, parasite of the Polychaeta marine worm Perinereis cultrifera; and 1 terrestrial gregarine, Gregarina acridiorum, parasite of the locust Locusta migratoria have been carried out. The discovery of two coexisting genomes matching the morphologically described species P. gigantea, along with another example involving G. acridiorum illustrates the magnitude of the upcoming taxonomic revisions, and the need to turn to molecular markers, likely on a genomic scale, to properly assess the diversity of gregarines. Furthermore, the first comparative genomics analyses including gregarines reveal their unsuspected genetic diversity across Apicomplexa. An apicomplexan scale analyses of the glideosome proteins was also performed. This model refers to a complex molecular structure at the origin of gliding, a signature movement of Apicomplexa that is essential for the manifestation of their pathogenicity. A detailed comparative analysis highlights its differential conservation at the apicomplexan scale, suggesting a diversity of adaptations to motility and host cell invasion issues. This study illustrates the importance of considering non-model, non-pathogenic, non-cultivatable apicomplexan to provide novel clues to the adaptive capabilities displayed by this ecologically and medically major group of parasites.
... Gliding has perhaps been best described for Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, both of which exhibit helical motion in three-dimensional (3D) matrices, as well as during host cell entry, that is driven by an actomyosin motor (10)(11)(12). Components of this motor, collectively called the "glideosome," are located within the space between the parasite inner membrane complex (IMC) and plasma membrane; they are generally conserved between apicomplexan zoites, including merozoites (11,13,14). According to the linear motor model, gliding is a substrate-dependent process. ...
Significance
Plasmodium malaria parasites use a unique substrate-dependent locomotion, termed gliding motility, to migrate through tissues and invade cells. Previously, it was thought that the small labile invasive stages that invade erythrocytes, merozoites, use this motility solely to penetrate target erythrocytes. Here we reveal that merozoites use gliding motility for translocation across host cells prior to invasion. This forms an important preinvasion step that is powered by a conserved actomyosin motor and is regulated by a complex signaling pathway. This work broadens our understanding of the role of gliding motility and invasion in the blood and will have a significant impact on our understanding of blood stage host–pathogen interactions and parasite biology, with implications for interventions targeting erythrocyte invasion.
... The TgMyoA is found tightly associated with the IMC in a hetero-oligomeric complex that was then called glideosome (Opitz & Soldati, 2002). In addition to TgMyoA, a myosin light chain (TgMLC1), two essential light chains (ELC1 and ELC2) and three additional partners, TgGAP40, TgGAP45 and TgGAP50 compose the glideosome (Gaskins et al, 2004) (Frénal et al, 2014). ...
Toxoplasma gondii is a cosmopolite obligate intracellular Apicomplexa parasite that infects a wide repertoire of warm-blooded animals and virtually all nucleated cells. About a third of the human population carries the persistent stage of T. gondii, and is known at risk for life-threatening toxoplasmosis in case of immune-dysfunction. The invasiveness of the T. gondii tachyzoite developmental stage is a key determinant for expansion of the parasite population and accounts for the initiation of acute tissue damages associated with the disease. The tachyzoite is a several micrometer size bow-shaped cell that displays a robust polarity and is equipped with a typical apical apparatus made of cytoskeletal arrangements and specific secretory vesicles. With these attributes, the tachyzoite contact the host cell surface with the apical side and enters within a second time-scale into a budding entry vesicle by injecting a protein complex into and beneath the facing plasma membrane. The complex, seen here as an invasive nanodevice, defines a tight zoite-cell interface that bridges both cells through a circular junction. This tight Zoite-Cell Junction (ZCJ) serves therefore as a door of entry but also as an anchor point to withstand the parasite invasive force required to actively enter the host cell. In addition, its tightness acts as a molecular sieve to select for components from the plasma membrane able to flow into the budding entry vesicle.The PhD thesis brings new insights on the forces underlying (i) the peculiar mode of locomotion called helical gliding of free tachyzoite (ii) the host cell invasion event in particular at the end of the process thereby introducing conceptual and experimental biophysics framework. The first part combines high-speed quantitative live microscopy with force microscopy and Reflection Interference Contrast Microscopy and use micropatterning. These quantitative approaches have allowed unveiling the spatiotemporal integration of a unique polar anchoring adhesion and the traction-spring-torque triad forces that set the Toxoplasma thrust force required for high-speed helical gliding.The second part of the PhD relies on the quantitative high speed live imaging and on a set of both parasite and host cell lines engineered to express fluorescent markers of interest, in particular related to the ZCJ element, together with innovative invasion assays designed to monitor in detail the poorly documented pinching off step of the budding entry vesicle. Indeed this membrane fission event promotes the birth of a bona fidae sub-cellular compartment enclosing the tachyzoite, and further remodeled to support parasite growth. These approaches have allowed identifying the peculiar rotation of the tachyzoite along the long axis which imposes a twisting motion on the parasite basal pole and directs closure of the circular invasive device therefore promoting both sealing and release of the entry vesicle. Importantly membrane fission occurs upstream the site of the nanodevice insertion and is independent of the host cell mechanoenzymes dynamins, a protein family primarily involved in pinching off of the endocytic pits and thus in endosome birth. Overall, the work supports the view that the tachyzoite has evolved a multifunction invasive nanodevice, which together with the final torque mimics the fission activity of the dynamins. Finally, monitoring distinct host cell plasma markers and their rapid reorganization upon the tachyzoite twist allowed proposing that the latter could also act as an initial mechanical trigger for the transition to the intracellular lifestyle.In conclusion, this PhD work has succeeded in implementing new biophysics-based concepts and techniques to start unraveling the biomechanics of the T. gondii tachyzoite, in particular in the context of essential behaviors including (i) the navigation on 2D and within 3D substrates and (ii) the host cell invasion process.
... The best studied group of IMC proteins are components of the motor complex that drives the locomotion of all motile parasite stages-also referred to as the "glideosome" ( Table 1) (Webb et al., 1996;Opitz and Soldati, 2002;Baum et al., 2006). This actin-myosin motor complex powers the motility needed for transmigration, gliding, invasion and potentially egress (Freńal et al., 2010); the physiological trademark of the motile merozoite, sporozoite and ookinete stages of parasite development. ...
Apicomplexan parasites, such as human malaria parasites, have complex lifecycles encompassing multiple and diverse environmental niches. Invading, replicating, and escaping from different cell types, along with exploiting each intracellular niche, necessitate large and dynamic changes in parasite morphology and cellular architecture. The inner membrane complex (IMC) is a unique structural element that is intricately involved with these distinct morphological changes. The IMC is a double membrane organelle that forms de novo and is located beneath the plasma membrane of these single-celled organisms. In Plasmodium spp. parasites it has three major purposes: it confers stability and shape to the cell, functions as an important scaffolding compartment during the formation of daughter cells, and plays a major role in motility and invasion. Recent years have revealed greater insights into the architecture, protein composition and function of the IMC. Here, we discuss the multiple roles of the IMC in each parasite lifecycle stage as well as insights into its sub-compartmentalization, biogenesis, disassembly and regulation during stage conversion of P. falciparum.
