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Biogenesis and secretion of micronemes in Toxoplasma gondii
DUBOIS, David Jean, SOLDATI-FAVRE, Dominique
DUBOIS, David Jean, SOLDATI-FAVRE, Dominique. Biogenesis and secretion of micronemes in Toxoplasma gondii. Cellular Microbiology, 2019, p. e13018
DOI : 10.1111/cmi.13018
Available at:
http://archive-ouverte.unige.ch/unige:114657
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may Dubois David (Orcid ID: 0000-0001-9814-1677)
Soldati-Favre Dominique (Orcid ID: 0000-0003-4156-2109) Hartland Elizabeth (Orcid ID: 0000-0003-4254-2863)
Biogenesis and secretion of micronemes in Toxoplasma gondii
David J. Dubois1*and Dominique Soldati-Favre1
*Correspondence: David Dubois E-mail: David.Dubois@unige.ch
1Department of Microbiology and Molecular Medicine, University of Geneva CMU, Geneva 4, Switzerland.
Keywords: Apicomplexa, Toxoplasma gondii, Plasmodium, microneme, secretory organelles, exocytosis, membranous fusion, signaling, invasion.
Abstract
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.
INTRODUCTION
The Apicomplexa phylum comprises a large group of protozoan parasites of significant public health and veterinary importance. The most notable members of this phylum for human health are Plasmodium spp., Cryptosporidium spp. and Toxoplasma gondii, the causative agents of malaria, cryptosporidiosis and toxoplasmosis, respectively. The ability of these obligate intracellular parasites to invade and egress from host cells in a tightly regulated manner is essential for their survival and dissemination. The Apicomplexa possess a unique set of specialised, apical secretory organelles, called micronemes and rhoptries that discharge their contents in a sequential manner (Carruthers et al., 1997). These parasites also contain a third type of secretory organelle, the dense granules, which are ubiquitously distributed in the cytosol and constitutively release their content to remodel the parasitophorous vacuole and reprogram the host cell (Hallee et al., 2018). During exocytosis, the micronemes release, among other proteins, adhesin complexes and perforins that critically participate in egress from infected cells, gliding motility, migration across organs and biological barriers and host cell invasion (Soldati et al., 2001). More restrictively, rhoptry discharge occurs only when parasites initiate the invasion process. The proteins located in the neck (RONs) of the organelle are associated to host cell invasion (Dubremetz, 2007, Boothroyd et al., 2008) while the ones in the bulb (ROPs) to the subversion of host cell functions (Bradley et al., 2007, Kemp et al., 2013). The rhoptries also discharge membranous materials that contribute to the formation of the parasitophorous vacuole membrane (PVM) (Dubremetz, 2007). The mechanisms by which these two organelles secrete their contents are still poorly understood.
MICRONEME DIVERSITY AND STRUCTURE
The apicomplexans exhibit complex life cycles involving differentiation into morphologically very distinct stages. The motile and invasive abilities of the zoites are critically dependent on micronemes or microneme-like organelles exocytosis. These abundant rod-like organelles clustered at the apical pole of the parasites were first described in the 60s by electron microscopy (EM) as part of the apical complex that morphologically characterizes the members of Apicomplexa (Garnham et al., 1962, Scholtyseck et al., 1970). The relative abundance of microneme organelles varies considerably between species and life stages.
Variation amongst species is unmistakable when comparing Eimeria or Sarcocystis to T.
gondii, as these parasites have many fold more micronemes than the latter (Figure 1) (Dubremetz et al., 2009). Their morphology also vary across the phylum. In T. gondii the micronemes are small rod shaped (Garnham et al., 1962), while they adopt a bottle-like structure in Plasmodium (Bannister et al., 2003, Schrevel et al., 2008). Stage-specific variation is also evident, where Plasmodium spp. merozoites have very few micronemes compared to ookinetes or sporozoites, similarly T. gondii tachyzoites possess fewer micronemes compared to bradyzoites and sporozoites (Dubey et al., 1998). The biological rationale for this considerable variation is likely correlated the ability to glide for various time length, the migratory distance, tissue specificity and accessibility of suitable host cells.
Micronemes also display diversity in terms of both subpopulations and degree of maturity.
