ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243-539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence.     

The arginine‐rich N‐terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii

Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host–pathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine‐rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells upregulate immunity‐related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole‐targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N‐terminal arginine‐rich domain, is critical to its function as a virulence determinant.     

The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.     

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

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.     

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C-terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C-terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase-related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.     

Localization of a Toxoplasma gondii Rhoptry Protein by Immunoelectron Microscopy During and After Host Cell Penetration

ABSTRACT We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (α249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

Identification of novel rhoptry proteins in Neospora caninum by LC/MS-MS analysis of subcellular fractions

Apicomplexan parasites possess an apical complex that is composed of two secretory organelles recognized as micronemes and rhoptries. Rhoptry contents are secreted into the parasitophorous vacuole during the host cell invasion process. Several rhoptry proteins have been identified in Toxoplasma gondii and seem to be involved in host-pathogen interactions and some of them are considered to be important virulence factors. Only one rhoptry protein, NcROP2, has been identified and extensively characterized in the closely related parasite Neospora caninum, and this has showed immunoprotective properties. Thus, with the aim of increasing knowledge of the rhoptry protein repertoire in N. caninum, a subcellular fractionation of tachyzoites was performed to obtain fractions enriched for this secretory organelle. 2-D SDS-PAGE followed by MS and LC/MS-MS were applied for fraction analysis and 8 potential novel rhoptry components (NcROP1, 5, 8, 30 and NcRON2, 3, 4, 8) and several kinases, proteases and phosphatases proteins were identified with a high homology to those previously found in T. gondii. Their existence in N. caninum tachyzoites suggests their involvement in similar events or pathways that occur in T. gondii. These novel proteins may be considered as targets that could be useful in the future development of immunoprophylactic measures.     

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.     

Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration.

We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (alpha 249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.     

Invasion factors are coupled to key signalling events leading to the establishment of infection in apicomplexan parasites

Invasion of host cells by apicomplexan parasites is initiated when specialized secretory organelles called micronemes discharge protein complexes onto the parasite surface in response to a rise in parasite intracellular calcium levels. The microneme proteins establish interactions with host cell receptors, engaging the parasite with the host cell surface, and signal for the immediate exocytosis of another set of secretory organelles named the rhoptries. The rhoptry proteins reprogram the invaded host cell and participate in the formation of the parasitophorous vacuole in which the intracellular parasite resides and replicates. Disengagement of the invading parasite from the host cell receptors involves the action of at least one parasite plasma membrane rhomboid protease, which is concomitantly implicated in a checkpoint that signals the parasite to switch from an invasive to a replicative mode.     

Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge

Host cell cholesterol is implicated in the entry and replication of an increasing number of intracellular microbial pathogens. Although uptake of viral particles via cholesterol-enriched caveolae is increasingly well described, the requirement of cholesterol for internalization of eukaryotic pathogens is poorly understood and is likely to be partly organism specific. We examined the role of cholesterol in active host cell invasion by the protozoan parasite Toxoplasma gondii. The parasitophorous vacuole membrane (PVM) surrounding T. gondii contains cholesterol at the time of invasion. Although cholesterol-enriched parasite apical organelles termed rhoptries discharge at the time of cell entry and contribute to PVM formation, surprisingly, rhoptry cholesterol is not necessary for this process. In contrast, host plasma membrane cholesterol is incorporated into the forming PVM during invasion, through a caveolae-independent mechanism. Unexpectedly, depleting host cell plasma membrane cholesterol blocks parasite internalization by reducing the release of rhoptry proteins that are necessary for invasion. Cholesterol back-addition into host plasma membrane reverses this inhibitory effect of depletion on parasite secretion. These data define a new mechanism by which host cholesterol specifically controls entry of an intracellular pathogen.     

Dynamics and 3D organization of secretory organelles of Toxoplasma gondii

Micronemes, rhoptries and dense granules are secretory organelles of Toxoplasma gondii crucial for host cell invasion and formation of the parasitophorous vacuole (PV). We examined whether their relative volumes change during the intracellular cycle. Stereological analysis of random ultrathin sections taken at 5min of interaction, 7 and 24h post-infection demonstrated that the relative volume of each type of organelle decreases just after the respective peak of secretion. Micronemes are radially arranged below the polar ring, while rhoptries converge to but only a few reach the inside of the conoid. In contrast to the apical and polarized organelles, dense granules were found scattered throughout the cytoplasm, with no preferential location in the parasite cell body. Extensive observation of random sections indicated that each organelle probably secretes in a different region. Micronemes secrete just below the posterior ring and probably require that the conoid is extruded. The rhoptries passing through the conoid secrete at a porosome-like point at the most apical region. Dense granules secrete laterally, probably at fenestrations in the inner membrane complex. Immunocytochemistry showed that there are no subpopulations of rhoptries or dense granules, as a single organelle can contain more than one kind of its specific proteins. The vacuolar-like profiles observed at the apical portion of parasites just after invasion were confirmed to be empty rhoptries, as they were positively labeled for rhoptry proteins. These findings contribute for a better understanding of the essential behavior of secretory organelles.     