... The actin and myosin are the essential components of glideosome apparatus of Plasmodium parasite which are located between the PPM and IMC. This apparatus is known to promote parasite gliding motility and enable the parasite egress from the host cell (Opitz & Soldati, 2002). Tubulins are essential subcellular components of eukaryotes which are involved in chromosome segregation, cytoskeletal architecture motility and transport phenomena (Cleveland & Sullivan, 1985;MacRae & Langdon, 1989). ...
Prefoldin (PFD) is a heterohexameric molecular chaperone which bind unfolded proteins and subsequently deliver them to a group II chaperonin for correct folding. Although there is structural and functional information available for humans and archaea PFDs, their existence and functions in malaria parasite remains uncharacterized. In the present review, we have collected the available information on prefoldin family members of archaea and humans and attempted to analyze unexplored PFD subunits of Plasmodium falciparum (Pf). Our review enhances the understanding of probable functions,
structure and mechanism of substrate binding of Pf prefoldin by comparing with the available information of its homologs in archaea and H. sapiens. Three PfPFD out of six and a Pf prefoldin-like protein are reported to be essential for parasite survival that signifies their importance in malaria parasite biology. Transcriptome analyses suggest that PfPFD subunits are up-regulated at the mRNA level during asexual and sexual stages of parasite life cycle. Our in silico analysis suggested several pivotal proteins like myosin E, cytoskeletal protein (tubulin), merozoite surface protein and ring exported protein
3 as their interacting partners. Based on structural information of archaeal and H. sapiens PFDs, P. falciparum counterparts have been modelled and key interface residues were identified that are critical for oligomerization of PfPFD subunits. We collated information on PFD-substrate binding and PFDchaperonin interaction in detail to understand the mechanism of substrate delivery in archaea and humans. Overall, our review enables readers to view the PFD family comprehensively.
... All genes known to be involved in the Plasmodium motor complex (Opitz & Soldati 2002;317 Baum et al. 2006), driving parasite gliding motion and enabling host cell invasion, were discovered in 318 P. ashfordi. These include: actin (ACT1), actin-like protein (ALP1), aldolase (FBPA), myosin A 319 (MyoA), myosin A tail interacting protein (MTIP), glideosome-associated protein 45 and 50 (GAP45 320 and GAP50), and thrombospondin related anonymous protein (TRAP) ( Table 2). ...
Malaria parasites ( Plasmodium spp.) include some of the world’s most widespread and virulent pathogens. Our knowledge of the molecular mechanisms these parasites use to invade and exploit hosts other than mice and primates is, however, extremely limited. It is therefore imperative to characterize transcriptome-wide gene expression from non-model malaria parasites and how this varies across host individuals. Here, we used high-throughput Illumina RNA-sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite Plasmodium ashfordi (lineage GRW2). By using a multi-step approach to filter out host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi. A total of 11 954 expressed transcripts were identified, and 7 860 were annotated with protein information. We quantified gene expression levels of all parasite transcripts across three hosts during two infection stages – peak and decreasing parasitemia. Interestingly, parasites from the same host displayed remarkably similar expression profiles during different infection stages, but showed large differences across hosts, indicating that P. ashfordi may adjust its gene expression to specific host individuals. We further show that the majority of transcripts are most similar to the human parasite Plasmodium falciparum, and a large number of red blood cell invasion genes were discovered, suggesting evolutionary conserved invasion strategies between mammalian and avian Plasmodium. The transcriptome of P. ashfordi and its host-specific gene expression advances our understanding of Plasmodium plasticity and is a valuable resource as it allows for further studies analysing gene evolution and comparisons of parasite gene expression.
... The loss of KinesinA and APR1 significantly reduces the induced secretion of adhesins Secretion of proteins from the micronemes is an important step for motility initiation as well as for invasion into host cells. For instance, transmembrane proteins including micronemal protein 2 (MIC2) and MIC2-associated protein (M2AP) are secreted onto the plasma membrane of the parasite (Carruthers and Sibley, 1997;Carruthers et al., 1999a;Huynh et al., 2003;Huynh and Carruthers, 2006), where they are proposed to link the actomyosin machinery to the host cell surface, such that activity of the motor complex generates productive parasite movement (Opitz and Soldati, 2002;Sibley, 2010). Consistent with this view, the knockdown of mic2 expression levels results in a greater proportion of non-motile parasites and parasites unable to sustain productive motility; these parasites also have a significant defect in host-cell invasion (Huynh and Carruthers, 2006). ...
The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here, we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and discovered that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites, and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and to invade host cells, as well as decreased secretion of effectors important for these processes. Together, the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.
... Le tachyzoïte est délimité par un complexe membranaire appelé pellicule, permettant de maintenir sa forme et dans laquelle est ancré le cytosquelette cortical (Baum et al. 2006). La pellicule est formée de la membrane plasmique entourant le parasite et sure la motilité parasitaire (Opitz et al. 2002). Il est constitué de la myosine A, de l'actine et de protéines d'ancrage à l'IMC appelées les protéines GAP (Glideosome Associated Proteins). ...