These subpopulations have been characterised as containing distinct localisations and protein compositions. Microneme diversity in T. gondii has been observed by super-resolution microscopy, which identified two distinct populations, both of which appear to be trafficked differently (Kremer et al., 2013). Additionally, distinctions have been made between the apical (concentrated at/beneath the conoid) and the peripheral/lateral (peripheral and down the parasite body) distribution of micronemes in T. gondii. These distinctions originate from
the observation that a protein called parafusin (discussed in a later chapter) exclusively co- localises with the apical micronemes (Matthiesen et al., 2001) and that the depletion of certain members of the endosomal tethering complexes (CORVET/HOPS complexes) leads to loss of certain microneme contents in the peripheral micronemes (Morlon-Guyot et al., 2018).
Plasmodium spp. present an assortment of micronemes and microneme-like organelles. It is important to emphasise that the classical definition of micronemes refers to apical organelles involved in invasion and motility, while the microneme-like organelles are seemingly exclusively involved in egress. Typically, sporozoites, merozoites and ookinetes possess micronemes, while merozoites have additional distinct microneme-like organelles named exonemes and mononemes that represent distinct organelles that contain subtilisin protease 1 (SUB1) and rhomboid protease 1 (ROM1), respectively (Singh et al., 2007, Yeoh et al., 2007). Osmiophilic bodies and egress vesicles represent an exclusive class of organelles identified within gametocytes. These two organelles have been shown to have numerous subpopulations and to contain a variety of gametocyte-specific proteins, as of yet exclusively involved in egress, some have been found to be sex-specific. (Deligianni et al., 2013, Olivieri et al., 2015, Kehrer et al., 2016a). Differences in the maturation status of Plasmodium micronemes has been documented through morphological changes, where spherical
“immature” micronemes evolve into bottle shaped micronemes in sporozoites (Schrevel et al., 2008).
MICRONEME CONTENTS AND CONSERVATION ACROSS THE PHYLUM
Micronemes contain a large array of proteins, which underpin many biological functions driving the escape from the vacuole, adhesion, motility, rhoptry secretion and host cell invasion. In this review, we will non-exhaustively discuss categories of microneme proteins based on their roles.
Perforin-like proteins (PLP) are distinct pore forming proteins that are contained within micronemes, upon release these proteins form complexes which compromise the integrity of the PV and host cell membrane, thereby enabling egress. PLPs are one of the key proteins, which are typically conserved amongst micronemes and microneme-like organelles. (Kafsack et al., 2009, Kafsack et al., 2010, Deligianni et al., 2013).
Adhesins, comprise a specific and dominant class of microneme proteins. These proteins can be classified by their adhesive repeat domains, which permit binding to a variety of host cell surface antigens comprising proteins and carbohydrates, such as collagen and sialic acid (Tomley et al., 2001). Adhesins are critically involved in host cell attachment, motility and invasion. In T. gondii, these adhesins are called MICs and usually assemble into complexes that form functional units (Huynh et al., 2003, Huynh et al., 2006). These complexes assemble during trafficking in the early compartments of the secretory pathway and coordinate the correct targeting of certain soluble subunits (Reiss et al., 2001, Harper et al., 2006). Several MICs are known to have an array of adhesive repeat domains that permit binding to a variety of host cell surface antigens comprising proteins and carbohydrates, such as collagen and sialic acid. The diversity of adhesins among the apicomplexans likely reflects parasite adaptation to environmental niches and host cell (Tomley et al., 2001, Boucher et al., 2015).
Upon microneme exocytosis, the adhesin complexes distribute on the parasite surface anchored in the parasite plasma membrane via the transmembrane spanning domain
containing proteins. The cytoplasmic C-terminus will interact and be translocated to the rear of the parasite by a phylum specific actomyosin system. This rearward translocation generate a forward movement, critical for gliding motility, cell traversal egress and host cell invasion (Frenal et al., 2013).
Intriguingly, rhoptry discharge relies on certain microneme proteins such as TgMIC8, Erythrocyte binding antigen-175 (PfEBA175). While this feature is conserved in both T.gondii and Plasmodium spp. the biological mechanisms which cause this are unknown (Kessler et al., 2008, Singh et al., 2010).
Invasion requires the highly conserved apicomplexan apical membrane antigen 1 (AMA1) to assemble with a complex of RONs to form the moving junction. This key structure brings the parasite and host plasma membrane in close proximity establishing the site of parasite entry into the host cell. Like for the adhesins, the actomyosin driven motility then enables the parasite to successfully invade the host cell (Mital et al., 2005, Bargieri et al., 2013, Lamarque et al., 2014).