Processing and secretion of ROP13: A unique Toxoplasma effector protein

Like most intracellular pathogens, Toxoplasma synthesizes and secretes an arsenal of proteins to successfully invade its host cell and hijack host functions for intracellular survival. The rhoptries are key secretory organelles that inject proteins into the host cell where they are positioned to co-opt host processes, although little is known regarding how these proteins exert their functions. We show here that the rhoptry protein ROP13 is synthesized as a pre-pro-protein that is processed in the parasite. Processing occurs at a conserved SφXE cleavage site as mutagenesis of glutamic acid to alanine at the P1 position disrupts ROP13 maturation. We also demonstrate that processing of the prodomain is not necessary for rhoptry targeting and secretion. While gene disruption reveals that ROP13 is not essential for growth in fibroblasts in vitro or for virulence in vivo, we find that ROP13 is a soluble effector protein that can access the cytoplasm of host cells. Exogenously expressed ROP13 in human cells remains cytosolic but also appears toxic, suggesting that over-expression of this effector protein is disrupting some function within the host cell.     

The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal

The rhoptries of Toxoplasma gondii are regulated secretory organelles involved in the invasion of host cells. Rhoptry proteins are synthesised as pre-pro-proteins that are processed first to pro-proteins upon entrance into the secretory pathway, then processed again to their mature forms late in the secretory pathway. The pro-mature processing site of the rhoptry protein ROP1 has been determined, paving the way for understanding the role of the pro region in rhoptry protein function. We demonstrate here that the ROP1 pro region is sufficient for targeting a reporter protein (amino acids 34-471 of the Trypanosoma brucei VSG117 protein) to the rhoptries. These results, together with our previous work showing that rhoptry targeting is unaffected by deletion of the pro region, indicate that the ROP1 protein contains at least two signals that can function in rhoptry targeting.     

The fine structure of secretion by Plasmodium knowlesi merozoites during red cell invasion

The secretory organelles of Plasmodium knowlesi were studied ultrastructurally to examine their mode of action during invasion. The formation of lamellar structures in merozoite rhoptries within late stage schizonts is prevented by the protease inhibitors chymostatin and leupeptin. Under normal conditions vesicles lined by 6-nm membranes are formed in rhoptries during erythrocyte invasion. Stereoscopic viewing of tilted sections shows that where the merozoite apex contacts the parasitophorous vacuole (PV) membrane during invasion, a domed elevation of the PV surface lies within the mouth of the rhoptry duct in contact with the secretory matrix. The membrane of the early invasion pit is thinner (6 nm) than the red cell membrane elsewhere, and sheets of lamellar material are frequently present on the invasion pit surface. These findings support the proposal that the rhoptry-microneme complex is capable of generating membranous material and inserting it into the red cell surface in a controlled manner to create the parasitophorous vacuole. On the basis of this model, measurements from serial sections show that the rhoptries could provide enough material to create a membrane lining the parasitophorous vacuole, and, with the contribution of the microspheres, could double it to accommodate the early ring stage of the parasite.     

The Fine Structure of Secretion by Plasmodium knowlesi Merozoites during Red Cell Invasion

The secretory organelles of Plasmodium knowlesi were studied ultrastructurally to examine their mode of action during invasion. The formation of lamellar structures in merozoite rhoptries within late stage schizonts is prevented by the protease inhibitors chymostatin and leupeptin. Under normal conditions vesicles lined by 6-nm membranes are formed in rhoptries during erythrocyte invasion. Stereoscopic viewing of tilted sections shows that where the merozoite apex contacts the parasitophorous vacuole (PV) membrane during invasion, a domed elevation of the PV surface lies within the mouth of the rhoptry duct in contact with the secretory matrix. The membrane of the early invasion pit is thinner (6 nm) than the red cell membrane elsewhere, and sheets of lamellar material are frequently present on the invasion pit surface. These findings support the proposal that the rhoptry-microneme complex is capable of generating membranous material and inserting it into the red cell surface in a controlled manner to create the parasitophorous vacuole. On the basis of this model, measurements from serial sections show that the rhoptries could provide enough material to create a membrane lining the parasitophorous vacuole, and, with the contribution of the microspheres, could double it to accommodate the early ring stage of the parasite.     

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.     

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.