Toxoplasma gondii (T. gondii) est un parasite intracellulaire obligatoire responsable d’une zoonose cosmopolite, la toxoplasmose. Chez les sujets sains, les parasites de type II persistent sous forme de kystes cérébraux, responsables de maladies mentales sévères et augmentant le risque d’apparition de maladies neurodégénératives. Le contrôle de la toxoplasmose chronique dépend de la sécrétion de l’interleukine pro-inflammatoire IL-12 par les cellules dendritiques (DCs) permettant l’activation des lymphocytes T CD8+ (LTs CD8+) cytotoxiques et la sécrétion de l’IFN-g. Les LTs CD8+ jouent un rôle clé dans l’élimination des cellules parasitées durant la phase aigüe de l’infection, mais aussi dans l’établissement d’une immunité protectrice à long terme. Afin de survivre et persister, T. gondii sécrète de nombreux facteurs de virulence qui modulent les réponses immunitaires de son hôte.Des travaux récents ont mis en évidence le rôle clé de la réponse UPR (Unfolded Protein Response) dans la régulation des réponses immunitaires. La réponse UPR est une réponse cytoprotectrice induite durant un stress cellulaire déclenché lors de variations dans l’homéostasie protéique et lipidique au sein du réticulum endoplasmique mais aussi lors d’un stress infectieux. Jusqu’à présent, l’influence de l’infection par T. gondii sur la réponse UPR n’est pas connue. Nous avons émis l’hypothèse que l’induction de l’UPR par T. gondii dans les DCs pourrait moduler leur capacité de présentation antigénique et la sécrétion de cytokines inflammatoires, impactant ainsi la dissémination et la persistance du parasite.En utilisant, des DCs déficientes pour certains effecteurs de l’UPR (IRE1a et XBP1s), nous avons examiné l’impact de leur activité sur les DCs infectées. In vitro, nos résultats ont montré que T. gondii active la voie IRE1a de l’UPR d’une manière dépendante de la voieMyD88 et contrôle ainsi la production des cytokines pro-inflammatoires IL-6 et IL-12 et la présentation antigénique d’antigènes parasitaires par le CMH-I. Dans les souris infectées,la voie IRE1a est spécifiquement activée dans la sous-population de cDC1 et régule l’activation des LTs CD8+, essentiels à la production de l’IFN-g. De plus, les souris déficientes pour IRE1a et XBP1 spécifiquement dans les DCs ne contrôlent pas la prolifération des parasites et succombent à l’infection aigüe. Notre étude révèle donc un rôle protecteur essentiel de cette voie dans les DCs pour combattre l’infection à T. gondii.
... T. gondii actively invades its host cells and replicates inside of a membrane-bound parasitophorous vacuole (PV) within the host cell cytoplasm [5]. Host cell invasion, PV formation and maintenance are mediated by a set of specialized secretory organelles known as micronemes, rhoptries, and dense granules [6][7][8][9]. While micronemes and rhoptries play roles in the initial stages of attachment and invasion, the dense granules secrete proteins called GRAs into the vacuolar space that participate in the remodeling and maintenance of the PV during intracellular replication [10][11][12][13][14][15][16]. ...
Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo.
... In T. gondii, MyoA, which is localized beneath the plasma member, controls gliding motility (Meissner et al., 2002). MyoA exists in complex called the glideosome, which includes several proteins (i.e., GAP40, GAP45, GAP50) that anchor the motor to the inner membrane complex (IMC) (Frenal et al., 2010;Opitz and Soldati, 2002). MyoA is distributed along the length of the parasite, except for the apical and basal poles. ...
Apicomplexan parasites of medical or veterinary interest are obligate intracellular parasites that display actin-myosin powered motility, control the secretion of adhesive proteins, and actively penetrate their host cells. Following an intracellular replicative phase, they emerge from their spent host cell by a process of active egress. The transition between these two modes of active motility, required for invasion/egress, versus a nonmotile state required for intracellular replication must be balanced to respond to environmental clues and promote efficient growth. These processes are regulated by two interrelated signaling pathways that control intracellular calcium and cyclic nucleotides. In turn, these second messengers coordinate the action of calcium-dependent and nucleotide-dependent kinases that control motility, secretion, invasion, egress, and also balance the transition to the quiescent replicative phase of the life cycle. Genetic and chemical biology approaches have uncovered the intricate control of these pathways and documented the essentiality of many of their key components. For example, secretion of adhesive microneme proteins and activation of motility requires the activity of protein kinase (PK) G, as well as downstream calcium dependent kinases such as CDPK1. In contrast, activation of PK A dampens calcium signaling and leads to a quiescent intracellular replicative phase. These two nucleotide dependent kinases are governed by cyclic nucleotides, the levels of which are controlled by a family of cyclases and phosphodiesterase. In addition to elucidating the intricacies of biological control, these studies identify several essential and novel candidates for development of selective inhibitors that may be used to block infection.
... Gliding is a substrate-dependent motility that is mediated by a protein complex, called the glideosome (Opitz and Soldati, 2002), which has an actomyosin motor that is composed of myosin A, myosin light chain, myosin essential chain and actin filaments (Boucher and Bosch, 2015). Motility is generated after the displacement of the myosin A head from the actin filament, which occurs when the extracellular domain of a transmembrane micronemal protein (TRAP in Plasmodium and MIC2 in T. gondii) strongly interacts with a receptor at the host cell surface or in the extracellular matrix. ...
Intracellular parasites from the genera Toxoplasma, Plasmodium, Trypanosoma, Leishmania and from the phylum Microsporidia are, respectively, the causative agents of toxoplasmosis, malaria, Chagas disease, leishmaniasis and microsporidiosis, illnesses that kill millions of people around the globe. Crossing the host cell plasma membrane (PM) is an obstacle these parasites must overcome to establish themselves intracellularly and so cause diseases. The mechanisms of cell invasion are quite diverse and include (1) formation of moving junctions that drive parasites into host cells, as for the protozoans Toxoplasma gondii and Plasmodium spp., (2) subversion of endocytic pathways used by the host cell to repair PM, as for Trypanosoma cruzi and Leishmania, (3) induction of phagocytosis as for Leishmania or (4) endocytosis of parasites induced by specialized structures, such as the polar tubes present in microsporidian species. Understanding the early steps of cell entry is essential for the development of vaccines and drugs for the prevention or treatment of these diseases, and thus enormous research efforts have been made to unveil their underlying biological mechanisms. This Review will focus on these mechanisms and the factors involved, with an emphasis on the recent insights into the cell biology of invasion by these pathogens.
... The importance of the actin in motility was recognized in both Eimeria (Jensen and Edgar, 1976;Russell and Sinden, 1982) and Plasmodium (Miller et al., 1979) and a later study in E. tenella showed that material secreted when sporozoites were allowed to glide on a substrate emanated from the apical tip (Entzeroth et al., 1989). Subsequently, a large number of studies, mainly in T. gondii, has led to definition of 'glideosomes' (Opitz and Soldati, 2002), protein complexes that lie between the parasite plasma membrane and the IMC and power substrate-dependent gliding motility (reviewed in detail by Frenal and Soldati-Favre, 2009). ...
Apicomplexans, including species of Eimeria, pose a real threat to the health and wellbeing of animals and humans. Eimeria parasites do not infect humans but cause an important economic impact on livestock, in particular on the poultry industry. Despite its high prevalence and financial costs, little is known about the cell biology of these 'cosmopolitan' parasites found all over the world. In this review, we discuss different aspects of the life cycle and stages of Eimeria species, focusing on cellular structures and organelles typical of the coccidian family as well as genus-specific features, complementing some 'unknowns' with what is described in the closely related coccidian Toxoplasma gondii.