Secreted-serine proteases (subtilisins) are key enzymes within micronemes, upon release these enzymes are involved in extensive processing of the adhesins. Cleaving adhesins through proteolysis acts to release bound host receptors, enabling forward motion. The constitutively secreted rhomboid proteases also perform this function (Dowse et al., 2004, Zhou et al., 2004, Baker et al., 2006, Lagal et al., 2010, Shen et al., 2014). Uniquely to Plasmodium SUB1 has been linked to facilitating egress from the parasitophorous vacuole (PV) (Tawk et al., 2013, Thomas et al., 2018). Table 1 provides a reference for specific highlighted proteins throughout this review, detailing localisation, role for microneme/rhoptries and conservation throughout apicomplexa.
MICRONEME ORGANELLE BIOGENESIS MATURATION AND TRAFFICKING.
The trafficking of proteins to the micronemes has been investigated and recently covered by a comprehensive review (Venugopal et al., 2018). Remarkably, the apicomplexans have repurposed the endosomal system to build the micronemes, rhoptries and dense granules.
Several classical endocytic trafficking regulators have been shown to be involved in apical secretory organelle formation including the Sortilin Receptor (Sortilin), the Dynamin related protein B (DrpB), Rab5A/C, Vacuolar Protein Sorting-associated proteins (VPS), Adaptor protein 1 (AP1) and BEACH domain-containing protein (BDCP) (Sangare et al., 2016).
The Sortilin, a conserved eukaryotic transmembrane Golgi receptor, mediates protein and vesicular trafficking between the Golgi, endosome, lysosomes and plasma membrane (Hermey, 2009). In T. gondii, conditional depletion of Sortilin leads to a severe defect in rhoptry and microneme biogenesis without apparently affecting the other organelles (Sloves et al., 2012). Intriguingly, a more recent study uncovered a broader role for Sortilin in Plasmodium in addition to its role in apical secretory organelle biogenesis. PfSortilin participates in the formation of the inner membrane complex consisting of joined flattened vesicles and to the the dense granules (Kono et al., 2013).
Dynamins consist of a family of large GTPase proteins typically involved in endocytic vesicle formation and trafficking (Konopka et al., 2006). T. gondii DrpB localises proximally to both Golgi and the endosome-like compartment (ELC) (Breinich et al., 2009), a subcompartment of the secretory pathway involved in the proteolytic maturation of MICs, ROPs and RONs (Parussini et al., 2010, Dogga et al., 2017). TgDrpB participates in rhoptry and microneme biogenesis and the expression of a dominant negative mutant of TgDrpB causes the secretory proteins to be constitutively diverted into the PV (Breinich et al., 2009).
Similarly, expression of dominant negative variants of the small GTPases Rab5A and 5C in
T. gondii leads to the constitutive secretion of rhoptry proteins, while only a subset of the MICs (MIC3, MIC8 and MIC11) were directed into the PV (Kremer et al., 2013).
Certain vacuolar protein sorting-associated proteins are members of the endosomal tethering complexes (HOPs/CORVET complexes). Depletion of VSPs in T. gondii causes extensive trafficking defects, notably the mistrafficking of microneme, rhoptry and dense granule proteins. Importantly, independent depletion of TgVPS8, 9, 11, 18, 39 and the clathrin adaptor protein 1 (TgAP1) all affect a subset of microneme proteins (typically MIC2, MIC6 and AMA1) that become detectable exclusively in the apical micronemes, while other microneme proteins are misdirected into alternative compartments. These examples of disrupting the classical endosomal trafficking further support the existence of two microneme populations engaged into different trafficking routes (Morlon-Guyot et al., 2015, Sakura et al., 2016, Venugopal et al., 2017, Morlon-Guyot et al., 2018).
