Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa)

Merozoites of the parasitic protozoon Sarcocystis muris (Apicomplexa) possess three types of characteristic organelles with electron dense contents named rhoptries, micronemes, and dense granules, which are supposed to be involved in the parasite-host cell interactions during and after invasion. Dense granules were purified from a merozoite homogenate by centrifugation on a sucrose density gradient. It was shown by SDS polyacrylamide gel electrophoresis that they contain a major protein of 21 kDa. Polyclonal antibodies raised against this protein were applied to ultrathin frozen and Lowicryl-K4M-embedded sections of the parasite before and after host cell invasion. Dense granules were distinctly labeled by immunogold before and after invasion. After host cell invasion the parasite is enclosed in a secondary parasitophorous vacuole which contains an electron-dense material. This deposition was heavily labeled by anti 21 kDa antibodies which clearly demonstrated that the dense granule contents is released into the secondary parasitophorous vacuole.     

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.     

Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Protein Trafficking to Apical Organelles of Malaria Parasites – Building an Invasion Machine

Malaria is caused by four species of apicomplexan protozoa belonging to the genus Plasmodium. These parasites possess a specialized collection of secretory organelles called rhoptries, micronemes and dense granules (DGs) that in part facilitate invasion of host cells. The mechanism by which the parasite traffics proteins to these organelles as well as regulates their secretion has important implications for understanding the invasion process and may lead to development of novel intervention strategies. In this review, we focus on emerging data about trafficking signals, mechanisms of biogenesis and secretion. At least some of these are conserved in higher eukaryotes, suggesting that rhoptries, micronemes and DGs are related to organelles such as secretory lysosomes that are well known to mainstream cell biologists.     

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.     

Protein trafficking to apical organelles of malaria parasites - building an invasion machine.

Malaria is caused by four species of apicomplexan protozoa belonging to the genus Plasmodium. These parasites possess a specialized collection of secretory organelles called rhoptries, micronemes and dense granules (DGs) that in part facilitate invasion of host cells. The mechanism by which the parasite traffics proteins to these organelles as well as regulates their secretion has important implications for understanding the invasion process and may lead to development of novel intervention strategies. In this review, we focus on emerging data about trafficking signals, mechanisms of biogenesis and secretion. At least some of these are conserved in higher eukaryotes, suggesting that rhoptries, micronemes and DGs are related to organelles such as secretory lysosomes that are well known to mainstream cell biologists.     

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca²⁺ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca²⁺, and we show that this Ca²⁺‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca²⁺ signalling plays in the invasion biology of apicomplexan parasites.     

Distribution of Cryptosporidium parvum sporozoite apical organelles during attachment to and internalization by cultured biliary epithelial cells

Although accumulating evidence supports an active role for host cells during Cryptosporidium parvum invasion of epithelia, our knowledge of the underlying parasite-specific processes triggering such events is limited. In an effort to better understand the invasion strategy of C. parvum, we characterized the presence and distribution of the apical organelles (micronemes, dense granules, and rhoptry) through the stages of attachment to, and internalization by, human biliary epithelia, using serial-section electron microscopy. Novel findings include an apparent organized rearrangement of micronemes upon host cell attachment. The apically segregated micronemes were apposed to a central microtubule-like filamentous structure, and the more distal micronemes localized to the periphery and apical region of the parasite during internalization, coinciding with the formation of the anterior vacuole. The morphological observations presented here extend our understanding of parasite-specific processes that occur during attachment to, and internalization by, host epithelial cells.     

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.     

Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis.