Malaria pathogenesis and parasite multiplication both depend on the ability of Plasmodium falciparum merozoites to invade human erythrocytes. Invasion is a complex multi-step process that is known to involve multiple P. falciparum proteins but dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed merozoites and erythrocytes and quantify the strength of ttachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified P. falciparum strains, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment phenotypes for the first time. Most of the interactions investigated did not affect the force needed to pull merozoites and erythrocytes apart, including loss of the major P. falciparum merozoite surface protein PfMSP1 and PfGAP45, part of the glideosome actinomyosin motor complex. The only factors that significantly reduced the strength of merozoite-erythrocyte attachment were ones that disrupted the function of members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families. While these assays also reinforced the known redundancy within these families, with the deletion of some ligands not impacting detachment force, it appears that the PfEBA/PfRH families play a central role in merozoite attachment, not the major merozoite surface protein PfMSP1.
Phylum apicomplexan consists of parasites, such as Plasmodium and Toxoplasma. These obligate intracellular parasites enter host cells via an energy-dependent process using specialized machinery, called the glideosome. In the present study, we used Plasmodium falciparum GAP50, a glideosome-associated protein, as a target to screen 951 different compounds from diverse chemical libraries. Using different screening methods, eight compounds (Hayatinine, Curine, MMV689758 (Bedaquiline), MMV1634402 (Brilacidin), and MMV688271, MMV782353, MMV642550, and USINB4-124-8) were identified, which showed promising binding affinity (KD < 75 μM), along with submicromolar range antiparasitic efficacy and selectivity index > 100 fold for malaria parasite. These eight compounds were effective against Chloroquine-resistant PfINDO and Artemisinin-resistant PfCam3.1R359T strains. Studies on the effect of these compounds at asexual blood stages showed that these eight compounds act differently at different developmental stages, indicating the binding of these compounds to other Plasmodium proteins, in addition to PfGAP50. We further studied the effects of compounds (Bedaquiline and USINB4-124-8) in an in vivoPlasmodium berghei mouse model of malaria. Importantly, the oral delivery of Bedaquiline (50 mg/kg b. wt.) showed substantial suppression of parasitemia, and three out of seven mice were cured of the infection. Thus, our study provides new scaffolds for the development of antimalarials that can act at multiple Plasmodium lifecycle stages.
Toxoplasma gondii is an obligate intracellular protozoan of worldwide distribution. It is effective in the infection of various homoeothermic animals of economic importance. The process of T. gondii invasion of host cells occurs in less than 20 s by the active mechanism of penetration. First, a mobile junction is formed due to the association between the apical end of the parasite and the host cell surface. Then, the secretion of invasive and docking proteins allows the formation of the mobile junction before the complete internalization of the parasite. Here, using high-resolution microscopy, it was described new morphological observations of the early events of host cell invasion by tachyzoites of T. gondii. Attempts were made to synchronize the interaction process using low temperatures and treatment of the host cells with cytochalasin D, a drug that interferes with the actin dynamics. Images were obtained showing that the parasite and the host cells seem to release small vesicles with diameters varying from 25 to 100 nm. Furthermore, tunneling nanotubes emerge from the host cell surface and interact with the parasite even at long distance. These observations add new details of adhesion and entry events, such as surface projections of the host cell plasma membrane, pseudopods, and nanotubes radiating from the host cell toward the parasite. In addition, scanning microscopy revealed intense vesiculation, with a morphological characteristic of extracellular microvesicles, during the entry of the tachyzoite into the host cell.
Eimeria necatrix is an obligate intracellular parasite that has a complex life cycle and causes significant economic losses to the poultry industry. To better understand the cellular invasion mechanism of E. necatrix and develop new measures against its infection, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to investigate protein abundance across different life cycle stages, including unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ-2). Our analysis identified a total of 3606 proteins, among which 1725, 1724, 2143 and 2386 were annotated by the Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and InterPro (IPR) databases, respectively. We also identified 388, 300 and 592 differentially abundant proteins in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. Further analysis revealed that 118 differentially abundant proteins were involved in cellular invasion and could be categorized into eight groups. These findings provide valuable insights into protein abundance across the different life cycle stages of E. necatrix and offer candidate proteins for future studies on cellular invasion and other biological processes. SIGNIFICANCE: Eimeria necatrix is an obligate intracellular parasite results in huge economic losses to the poultry industry. Understanding proteomic variations across the life cycle stages of E. necatrix may offer proteins related to cellular invasion of E. necatrix, and provide resources for the development of new treatment and prevention interventions against E. necatrix infection. The current data provide an overall summary of the protein abundance across the three life cycle stages of E. necatrix. We identified differentially abundant proteins potential related to cellular invasion. The candidate proteins we identified will form the basis of future studies for cellular invasion. This work also will help in the development of novel strategies for coccidiosis control.
The protozoa Toxoplasma gondii and Plasmodium spp., are preeminent members of the Apicomplexa parasitic phylum in large part due to their public health and economic impact. Hence, they serve as model unicellular eukaryotes with which to explore the repertoire of molecular and cellular strategies that specific developmental morphotypes deploy to timely adjust to their host(s) in order to perpetuate. In particular, host tissue- and cell-invasive morphotypes termed zoites alternate extracellular and intracellular lifestyles, thereby sensing and reacting to a wealth of host-derived biomechanical cues over their partnership. In the recent years, biophysical tools especially related to real time force measurement have been introduced, teaching us how creative are these microbes to shape a unique motility system that powers fast gliding through a variety of extracellular matrices, across cellular barriers, in vascular systems or into host cells. Equally performant was this toolkit to start illuminating how parasites manipulate their hosting cell adhesive and rheological properties to their advantage. In this review, besides highlighting major discoveries along the way, we discuss the most promising development, synergy and multimodal integration in active noninvasive force microscopy methods. These should in the near future unlock current limitations and allow capturing, from molecules to tissues, the many biomechanical and biophysical interplays over the dynamic host and microbe partnership. This article is protected by copyright. All rights reserved.
Phylum apicomplexan consists of parasites like Plasmodium and Toxoplasma . These obligate intracellular parasites enter host cells via an energy-dependent process using a specialized machinery called glideosome. In the present study, we used Plasmodium falciparum GAP 50, a glideosome-associated protein as a target to screen 951 different compounds from diverse chemical libraries. Using different screening methods, eight compounds, Hayatinine, Curine, MMV689758 (Bedaquiline), MMV1634402 (Brilacidin), and MMV688271, MMV782353, MMV642550, and USINB4-124-8 were identified which showed promising binding affinity (KD < 75 µM) along with sub-micromolar range anti-parasitic efficacy and selectivity index for malaria parasite > 100 fold. These eight compounds were effective against the chloroquine-resistant Pf INDO and artemisinin-resistant, Pf Cam 3.1 R359T strain. Studies on the effect of these compounds at asexual blood stages showed that these eight compounds act differently at different developmental stages, indicating the binding of these compounds to other Plasmodium proteins besides binding to Pf GAP50. We further studied the effect of compounds in vivo P. berghei mouse model of malaria. Importantly, orally delivered Bedaquiline (50 mg/Kg b. wt.) showed substantial suppression of parasitemia, and three out of seven mice were cured of the infection. Thus, our study provides new scaffolds for the development of antimalarials that may act at multiple Plasmodium life cycle stages.