Recently, a transporter family 1 protein (TFP1) has been identified as an essential putative transporter localised to the micronemes. Conditional knockdown of TgTFP1 leads to a significant drop in the number of microneme organelles and those left exhibit a severe morphological alteration, forming large ovoid structures instead of adopting the typical rod shape (Hammoudi et al., 2018). At this point, the nature of the substrate(s) transported by either TFP1 or the related transporters present at the rhoptry membranes (TFP2, TFP3) and at the Golgi (TFP4) is not known. The closest homologue of TFP1 in the malaria parasites is a member of the Major Facilitator Superfamily (MFS) (Pao et al., 1998, Madej et al., 2014), which was previously characterized in P. falciparum, P. yoelii and P. berghei as pantothenate transporter (PAT) and localized to the micronemes (Kehrer et al., 2016b). Functional dissection of the PbPAT failed to support a role as a pantothenate transporter, rather the loss of this protein lead to a defect in microneme and osmiophilic body fusion to the plasma membrane in sporozoites and gametocytes, respectively. These observations provide
compelling evidence that both microneme and microneme-like organelles require specific substrates to ensure organelle maintenance and maturation. Similarly, in Plasmodium, the microgamete surface protein (MiGS) is another protein linked to organelle integrity.
Identified in P. yoelii, MiGS contains an aspartyl protease-like domain and is exclusively expressed in male gametocytes, localising to the male osmiophilic body (MOB) and microgamete surface. The knockout of PyMiGS leads to a severe reduction in MOB formation and in the inability of the male parasites to exflagellate (Tachibana et al., 2018).
Micronemes are positioned adjacent to the subpellicular microtubules, a single layer of microtubules arranged just beneath the cell membrane and inner membrane complex (IMC), a cytoskeletal structure consisting of flattened vesicles (Kono et al., 2013) (Figure 2A). This has been compellingly observed through high-resolution microscopy in T.gondii and is supported by the fact that microtubule destabilisation leads to spatial redistribution of the micronemes (Leung et al., 2017). This feature appears to be consistent in Plasmodium merozoites (Bannister et al., 2003). To this date, few proteins have been directly identified in positioning micronemes or trafficking them along microtubules, however a recent investigation on a double knockout of Kinesin A and Apical polar ring1 (APR1) showed alteration of the distribution of micronemes along the cortical microtubules (Leung et al., 2017). The dynein light chain 8a (TgDLC8a) is a key component of a microtubule motor complex and proves to be a promising candidate for transporting micronemes to the site of exocytosis. TgDLC8a has been localised to the apical tip, spindle poles, centrioles and basal end (Hu et al., 2006) and in an independent study to the apical region only (Qureshi et al., 2013). Conditional knockdown of TgDLC8a leads to a significant defect in microneme exocytosis. Remarkably, the phenotypic consequences of TgDLC8a depletion is peculiar as egress, motility and attachment are normal while induced microneme secretion and invasion are severely impacted. TgDLC8a is seemingly involved in the apical replenishment of
micronemes, in order to sustain successive waves of microneme exocytosis (Frénal et al.
unpublished).
SIGNALING CASCADE LEADING TO EXOCYTOSIS OF MICRONEMES
Microneme exocytosis is carried out in a highly coordinated manner, the proposed signalling cascade leading to microneme exocytosis initiates with the stimulation of a guanylate cyclase (GC) (Moon et al., 2009, Brown et al., 2018) to produce cGMP and activate the protein kinase G (PKG) (Brown et al., 2017). PKG presumably activates phosphatidylinositol 4- kinase (PI4K), which phosphorylates phosphoinositol (PI) into phosphatidylinositol 4- phosphate (PI4P), which is further converted into phosphatidylinositol 4,5-bisphosphate (PI4,5P2) via the action of phosphatidylinositol 4,5-bisphosphate kinase (PI4P5K) (Brochet et al., 2014). In T. gondii, phosphoinositide phospholipase C (TgPI-PLC), is positioned at the parasite periphery where it presumably cleaves PI4,5P2 to produce inositol-trisphosphate (IP3) and diacylglycerol (DAG) (Fang et al., 2006, Bullen et al., 2016a). IP3 is believed to stimulate intracellular calcium release from undefined storage compartment(s), which activates the CDPKs. Several reviews cover the implications of TgCDPK1 and TgCDPK3 as well the extensive studies into the Plasmodium CDPKs (Nagamune et al., 2008, Lourido et al., 2015, Brochet et al., 2016). Although their substrates have not been formally identified, these CDPKs upon calcium flux are likely responsible for the phosphorylation of many proteins involved in microneme exocytosis and the actomyosin system activation (Solyakov et al., 2011, Treeck et al., 2011).
