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.     

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.     

Signalling mechanisms involved in apical organelle discharge during host cell invasion by apicomplexan parasites

Malaria is caused by Plasmodium parasites, which belong to the phylum apicomplexa. The characteristic feature of apicomplexan parasites is the presence of apical organelles, referred to as micronemes and rhoptries, in the invasive stages of the parasite life cycle. Survival of these obligate intracellular parasites depends on successful invasion of host cells, which is mediated by specific molecular interactions between host receptors and parasite ligands that are commonly stored in these apical organelles. The timely release of these ligands from apical organelles to the parasite surface is crucial for receptor engagement and invasion. This article is a broad overview of the signalling mechanisms that control the regulated secretion of apical organelles during host cell invasion by apicomplexan parasites.     

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.     

Plasmodium rhoptries: how things went pear-shaped

Plasmodium parasites have three sets of specialised secretory organelles at the apical end of their invasive forms--rhoptries, micronemes and dense granules. The contents of these organelles are responsible for or contribute to host cell invasion and modification, and at least four apical proteins are leading vaccine candidates. Given the unusual nature of Plasmodium invasion, it is not surprising that unique proteins are involved in this process. Nowhere is this more evident than in rhoptries. We have collated data from several recent studies to compile a rhoptry proteome. Discussion is focussed here on rhoptry content and function.     

Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis

Invasion and egress are two key steps in the lytic cycle of Apicomplexa that are governed by the sequential discharge of proteins from two apical secretory organelles called micronemes and rhoptries. In Toxoplasma gondii, the biogenesis of these specialized organelles depends on the post Golgi trafficking machinery, forming an endosomal‐like compartment (ELC) resembling endomembrane systems found in eukaryotes. In this study, we have characterized four phylogenetically related Transporter Facilitator Proteins (TFPs) conserved among the apicomplexans. TFP1 localises to the micronemes and ELC, TFP2 and TFP3 to the rhoptries and TFP4 to the Golgi. TFP1 crucially contributes to parasite fitness and survival while the other members of this family are dispensable. Conditional depletion of TFP1 impairs microneme biogenesis and leads to a complete block in exocytosis, which hampers gliding motility, attachment, invasion and egress. Morphological investigations revealed that TFP1 participates in the condensation of the microneme content, suggesting the transport of a relevant molecule for maintaining the intraluminal microenvironment necessary for organelle maturation and exocytosis. In absence of TFP2, rhoptries adopt a considerable elongated shape, but the abundance, processing or secretion of the rhoptry content are not affected. These findings establish the relevance of TFPs in organelle maturation of T. gondii.     

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.     

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.     

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.     

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.     

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.     

An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.     

Biogenesis and secretion of micronemes in Toxoplasma gondii

One of the hallmarks of the parasitic phylum of Apicomplexa is the presence of highly specialised, apical secretory organelles, called the micronemes and rhoptries that play critical roles in ensuring survival and dissemination. Upon exocytosis, the micronemes release adhesin complexes, perforins, and proteases that are crucially implicated in egress from infected cells, gliding motility, migration across biological barriers, and host cell invasion. Recent studies on Toxoplasma gondii and Plasmodium species have shed more light on the signalling events and the machinery that trigger microneme secretion. Intracellular cyclic nucleotides, calcium level, and phosphatidic acid act as key mediators of microneme exocytosis, and several downstream effectors have been identified. Here, we review the key steps of microneme biogenesis and exocytosis, summarising the still fractal knowledge at the molecular level regarding the fusion event with the parasite plasma membrane.     

A transient forward-targeting element for microneme-regulated secretion in Toxoplasma gondii.