Rhoptries and micronemes are essential for host cell invasion and survival of all apicomplexan parasites, which are composed of numerous obligate intracellular protozoan pathogens including Plasmodium falciparum (malaria) and Toxoplasma gondii (toxoplasmosis) that infect humans and animals causing severe diseases. We identified Toxoplasma gondii TgSORT as an essential cargo receptor, which drives the transport of rhoptry (ROP) and microneme (MIC) proteins to ensure the biogenesis of these secretory organelles. The luminal ectodomain of 752 amino acid long situated at the N-terminus end of TgSORT has been described to bind to MIC and ROP proteins. Here, we present an optimized protocol for expression of the entire luminal ectodomain of TgSORT (Tg-NSORT) in the yeast Pichia pastoris. Optimization of its coding sequence, cloning and transformation of the yeast P. pastoris allowed the secretion of Tg-NSORT. The protein was purified and further analyzed by negative staining electron microscopy. In addition, molecular modeling using AlphaFold identified key differences between human and T gondii sortilin. The structural features that are only present in T. gondii and other apicomplexan parasites were highlighted. Elucidating the roles of these specific structural features may be useful for designing new therapeutic agents against apicomplexan parasites.
The pathogenesis of Toxoplasma gondii is mainly due to tissue damage caused by the repeating lytic cycles of the parasite. Many proteins localized to the pellicle of the parasite, particularly kinases, have been identified as critical regulators of the Toxoplasma lytic cycle. However, little is known about the associated protein phosphatases. Phosphatase of regenerating liver (PRL), a highly conserved tyrosine phosphatase, is an oncogene that plays pivotal roles in mammalian cells and typically associates with membranes via a conserved prenylation site. PRL in Toxoplasma has a predicted prenylation motif in the C-terminus like other homologs. We have determined that TgPRL localizes to the plasma membrane and that disruption of TgPRL results in a defect in the parasite’s ability to attach to host cells. This function is dependent both on TgPRL’s membrane localization and phosphatase activity. Importantly, in vivo experiments have shown that while mice infected with parental strain parasites die within days of infection, those infected with parasites lacking TgPRL not only survive but also develop immunity that confers protection against subsequent infection with wild-type parasites. Immunoprecipitation experiments revealed that the PRL-CNNM (Cyclin M) complex, which regulates intracellular Mg ²⁺ homeostasis in mammalian cells, is also present in Toxoplasma . Consistent with this interaction, parasites lacking TgPRL had higher intracellular Mg ²⁺ levels than the parental or complemented strains, suggesting TgPRL is involved in regulating intracellular Mg ²⁺ homeostasis. Thus, TgPRL is a vital regulator of the Toxoplasma lytic cycle and virulence, showing its potential as a target of therapeutic intervention.
IMPORTANCE
Infection with Toxoplasma gondii can lead to severe and even life-threatening diseases in people with compromised or suppressed immune systems. Unfortunately, drugs to combat the parasite are limited, highly toxic, and ineffective against the chronic stage of the parasite. Consequently, there is a strong demand for the discovery of new treatments. A comprehensive understanding of how the parasite propagates in the host cells and which proteins contribute to the parasite’s virulence will facilitate the discovery of new drug targets. Our study meets this objective and adds new insights to understanding the lytic cycle regulation and virulence of Toxoplasma by determining that the protein phosphatase TgPRL plays a vital role in the parasite’s ability to attach to host cells and that it is essential for parasite virulence.
This brief review is devoted to a discussion of the possible gliding mechanisms of raphid pennate diatoms. This locomotion method is carried out without the participation of flagella, cilia and any deformations of the plasmalemma. One of the main hypotheses considered in recent years assumes participation of the actin‐myosin system in the gliding. Such a system would require a connection by transmembrane components connecting with polymeric substances excreted through the raphe, and such transmembrane complexes for diatoms have not yet been directly observed, in contrast to a similar type of motility system in the parasitic protozoa Apicomplexa . However, there is little doubt that the actin‐myosin system of the diatoms is required for motility, at the very least for transporting mucilage‐containing vesicles that are essential and necessary for successful adhesion and further gliding of diatom cells along a substrate. Clearly, elucidating the mechanism of gliding will require the understanding of various chemical and mechanical properties of the mucilage, as well as in better determining the control systems for the secretion of mucilage strands and the factors that regulate them.
Ocular toxoplasmosis is a retinitis –almost always accompanied by vitritis and choroiditis– caused by intraocular infection with Toxoplasma gondii. Depending on retinal location, this condition may cause substantial vision impairment. T. gondii is an obligate intracellular protozoan parasite, with both sexual and asexual life cycles, and infection is typically contracted orally by consuming encysted bradyzoites in undercooked meat, or oocysts on unwashed garden produce or in contaminated water. Presently available anti-parasitic drugs cannot eliminate T. gondii from the body. In vitro studies using T. gondii tachyzoites, and human retinal cells and tissue have provided important insights into the pathogenesis of ocular toxoplasmosis. T. gondii may cross the vascular endothelium to access human retina by at least three routes: in leukocyte taxis; as a transmigrating tachyzoite; and after infecting endothelial cells. The parasite is capable of navigating the human neuroretina, gaining access to a range of cell populations. Retinal Müller glial cells may be preferred initial host cells. T. gondii infection of the retinal pigment epithelial cells alters the secretion of growth factors and induces proliferation of adjacent uninfected epithelial cells. This increases susceptibility of the cells to parasite infection, and may be the basis of the characteristic hyperpigmented toxoplasmic retinal lesion. Infected epithelial cells also generate a vigorous immunologic response, and influence the activity of leukocytes that infiltrate the retina. A range of T. gondii genotypes are associated with human ocular toxoplasmosis, and individual immunogenetics –including polymorphisms in genes encoding innate immune receptors, human leukocyte antigens and cytokines– impacts the clinical manifestations. Research into basic pathogenic mechanisms of ocular toxoplasmosis highlights the importance of prevention and suggests new biological drug targets for established disease.