In parallel, DAG is converted into phosphatidic acid (PA) via the action of a diacylglycerol kinase 1 (DGK1) at the inner leaflet of the parasite plasma membrane. This transient production of PA is sensed by the plekstrin homology (PH) domain containing protein (TgAPH) which is acylated at the surface of micronemes and ultimately mediates microneme
exocytosis via an unknown mechanism (Bullen et al., 2016a, Darvill et al., 2018). (Figure 2B)
PREPARING MICRONEMES FOR SECRETION
Due to the presence of the IMC that limits access to the plasma membrane micronemes fuse at the apical region of parasite (Figure 2A). In coccidians, a proposed model speculates that the apically positioned micronemes are channelling through the conoid, a hollow apical cone consisting of tightly wound tubulin fibres and then fuse at the tip of the parasites. Microneme secretion has been documented to occur in consecutive waves that are correlated with intracellular fluctuations in Ca2+ (Del Carmen et al., 2009). Furthermore, in coccidians the conoid protrudes and retracts during the process of microneme secretion. For T. gondii, this model proposes an intriguing hypothesis that micronemes are stored on the subpellicular microtubules and prior to exocytosis are docked onto the intra-conoidal microtubules, two short microtubules within centre of the conoid to then fuse with the plasma membrane at the apical tip (Figure 2A). The subsequent retractions of the conoid would possibly allow for the replenishment of the stored subpellicular micronemes. This model is supported in part by the observation of micronemes positioned inside the conoid by ion beam scanning electron microscopy and 3D reconstitution (Figure 3). Alternatively, others postulate that the micronemes are fusing near the apical polar rings, an electron dense ring-like structures just beneath the conoid, which functions as microtubule organising centre (Paredes-Santos et al., 2012). Plasmodium spp. lack a conoid, hence fusion would occur near the apical polar rings (Morrissette et al., 2002). In the malaria merozoites, a model has been proposed where the micronemes would fuse with the rhoptry neck and be concomitantly secreted (Bannister et al., 2003). This option is not plausible for T. gondii where microneme and rhoptries are
discharged in a temporarily sequential manner (Carruthers et al., 1997). Furthermore, microneme secretion is unaffected when rhoptries are mis-positioned (Mueller et al., 2013).
Little is understood about the prerequisite changes, which prepare micronemes for secretion.
Nonetheless, certain micronemal peripheral proteins and apical ring structures have been implicated in this process.TgRNG2 is an apical polar ring protein previously reported to contribute to microneme exocytosis without compromising the structural integrity of the conoid. Intriguingly, the microneme secretion defect observed upon TgRNG2 depletion occurs exclusively when parasites are stimulated with Zaprinast, an inhibitor of phosphodiesterases (Collins et al., 2013) and not in the presence of the calcium ionophore A23187 (Katris et al., 2014). Currently is not clear whether RNG2 provides a signalling platform or plays a more structural or mechanical role.
Centrins are highly conserved eukaryotic calcium binding proteins, typically associated with centrioles or flagellum (Salisbury, 1995). T. gondii possesses three genes coding for centrins.
Centrin 1 (TgCEN1) localises to the centrioles/centrioles, Centrin 2 (TgCEN2) to the apical and basal pole, while Centrin 3 (TgCEN3) localises to the centrosome and faintly to the conoid (Hu et al., 2006). TgCEN2 has previously been involved in the constriction of the basal pole (Frenal et al., 2017). TgCEN2 has recently generated renewed interest with studies indicating that its depletion causes a defect in microneme secretion, consequently leading to severe defects in attachment, motility, invasion, and egress. These findings suggest that microneme secretion could be directly influenced by intracellular Ca2+ level rather than through CDPKs dependent phosphorylation cascades (Leung et al., 2018).
Phosphoglucomutase paralogs, also known as Parafusins have been a focus of interest based on identified but unclear role in Ca2+ mediated exocytosis (Plattner et al., 2005). In Paramecium, Parafusins coat the secretory organelles called tricocysts and are released just prior to exocytosis (Zhao et al., 1998, Liu et al., 2011). T. gondii possesses two parafusin
paralogs, parafusin related protein 1 (TgPRP1) and phosphoglucomutase 2 (TgPGM2).
