Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane‐bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K⁺) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca²⁺), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca²⁺ are not completely understood. Here, we show that a P. falciparum Ca²⁺‐dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca²⁺‐dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K⁺ environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K⁺ environment resulted in inhibition of secretion of microneme protein apical merozoite antigen‐1 (PfAMA‐1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca²⁺‐dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood‐stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.     

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.     

TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells

The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca²⁺-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca²⁺ ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K⁺ concentration of the host cell. Our results therefore suggest that TgCDPK3''s role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca²⁺ signaling in specific ionic environments to upregulate multiple processes required for gliding motility.     

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.     

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.     

Analysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules

Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca²⁺ signaling although the temporal dynamics of Ca²⁺ fluxes and down‐stream second messenger pathways are poorly understood. Here, we use a genetically encoded biosensor, GFP‐Calmodulin‐M13–6 (GCaMP6), to capture Ca²⁺ flux in live Toxoplasma and investigate the role of Ca²⁺ signaling in egress and motility. Our analysis determines how environmental cues and signal activation influence intracellular Ca²⁺ flux, allowing placement of effector molecules within this pathway. Importantly, we have identified key interrelationships between cGMP and Ca²⁺ signaling that are required for activation of egress and motility. Furthermore, we extend this analysis to show that the Ca²⁺ Dependent Protein Kinases–TgCDPK1 and TgCDPK3—play a role in signal quenching before egress. This work highlights the interrelationships of second messenger pathways of Toxoplasma in space and time, which is likely required for pathogenesis of all apicomplexan species.     

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

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

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.     

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.     

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca²⁺ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.     

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.     

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 .     

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.     

Intersection of endocytic and exocytic systems in Toxoplasma gondii

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome‐like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant‐like features suggest T. gondii endocytic trafficking involves transit through early and late endosome‐like compartments (ELCs) and potentially the trans‐Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin‐related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post‐invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host‐derived proteins, suggesting that DrpB is not required for parasite endocytosis.      

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis

The inner membrane complex and the apical secretory organelles are defining features of apicomplexan parasites. Despite their critical roles, the mechanisms behind the biogenesis of these structures in the malaria parasite Plasmodium falciparum are still poorly defined. We here show that decreasing expression of the P. falciparum homologue of the conserved endolysomal escorter Sortilin‐VPS10 prevents the formation of the inner membrane complex and abrogates the generation of new merozoites. Moreover, protein trafficking to the rhoptries, the micronemes, and the dense granules is disrupted, which leads to the accumulation of apical complex proteins in the endoplasmic reticulum and the parasitophorous vacuole. We further show that protein export to the erythrocyte and transport through the constitutive secretory pathway are functional. Taken together, our results suggest that the malaria parasite P. falciparum Sortilin has potentially broader functions than most of its other eukaryotic counterparts.     

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.     

Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

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.