Apicomplexan parasites of the genus Eimeria are organisms which invade the intestinal tract, causing coccidiosis, an enteric disease of major economic importance worldwide. The disease causes high morbidity ranging from an acute, bloody enteritis with high mortality, to subclinical disease. However, the presence of intestinal lesions depends on the Eimeria species. The most important poultry Eimeria species are: E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, E. mitis, and E. praecox. Key points to better understanding the behavior of this species are the host-parasite interactions and its life cycle. The present paper reviews the literature available regarding the life cycle and the initial host-parasite interaction. More studies are needed to better understand these interactions in poultry Eimerias, taking into account that almost all the information available was generated from other apicomplexan parasites that generate human disease.
Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two stages and functionally characterized. Localized beneath the cell surface, it includes actin filaments with unconventional myosin motors housed within a multi-member glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known on the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion-deadhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite in 3D conditions. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital gliding capacity.
The Apicomplexa are named for their unique apical secretory organelles: the micronemes, the rhoptries, and the dense granules (DGs). The contents of these organelles are critical for the successful invasion and intracellular survival of Toxoplasma gondii. Microneme proteins are secreted in a calcium-dependent manner and are critical for adhesion and bridging with the host, as well as parasite egress from host cells. Rhoptry neck molecules, with microneme proteins, form the moving junction that enables the parasite to enter the host, while other rhoptry proteins are important for modulation of host cell signaling and formation of the parasitophorous vacuole. DG proteins play important roles in the interaction with the host as well as survival within the host. This chapter provides a detailed overview of the functions of Toxoplasma secretory proteins and their roles in invasion, egress, and host cell parasitism.
Toxoplasma gondii is a remarkable species with a rich cell, developmental, and population biology. It is also sometimes responsible for serious disease in animals and humans and the stages responsible for such disease are relatively easy to study in vitro or in laboratory animal models. As a result of all this, Toxoplasma has become the subject of intense investigation over the last several decades, becoming a model organism for the study of the phylum of which it is a member, Apicomplexa. This has led to an ever-growing number of investigators applying an ever-expanding set of techniques to dissecting how Toxoplasma “ticks” and how it interacts with its many hosts. In this perspective piece I first wind back the clock 30 years and then trace the extraordinary pace of methodologies that have propelled the field forward to where we are today. In keeping with the theme of this collection, I focus almost exclusively on the parasite, rather than host side of the equation. I finish with a few thoughts about where the field might be headed—though if we have learned anything, the only sure prediction is that the pace of technological advance will surely continue to accelerate and the future will give us still undreamed of methods for taking apart (and then putting back together) this amazing organism with all its intricate biology. We have so far surely just scratched the surface.
This book discusses in detail the structural, evolutionary and functional role of actin and its regulatory proteins in gliding motility in apicomplexan organisms, a unique phenomenon found in actin-myosin cytoskeletal elements.
The book also explores the potential of different actin regulators, namely formin, profilin, actin depolymerization factor (ADF), capping proteins (CPα and CPβ), cyclase-associated protein (CAP) and coronin 13–24 as potential drug targets against malaria. As the chief components of the gliding motor, the actin-regulator proteins are characterized by unique features that make them promising targets for structure-based drug design.
Lastly, the book proposes a mathematical model, based on kinetic data mining, to help understand the most vital regulators for actin polymerization dynamics.
The micronemal protein 2 (MIC2) of Toxoplasma gondiishares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These
molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and
membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two
conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to
T. gondii micronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence
is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species.
The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the
two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes
ofT. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional
relationship that links these organelles with the endoplasmic reticulum of the parasite.
Application of Fourier analysis techniques to images of isolated, frozen-hydrated subpellicular microtubules from the protozoan parasite Toxoplasma gondii demonstrates a distinctive 32 nm periodicity along the length of the microtubules. A 32 nm longitudinal repeat is also observed in the double rows of intramembranous particles seen in freeze-fracture images of the parasite's pellicle; these rows are thought to overlie the subpellicular microtubules. Remarkably, the 32 nm intramembranous particle periodicity is carried over laterally to the single rows of particles that lie between the microtubule-associated double rows. This creates a two-dimensional particle lattice, with the second dimension at an angle of approximately 75 degrees to the longitudinal rows (depending on position along the length of the parasite). Drugs that disrupt known cytoskeletal components fail to destroy the integrity of the particle lattice. This intramembranous particle organization suggests the existence of multiple cytoskeletal filaments of unknown identity. Filaments associated with the particle lattice provide a possible mechanism for motility and shape change in Toxoplasma: distortion of the lattice may mediate the twirling motility seen upon host-cell lysis, and morphological changes observed during invasion.
Many protozoans of the phylum Apicomplexa are invasive parasites that exhibit a substrate-dependent gliding motility. Plasmodium (malaria) sporozoites, the stage of the parasite that invades the salivary glands of the mosquito vector and the liver of the vertebrate host, express a surface protein called thrombospondin-related anonymous protein (TRAP) that has homologs in other Apicomplexa. By gene targeting in a rodent Plasmodium, we demonstrate that TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporozoite gliding motility in vitro. This suggests that in Plasmodium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.
Here, we describe the complete deduced amino acid sequence of three unconventional myosins identified in the protozoan parasite Toxoplasma gondii. Phylogenetic analysis reveals that the three myosins represent a novel, highly-divergent class addition to the myosin superfamily. Toxoplasma gondii myosin-A (TgM-A) is a remarkably small approximately 93 kDa myosin that shows a striking departure from typical myosin heavy chain structure in having a head and tail domain but no discernible neck domain. In other myosins, the neck is defined by one or more IQ motifs that serve as potential light chain binding domains. No IQ motifs are apparent in TgM-A. The tail domain of TgM-A encompasses only 57 amino acid residues and is characterized by its highly basic charge (pI = 10.8). The other two Toxoplasma myosins, TgM-B and TgM-C appear to be the product of differential RNA splicing with TgM-B yielding a protein of approximately 114 kDa and TgM-C a protein of approximately 125 kDa. These two myosins are identical throughout their head domain and neck domain which contains a single IQ motif. TgM-B and C share the proximal 245 residues of their tail domain and then diverge in their tail structure distally. The tails, like that of TgM-A, share no homology to any other myosin tails apart from a highly basic charge. The identification of yet another class of unconventional myosins, including a myosin as novel in structure as the 93 kDa TgM-A, continues to underscore the diversity of this family of molecular motors.
The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound.
It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.
We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 microM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.
One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca(2+) levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca(2+)-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+](i). Furthermore, sequential addition experiments with ethanol and other Ca(2+)-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+](i) by mobilizing Ca(2+) from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+](i) in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca(2+)-elevating effects of alcohol in higher eukaryotes.
Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.
Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins
that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide
genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion.
We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails
interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related
proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.
The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.
Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.
Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.
MIC2 is an adhesive protein that participates in host cell invasion by the obligate intracellular parasite Toxoplasma gondii. Earlier studies established that MIC2 is secreted into the culture medium by extracellular parasites and that release is coincident with proteolytic modification. Since little is known about proteolytic processing of proteins secreted by T. gondii, we undertook this study to investigate the proteolytic events that accompany secretion of MIC2. We demonstrate that the C-terminal domain of MIC2 is removed by a protease, termed MPP1, when MIC2 is released into the culture supernatant. Additionally, prior to release, a second protease, termed MPP2, trims the N terminus of MIC2, resulting in the release of heterogeneously sized species of MIC2. Although MPP1 activity was unaffected by any of the protease inhibitors tested, MPP2 activity was blocked by a subset of serine and cysteine protease inhibitors. These results establish that MIC2 is proteolytically modified at multiple sites by two distinct enzymes that probably operate on the parasite surface.
The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that
contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge
from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that
contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide
bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeriaspecies, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of
all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus
only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal
apple domain.
Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid
events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca2+]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process
termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly
lose infectivity (termed ionophore-induced death [IID]). From among several Iie− mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The
association between the Iie− and Iid− phenotypes is supported by the observation that two-thirds of mutants selected as Iid− are also Iie−. Characterization of three distinct classes of IIE and IID mutants revealed that the Iie− phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized
just prior to egress. Iie−parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming
that this step is deficient in the Iie− mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading
to parasite egress. The fact that Iie−mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and
IIE.
The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known
to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive
protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for
calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at
the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity
was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926
at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected
in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion.
The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.
Microneme organelles are found in the apical complex of all apicomplexan parasites and play an important role in the invasion process. The recent identification of microneme proteins from different apicomplexan genera has revealed a striking conservation of structural domains, some of which show functional complementation across species. This supports the idea that the mechanism of host cell invasion across the phylum is conserved not only morphologically, but also functionally at the molecular level. Here, we review and summarize these recent findings.
Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
The ability of intracellular parasites to monitor the viability of their host cells is essential for their survival. The protozoan
parasite Toxoplasma gondii actively invades nucleated animal cells and replicates in their cytoplasm. Two to 3 days after infection, the parasite-filled
host cell breaks down and the parasites leave to initiate infection of a new cell. Parasite egress from the host cell is triggered
by rupture of the host plasma membrane and the ensuing reduction in the concentration of cytoplasmic potassium. The many other
changes in host cell composition do not appear be used as triggers. The reduction in the host cell [K+] appears to activate a phospholipase C activity inToxoplasma that, in turn, causes an increase in cytoplasmic [Ca2+] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca2+ blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K+] and parasite phospholipase C activation. The increase in [Ca2+]C brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent
protein phosphatase calcineurin.
Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.
Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.
In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.
TgMIC6, TgMIC7, TgMIC8 and TgMIC9 are members of a novel family of transmembrane proteins localized in the micronemes of the protozoan parasite Toxoplasma gondii. These proteins contain multiple epidermal growth factor-like domains, a putative transmembrane spanning domain and a short cytoplasmic tail. Sorting signals to the micronemes are encoded in this short tail. We established previously that TgMIC6 serves as an escorter for two soluble adhesins, TgMIC1 and TgMIC4. Here, we present the characterization of TgMIC6 and three additional members of this family, TgMIC7, -8 and -9. Consistent with having sorting signals localized in its C-terminal tail, TgMIC6 exhibits a classical type I membrane topology during its transport along the secretory pathway and during storage in the micronemes. TgMIC6 is processed at the N-terminus, probably in the trans-Golgi network, and the cleavage site has been precisely mapped. Additionally, like other members of the thrombospondin-related anonymous protein family, TgMIC2, TgMIC6 and TgMIC8 are proteolytically cleaved near their C-terminal domain upon discharge by micronemes. We also provide evidence that TgMIC8 escorts another recently described soluble adhesin, TgMIC3. This suggests that the existence of microneme protein complexes is not an exception but rather the rule. TgMIC6 and TgMIC8 are expressed in the rapidly dividing tachyzoites, while TgMIC7 and TgMIC9 genes are predominantly expressed in bradyzoites, where they presumably also serve as escorters.
The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites.
Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.
In the past few years genetic, biochemical, and cytolocalization data have implicated members of the myosin superfamily of
actin-based molecular motors in a variety of cellular functions including membrane trafficking, cell movements, and signal
transduction. The importance of myosins is illustrated by the identification of myosin genes as targets for disease-causing
mutations. The task at hand is to decipher how the multitude of myosins function at both the molecular and cellular level—a
task facilitated by our understanding of myosin structure and function in muscle.
We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13 400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immuno-electron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.
Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii.
Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.
The sporozoites of Eimeria tenella and Eimeria acervulina show bending, pivoting and gliding motility. All these types of motility occur intermittently and with decreasing frequency during the life of a sporozoite. Gliding is the only locomotive action expressed by these sporozoites and is only seen when the sporozoites are in contact with the substratum. All gliding sporozoites adopt a set pattern of body’ attitudes which suggests that locomotion involves a fixed body shape.
The microtubule inhibitors, colchicine, griseofulvin, vinblastine sulphate and nocodazole, have no effect on sporozoite motility. Ultrastructural examination reveals, in addition, that they have no effect on the subpellicular microtubules. The microfilament inhibitor, cytochalasin B, completely, and reversibly, inhibits pivoting and gliding but bending is only slightly depressed by the drug. High magnesium ion concentration inhibits all motility completely.
The cell membrane was readily labelled with fluorescein isothiocyanate-conjugated cationized ferritin, the label was rapidly capped and shed from the posterior of the sporozoite. This capping reaction takes place only during sporozoite locomotion. The membrane label was seen to ‘move’ backwards relative to the sporozoite at the same rate as the sporozoite moved forwards relative to the substratum. The substratum and the leading edge of the cap remained static relative to each other. Both capping and locomotion are sensitive to low temperature and cytochalasin B.
From these results a theory of sporozoite motility is postulated. The sporozoites adhere to the substratum by surface ligands. This ligand/substratum complex is then capped along the fixed spiral of the sporozoite body by a microfilament-based contractile system.
This proposed model for motility of coccidia sporozoites is consistent with all current observations on cell invasion by the sporozoa and therefore suggests that locomotion is an integral component of host cell invasion in this group of parasites.
Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. We demonstrate that invasion is critically dependent on actin filaments in the parasite, but not the host cell. Invasion into cytochalasin D (CD)-resistant host cells was blocked by CD, while parasite mutants invaded wild-type host cells in the presence of drug. CD resistance in Toxoplasma was mediated by a point mutation in the single-copy actin gene ACT1. Transfection of the mutant act1 allele into wild-type Toxoplasma conferred motility and invasion in the presence of CD. We conclude that host cell invasion by Toxoplasma, and likely by related Apicomplexans, is actively powered by an actin-based contractile system in the parasite.
The cDNA encoding the Toxoplasma gondii microneme protein MIC1 and the corresponding gene have been cloned and sequenced. The MIC1 gene contains three introns. The cDNA encodes a 456 amino acid (aa) sequence, with a typical signal sequence and no other trans-membrane domain. The protein contains a tandemly duplicated domain with conservation of cysteines and presents distant homology with the Plasmodium sp. microneme protein TRAP-SSP2. The MIC1 protein from tachyzoite lysates and a PMAL recombinant expressing the N-terminal duplicated domain of the protein bound to the surface of putative host cells, suggesting a possible involvement of MIC1 in host cell binding/recognition.
Invasion of vertebrate cells by the protozoan Toxoplasma gondii is accompanied by regulated protein secretion from three distinct parasite organelles called micronemes, rhoptries, and dense granules. We have compared the kinetics of secretion from these different compartments during host cell invasion using immunofluorescence, immunoelectron microscopy, and quantitative immunoassays. Binding to the host cell triggered apical release of the micronemal protein MIC2 at the tight attachment zone that forms between the parasite and the host cell. In a second step, invagination of the host cell plasma membrane was initiated by discharge of the rhoptry protein ROP1 to form a nascent parasitophorous vacuole (PV). ROP1 was fully discharged into the vacuole by the time invasion was complete. In contrast to these very rapid early events, release of the dense granule markers GRA1 and NTPase was delayed until after the parasite was fully within the PV, eventually peaking at 20 min post-invasion. The sequential triggering of secretion from different organelles implies that their release is governed by separate signals and that their contents mediate distinct phases of intracellular parasitism.
We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13,400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immunoelectron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.
An important group of animal and human pathogens, belonging to the phylum Apicomplexa, employs a novel form of motility, known as gliding, to move on solid substrates and to enter host cells. Gliding is dependent on the parasite cytoskeleton and involves a conserved family of secretory adhesins.
In the past few years genetic, biochemical, and cytolocalization data have implicated members of the myosin superfamily of actin-based molecular motors in a variety of cellular functions including membrane trafficking, cell movements, and signal transduction. The importance of myosins is illustrated by the identification of myosin genes as targets for disease-causing mutations. The task at hand is to decipher how the multitude of myosins function at both the molecular and cellular level-a task facilitated by our understanding of myosin structure and function in muscle.
Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore-stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin-myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus-coupled secretion system responsible for releasing adhesins involved in cell attachment.
The invasive stages of Toxoplasma gondii, an Apicomplexan parasite, actively invade their host cells in an actin-dependent way. However, despite containing biochemically significant amounts of actin, actin filaments have never been observed in these parasites. Jasplakinolide, a membrane-permeable actin-polymerizing and filament-stabilizing drug, induced the polymerization of actin filaments at the anterior end of each tachyzoite in association with the conoid, where they formed, in many cases, a prominent membrane-enclosed apical projection reminiscent of acrosomal processes of invertebrate sperm. These jasplakinolide-induced filaments decorated with myosin subfragment 1, demonstrating unequivocally that they were indeed actin. Jasplakinolide-treated tachyzoites were unable to invade host cells, but once the drug was removed the parasites were able to enter host cells. Actin polymerization at the apical end of the parasite is consistent with the role of the apical end in host-cell invasion powered by a jackhammer-like extension and retraction of the conoid complex coupled to the secretion and rearward capping of surface proteins.
Apicomplexa constitute one of the largest phyla of protozoa. Most Apicomplexa, including those pathogenic to humans, are obligate intracellular parasites. Their extracellular forms, which are highly polarized and elongated cells, share two unique abilities: they glide on solid substrates without changing their shape and reach an intracellular compartment without active participation from the host cell. There is now ample ultrastructural evidence that these processes result from the backward movement of extracellular interactions along the anteroposterior axis of the parasite. Recent work in several Apicomplexa, including genetic studies in the Plasmodium sporozoite, has provided molecular support for this 'capping' model. It appears that the same machinery drives both gliding motility and host cell invasion. The cytoplasmic motor, a transmembrane bridge and surface ligands essential for cell invasion are conserved among the main apicomplexan pathogens.
Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.
Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.
The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.
Electron microscopic examination of detergent-extracted Toxoplasma tachyzoites reveals the presence of a mechanically stable cytoskeletal structure associated with the pellicle of this parasite. This structure, composed of interwoven 8-10 nm filaments, is associated with the cytoplasmic face of the pellicle and surrounds the microtubule-based cytoskeleton. Two protein components of this network, TgIMC1 and TgIMC2, were identified. Both are novel proteins, but have a resemblance to mammalian filament proteins in that they are predicted to have extended, coiled-coil domains. TgIMC1 is also homologous to articulins, the major components of the membrane skeleton of algae and free-living protists. A homologue of TgIMC1 in the related malaria parasite Plasmodium falciparum was also identified suggesting the presence of structurally similar membrane skeletons in all apicomplexan parasites. We suggest that the subpellicular network, formed by TgIMC1 and 2 in Toxoplasma gondii and related parasites, plays a role in the determination of cell shape and is a source of mechanical strength.
Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.
Toxoplasma gondii is an obligate intracellular protozoan that infects an astonishing variety of vertebrate hosts including humans. Classified in the phylum Apicomplexa, T. gondii causes an opportunistic disease, toxoplasmosis, in individuals with immune dysfunction and congenital disease in infected infants. Re-emergence of toxoplasmosis as a life-threatening disease in patients with AIDS is anticipated in the wake of emerging multi-drug resistant strains of HIV. In immunodeficient patients, the available evidence suggests that tissue pathology associated with T. gondii infection is due to parasite-directed lytic destruction of individual host cells. The Toxoplasma lytic cycle begins when the parasite actively invades a target cell. In association with invasion, T. gondii sequentially discharges three sets of secretory organelles beginning with the micronemes, which contain adhesive proteins involved in parasite attachment to a host cell. Deployed as protein complexes, several micronemal proteins possess vertebrate-derived adhesive sequences that function in binding receptors on the surface of a target cell. Each protein in these adhesive complexes fulfills a specific role in movement through the secretory pathway, targeting to the micronemes, or adhesion. It is anticipated that these adhesive complexes recognize a variety of host receptors, including some that are expressed on multiple cell types, and that this diversity in host cell receptors contributes to the remarkably broad tissue- and host-range of T. gondii.