TgPRP1 has been localised to the apical micronemes (Matthiesen et al., 2001). Independent disruption of each paralog or double knockout is reported to be dispensable for parasite survival, nevertheless lead to a severe disruption in Ca2+ induced microneme secretion while stimulation of exocytosis using other agonists showed no defect (Saha et al., 2017).
MICRONEME FUSION WITH THE PLASMA MEMBRANE
Membrane fusion, the merging of two independent membranes is a kinetically costly process that needs to be selectively controlled in a spatio-temporal manner and consequently involves complex signalling lipids, membrane remodelling, fusion and scaffolding protein interactions (Ungar et al., 2003, Rizo et al., 2012, Dubuke et al., 2016). Phosphatidylinositides are key phospholipids involved in targeted membrane signalling. The exquisite control of this system occurs through tightly regulation of synthesis, inter-conversion and degradation of these lipid classes. The generation of these unique and rare lipid classes allows for targeted lipid-protein interactions, typically mediated by PH domain containing proteins (Lemmon, 2008, Moravcevic et al., 2012). Apicomplexans appear to have conserved this mode of phosphatidylinositides signalling (Bullen et al., 2016b, Wengelnik et al., 2018). TgAPH is viewed as a PA sensor critical for microneme exocytosis. A deeper dissection of the structure-function relationship of TgAPH and PfAPH revealed that the PH domain tightly binds to two PA molecules and bears an essential highly charged upstream linker residue, postulated to further associate with the membranes to presumably bring in close proximity the fusion machinery (Darvill et al., 2018).
SNAREs are widely conserved eukaryotic proteins known to mediate vesicle fusion, thus are likely involved to participate in microneme exocytosis. SNAREs function through the association of reciprocal transmembrane SNAREs (SNARE-like) proteins on each of the
fusing membranes. Cofactors are then subsequently recruited to the SNAREs forming a functional fusion complex. Force generated by this complex bends the membrane leading to the accumulation of cone shaped lipids (ceramide, PA, DAG and lysophospholipids) within the clefts, further enabling membrane curvature (Stace et al., 2006, Lemmon, 2008).
Ultimately, the complex generates sufficient force and proximity to fuse the membranes (Lemmon, 2008, Moravcevic et al., 2012). Many of these fusion related proteins are known to harbour C2 domains, capable of binding to Ca2+ and/or phospholipids. The double C2, Ca2+-binding domain containing proteins (DOC) are also lipid-binding proteins predominantly reported to be interacting with membrane fusion machinery (Pinheiro et al., 2016, Michaeli et al., 2017). Numerous eukaryotic fusion related proteins can be identified in the apicomplexan genomes, yet only three proteins have been characterized so far. DOC2.1 is present in both T. gondii and P. falciparum and implicated in microneme secretion.
TgDOC2.1 was initially identified through the functional complementation of a chemical mutagenesis-based screening mutant exhibiting severe invasion, egress, motility and microneme secretion defects during the temperature restrictive condition. The role of PfDOC2.1 was assessed by N-terminal fusion of a destabilisation domain at the endogenous locus, which partially degraded PfDOC2.1 in the absence of shield and caused mild reductions in invasion and microneme secretion, while egress was apparently unaffected (Farrell et al., 2012). Biochemical examination of PfDOC2.1 revealed that it binds Ca2+ only in the presence of lipids (Jean et al., 2014). While the localisation of TgDOC2.1 is not known, PfDOC2.1 anti-sera revealed a punctate cytosolic and/or plasma membrane staining (Jean et al., 2014).
Furthermore, ferlins belong to a family of multiple C2 domain containing proteins, responsible for an ancient eukaryotic mechanism of membrane fusion predating SNARE machinery and widely documented to be involved in exocytosis (Lek et al., 2012). T. gondii
possesses three putative ferlins, Ferlin 2 (TgFER2) has recently been localised to the apical (enrichment in the conoid) and basal poles. Parasites depleted in TgFER2 fail to secrete rhoptry contents while microneme secretion is unaffected. It is plausible that TgFER1 and possibly the more divergent TgFER3 could be involved in microneme exocytosis (Coleman et al., 2018). A Ferlin and Ferlin-like protein are present in Plasmodium spp., a recent investigation in P. berghei investigated Ferlin-Like Protein (FLP). FLP is reported to be most similar to TgFER1. FLP localises as punctate vesicles in both the asexual erythrocytic stages and gametocytes, yet did not definitively co-localise with known markers of egress related organelles or osmiophilic bodies. Nevertheless depletion in gametocytes leads to parasites unable to egress from the RBC (Obrova et al., 2018).
