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.     

Evolutionary repurposing of endosomal systems for apical organelle biogenesis in Toxoplasma gondii

It is very difficult to define an endocytic system in Toxoplasma gondii. The parasite does not appear to take up exogenous materials via classical endocytosis. The presence of Rab5 and Rab7, classical markers of endocytic compartments, and their decoration of endomembranous structures suggest, however, that an endosomal-like system may operate. Additionally, new findings reveal that dynamin and the transmembrane type-I receptor sortilin are involved in the biogenesis of T. gondii micronemes and rhoptries, unique apical secretory organelles required for parasite migration and host–cell invasion, manipulation and egress. Evidence suggests that the parasite uses an endosomal-like system to traffic and sort proteins to rhoptries and micronemes via the endoplasmic reticulum and Golgi. In this review, I discuss recent findings suggesting that T. gondii and other apicomplexans have reduced their endosomal system and repurposed the evolutionarily conserved regulators of the system to build the apical secretory organelles. This review is also intended to serve as a resource for future investigations of apicomplexan biology and evolution.     

Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection

Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles. TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.     

Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii

The micronemal protein 2 (MIC2) of Toxoplasma gondii shares 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 of T. 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.     

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.     

Assembly and export of a Toxoplasma microneme complex in Giardia lamblia

The microneme proteins of Toxoplasma gondii belong to a large family of adhesins of apicomplexan parasites involved in motility and host cell invasion. During secretory transport, soluble micronemes associate with membrane-bound carriers/escorters and become exposed on the parasite surface as complexes with an array of adhesive domains. Previously, we have exploited the intestinal protozoan Giardia lamblia as an expression system to produce correctly folded and unglycosylated monomeric surface proteins of T. gondii. Here, we report assembly and export of a trimeric microneme (MIC1/4/6) adhesin complex from Toxoplasma. Co-expressed, recombinant microneme proteins were used to investigate structural requirements for microneme complex formation. In addition, export of a microneme subunit induced development of novel Golgi-like compartments demonstrating the existence of post endoplasmic reticulum structures involved in constitutive secretion in this 'Golgi-less' cell. Recreation of the trimeric microneme escorter-cargo system in Giardia is a versatile tool to analyse universal requirements for complex assembly, receptor-ligand interactions and Golgi neogenesis in the basal Giardia secretory system.     

The Role of Clathrin in Post-Golgi Trafficking in Toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.     

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.     

The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin‐based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.     

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.     

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.     

Comparative genomics of the Rab protein family in Apicomplexan parasites

Rab genes encode a subgroup of small GTP-binding proteins within the ras super-family that regulate targeting and fusion of transport vesicles within the secretory and endocytic pathways. These genes are of particular interest in the protozoan phylum Apicomplexa, since a family of Rab GTPases has been described for Plasmodium and most putative secretory pathway proteins in Apicomplexa have conventional predicted signal peptides. Moreover, peptide motifs have now been identified within a large number of secreted Plasmodium proteins that direct their targeting to the red blood cell cytosol, the apicoplast, the food vacuole and Maurer's clefs; in contrast, motifs that direct proteins to secretory organelles (rhoptries, micronemes and microspheres) have yet to be defined. The nature of the vesicle in which these proteins are transported to their destinations remains unknown and morphological structures equivalent to the endoplasmic reticulum and trans-Golgi stacks typical of other eukaryotes cannot be visualised in Apicomplexa. Since Rab GTPases regulate vesicular traffic in all eukaryotes, and this traffic in intracellular parasites could regulate import of nutrient and drugs and export of antigens, host cell modulatory proteins and lactate we compare and contrast here the Rab families of Apicomplexa.     

A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex

Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection.     

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.     

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.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

Structural Mechanisms for Regulation of Membrane Traffic by Rab GTPases

In all eukaryotic organisms, Rab GTPases function as critical regulators of membrane traffic, organelle biogenesis and maturation, and related cellular processes. The numerous Rab proteins have distinctive yet overlapping subcellular distributions throughout the endomembrane system. Intensive investigation has clarified the underlying molecular and structural mechanisms for several ubiquitous Rab proteins that control membrane traffic between tubular-vesicular organelles in the exocytic, endocytic and recycling pathways. In this review, we focus on structural insights that inform our current understanding of the organization of the Rab family as well as the mechanisms for membrane targeting and activation, interaction with effectors, deactivation and specificity determination.     

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3 and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.     

Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14‐fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC‐MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.     

EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats

Micronemes are specialised secretory organelles that release their proteins by a stimulus-coupled exocytosis that occurs when apicomplexan parasites make contact with target host cells. These proteins play crucial roles in motility and invasion, most likely by mediating adhesion between parasite and host cell surfaces and facilitating the transmission of dynamic forces generated by the parasite actinomyosin cytoskeleton. Members of the TRAP family of microneme proteins are characterised by having extracellular domains containing one or more types of cysteine-rich, adhesive modules, highly-conserved transmembrane regions and cytosolic tails that contain one or more tyrosines, stretches of acidic residues and a single tryptophan. In this paper, we describe a novel member of the TRAP family, EtMIC4, a 218 kDa microneme protein from Eimeria tenella. EtMIC4 contains 31 epidermal growth factor (EGF) modules, 12 thrombospondin type-1 (TSP-1) modules and a highly acidic, proline and glycine-rich region in its extracellular region, plus the conserved transmembrane and cytosolic tail. Like EtMIC1, another TRAP family member from E. tenella, EtMIC4 is expressed in sporozoites and all the merozoite stages of the parasite, suggesting that this parasite has a strong requirement for TSP-1 modules. Unlike the other microneme proteins so far studied in E. tenella, EtMIC4 appears to be found constitutively on the sporozoite surface as well as within the micronemes.