The dense granules of the intracellular protozoan Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole (PV) in which the parasite develops. The biogenesis of dense granules as well as the regulatory mechanisms controlling their specific exocytosis are still poorly understood. In this paper, we analyzed the secretory pathway of dense granule proteins (GRA proteins) in extracellular T. gondii through the effects of brefeldin A (BFA). Ultrastructural studies of BFA-treated parasites showed disassembly of the Golgi apparatus and accumulation of GRA proteins in a dilated vacuolar system connected to the nuclear envelope. BFA reversibly blocked the intracellular transport of the newly synthesized GRA proteins in a dose-dependent manner (blockade of 95% at 1 microg/ml of BFA). By contrast, discharge of GRA proteins from preformed dense granules was unaffected by BFA over a course of 60 min incubation. GRA protein secretion was dependent on incubation temperature as it only occurred above 26 degrees C and it could be stimulated by external factors. This stimulus might be provided by factor(s) present in the serum of the extracellular medium, as incubation of parasites in serum-free medium resulted in a dramatic decrease in protein secretion. Exocytosis can be restored in a dose-dependent fashion by serum addition (maximal stimulatory activity in the 30-200 kDa range) and was optimal at an extracellular pH of 6.5. Altogether, these results demonstrate that GRA proteins are exported through the Golgi apparatus via the classical secretory pathway and can be experimentally discharged from storage dense granules as regulated secretory proteins in response to specific stimulation, arguing in favor of a regulated component for dense granule exocytosis in T. gondii.     

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.     

Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography

Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.     

The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide

Host cell invasion is a key step in the life cycle of the intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis. Attachment and invasion by this parasite is dependent on secretion of proteins from the micronemes, cigar-shaped organelles found in the apical end of the parasite. Although many of these proteins contain adhesive motifs suggestive of a role in parasite attachment, a growing subset of microneme proteins (MICs) do not possess adhesive sequences implying that they have alternative roles. We have identified a novel 16 kDa microneme protein, TgMIC11, that is conserved among several coccidian parasites. As it traffics through the secretory system, TgMIC11 is modified by two successive proteolytic events to remove an internal propeptide, resulting in the mature protein that consists of an alpha-chain and beta-chain tethered by a single disulfide bond. Dual staining immunofluorescence confirmed that TgMIC11 localises to the apical micronemes and, like other micronemal proteins, it is also secreted in a calcium dependent manner. This is the first microneme protein characterised to date in the phylum Apicomplexa that possesses this unique structure and undergoes maturation by removal of an internal propeptide.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Targeting and subcellular localization of Toxoplasma gondii catalase. Identification of peroxisomes in an apicomplexan parasite

We sought to identify and characterize peroxisomes in the apicomplexan parasite Toxoplasma gondii. To initiate this process, we first cloned and sequenced the gene for T. gondii catalase (EC 1. 11.1.6), a marker enzyme for peroxisomes in eukaryotic cells. The gene predicts a protein of 57.2 kDa and 502 amino acids and has a strong homology to other eukaryotic catalases. A polyclonal antiserum raised against a glutathione S-transferase fusion protein recognized a single band with a molecular mass of 63 kDa by immunoblot. By immunofluorescence T. gondii catalase is present primarily in a punctate staining pattern anterior to the parasite nucleus. This compartment is distinguishable from other parasite organelles, namely micronemes, rhoptries, dense granules, and the apicoplast. Cytochemical visualization of catalase using diaminobenzidine precipitation gives a vesicular staining pattern anterior to the nucleus at the light level and round, vesicular structures with an estimated diameter of 100-300 nm by electron microscopy. T. gondii catalase has a putative C-terminal peroxisomal targeting signal in the last 3 amino acids (-AKM). Expression of T. gondii catalase in mammalian cells results in peroxisomal localization, whereas a construct lacking the targeting signal remains in the cytosol. Furthermore, addition of -AKM to the C terminus of chloramphenicol acetyltransferase is sufficient to target this protein to peroxisomes. These results provide the first evidence for peroxisomes in Apicomplexan parasites.     

Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii

The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural differences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5-4 microm, than those of T. gondii which were smooth and 0.5 microm thick.     

Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.     

Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.     

The Protozoan Parasite Toxoplasma gondii Targets Proteins to Dense Granules and the Vacuolar Space Using Both Conserved and Unusual Mechanisms

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.