In silico identification of specialized secretory-organelle proteins in apicomplexan parasites and in vivo validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

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.     

The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii--host cell interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.     

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.     

Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium sp., are obligate intracellular protozoa. They enter into a host cell by attaching to and then creating an invagination in the host cell plasma membrane. Contact between parasite and host plasma membranes occurs in the form of a ring-shaped moving junction that begins at the anterior end of the parasite and then migrates posteriorly. The resulting invagination of host plasma membrane creates a parasitophorous vacuole that completely envelops the now intracellular parasite. At the start of this process, apical membrane antigen 1 (AMA1) is released onto the parasite surface from specialized secretory organelles called micronemes. The T. gondii version of this protein, TgAMA1, has been shown to be essential for invasion but its exact role has not previously been determined. We identify here a trio of proteins that associate with TgAMA1, at least one of which associates with TgAMA1 at the moving junction. Surprisingly, these new proteins derive not from micronemes, but from the anterior secretory organelles known as rhoptries and specifically, for at least two, from the neck portion of these club-shaped structures. Homologues for these AMA1-associated proteins are found throughout the Apicomplexa strongly suggesting that this moving junction apparatus is a conserved feature of this important class of parasites. Differences between the contributing proteins in different species may, in part, be the result of selective pressure from the different niches occupied by these parasites.     

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.     

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii

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.     

TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2

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 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.     

Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.     

Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.     

Toxoplasma gondii uses unusual sorting mechanisms to deliver transmembrane proteins into the host-cell vacuole

A critical step in infection by the apicomplexan parasite Toxoplasma gondii is the formation of a membrane-bound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.     

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells

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.     

Location, location, location: trafficking and function of secreted proteases of Toxoplasma and Plasmodium

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.     

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii.

Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.