BACKGROUND INFORMATION: Accurate sorting of proteins to the three types of secretory granules in Toxoplasma gondii is crucial for successful cell invasion by this obligate intracellular parasite. As in other eukaryotic systems, propeptide sequences are a common yet poorly understood feature of proteins destined for regulated secretion, which for Toxoplasma occurs through two distinct invasion organelles, rhoptries and micronemes. Microneme discharge during parasite apical attachment plays a pivotal role in cell invasion by delivering adhesive proteins for host receptor engagement. RESULTS: We show here that the small micronemal proprotein MIC5 (microneme protein-5) undergoes proteolytic maturation at a site beyond the Golgi, and only the processed form of MIC5 is secreted via the micronemes. Proper cleavage of the MIC5 propeptide relies on an arginine residue in the P1' position, although P1' mutants are still cleaved to a lesser extent at an alternative site downstream of the primary site. Nonetheless, this aberrantly cleaved species still correctly traffics to the micronemes, indicating that correct cleavage is not necessary for micronemal targeting. In contrast, a deletion mutant lacking the propeptide was retained within the secretory system, principally in the ER (endoplasmic reticulum). The MIC5 propeptide also supported correct trafficking when exchanged for the M2AP propeptide, which was recently shown to also be required for micronemal trafficking of the TgMIC2 (T. gondii MIC2)-M2AP complex [Harper, Huynh, Coppens, Parussini, Moreno and Carruthers (2006) Mol. Biol. Cell 17, 4551-4563]. CONCLUSION: Our results illuminate common and unique features of micronemal propeptides in their role as trafficking facilitators.     

Molecular interactions and signaling mechanisms during erythrocyte invasion by malaria parasites

Invasion of erythrocytes by Plasmodium merozoites is a complex process that is mediated by specific molecular interactions. Here, we review recent studies on interactions between erythrocyte binding antigens (EBA) and PfRH proteins from the parasite and erythrocyte receptors involved in invasion. The timely release of these parasite ligands from internal organelles such as micronemes and rhoptries to the merozoite surface is critical for receptor-engagement leading to successful invasion. We review information on signaling mechanisms that control the regulated secretion of parasite proteins during invasion. Erythrocyte invasion involves the formation and movement of a junction between the invading merozoite and host erythrocyte. We review recent studies on the molecular composition of the junction and the molecular motor that drives movement of the junction.     

Protein trafficking inside Toxoplasma gondii

The accurate targeting of proteins to their final destination is an essential process in all living cells. Apicomplexans are obligate intracellular protozoan parasites that possess a compartmental organization similar to that of free-living eukaryotes but can be viewed as professional secretory cells. Establishment of parasitism involves the sequential secretion from highly specialized secretory organelles, including micronemes, rhoptries and dense granules. Additionally, apicomplexans harbor a tubular mitochondrion, a nonphotosynthetic plastid organelle termed the apicoplast, acidocalcisomes and an elaborated inner membrane complex composed of flattened membrane cisternae that are derived from the secretory pathway. Given the multitude of destinations both inside and outside the parasite, the endoplasmic reticulum/Golgi of the apicomplexans constitutes one of the most busy roads intersections in eukaryotic traffic.     

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.     

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal‐Like Compartments and the Golgi Complex

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post‐Golgi and it has been speculated that they pass through endosomal‐like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well‐established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant‐like secretory pathway .     

Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10

Hoff, E. F., Cook, S. H., Sherman, G. D., Harper, J. M., Ferguson, D. J. P., Dubremetz, J. F., and Carruthers, V. B. 2001. Toxoplasma gondii: Molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology, 97, 77-88. During host cell invasion, Toxoplasma gondii secretes proteins from specialized organelles (micronemes and rhoptries) located at the apical end of the parasite. The contents of the micronemes appear to be crucial to T. gondii invasion, as inhibition of microneme secretion prevents parasite entry into host cells. Here we describe a new T. gondii microneme protein, TgMIC10. Molecular characterization of a full-length TgMIC10 cDNA revealed that TgMIC10 lacks homology to any previously characterized proteins, although a homologue, NcMIC10, was identified in a closely related parasite, Neospora caninum. TgMIC10 has an unusually long secretory leader sequence of 58 amino acids; the mature TgMIC10 is 18 kDa, possesses nine diglutamic acid repeats and an imperfect repeat sequence (RK(R/Y)HEEL), and is entirely devoid of cysteines. Antibodies raised against recombinant TgMIC10 recognized the native TgMIC10 and localized the protein to the micronemes in indirect immunofluorescence and immunoEM experiments. Comparison of immunofluorescence images indicates that TgMIC10 expression is higher in T. gondii tachyzoites, which are responsible for active infection, than in bradyzoites, which are responsible for latent infection.