INSIGHTS FROM OTHER ORGANISMS
Exocytosis of micronemes in apicomplexans currently comprises a patchwork of relevant and yet not comprehensively linked factors. Significant parallels could be gleaned from the in- depth work studying neurosecretion within synapses (Ramakrishnan et al., 2012, Ulloa et al., 2018) or alternative studies into related alveolates and their respective extrusomes (Plattner, 2017).
SNARE assembly complexes of neuronal cells provide valuable insights for the apicomplexan system. Neuronal cells activity involves rapid and co-ordinated exocytosis.
These events require the recruitment of adaptor proteins that facilitate fusion. Tethering complexes are documented to perform such a function, enhancing fusogenic potential.
Proteins such as Sec1/Munc18-like (SM) proteins are well-documented parts of multi- subunit tethering complexes (Archbold et al., 2014, D'Agostino et al., 2017). Considering the rapid fusion events during induced microneme secretion, it is highly likely that similar co- factors would be involved. Similarly, searching for orthologues contributing to SNARE
recruitment-assembly-disassembly would be an interesting and valid avenue of investigation.
However, they would be equally likely to influence other membrane fusion events. The Apicomplexa are divergent from most eukaryotic model organisms, hence drawing close parallels is difficult, however work on the closely related Alveolates can provide better candidates for identifying proteins with conserved biological function (Gubbels et al., 2012).
Ciliates in particular have quite well characterised extrusomes, which depending on species can be involved in predation or defence (Plattner, 2017). These extrusomes are morphologically similar to the rhoptries, yet the regulation of their secretion shares striking similarities to micronemes (Plattner, 2017). Furthermore, numerous chemical mutagenesis and silencing screens have identified biogenesis and non-discharge mutants (Vayssie et al., 2000). Consequently, these resources provide a wealth of untapped resources that can be used to investigate the fundamental biology of the specialized apical secretory organelles.
CONCLUDING REMARKS
Apicomplexans have evolved these unique secretory organelles to assist in the key entry and exit steps of their respective parasitic lifecycle. The mechanisms governing biogenesis, signalling and exocytosis of these atypical organelles is composed of a fractal series of key information and few known factors. The immediate challenge is to connect these factors to fill the mechanistic gaps. A clearly discernible theme is the distinct interplay between classical eukaryotic proteins and unique apicomplexan proteins controlling these processes.
Maturation and exocytosis remain the most poorly understood of these subjects, therefore, some of the many interesting avenues of research topics to broach would be the fusion event itself and the mechanisms underpinning the controlled secretion and replenishment of micronemes at the apical tip of the parasites.
ACKNOWLEDGEMENTS
We are grateful to Drs Jean Francois Dubremetz, David Ferguson and Bohumil Maco for generously sharing EM pictures. We thank past and present lab members for their contributions and the Swiss National Science Foundation (FN3100A0-116722 to DSF) for supporting the work on the biology of Toxoplasma gondii. D.J.D is supported by IZRJZ3164183 (to Dr. Karine Frenal). Results incorporated in study received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant agreement no. 695596.
CONFLICT OF INTEREST
We declare to have no conflicts of interest.
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Figure 1. Varied microneme abundance in Apicomplexa.
Electron microscopy images of A. Sarcocystis muris cystozoite (Scale bar: 2μm) and B.
Toxoplasma gondii bradyzoite (Scale bar: 500nm). Micronemes (M) Image A. provided by JF. Dubremetz B. Reproduced with permission (Dubremetz et al., 2009).
Figure 2. Schematic diagram of signalling and exocytosis related proteins.
A. Schematic representation of Toxoplasma gondii tachyzoite, highlighting exocytosis related proteins. B. Diagram of signalling cascade. C. Zoom in on microneme fusion with parasite plasma membrane.
Parasite plasma membrane (PPM), Inner membrane complex (IMC), Apical polar ring (APR), Acylated plekstrin homology domain containing protein (APH), Phosphatidic acid (PA), Diacylglycerol (DAG), Diacylglycerol kinase 1 (DGK), Inositol-trisphosphate (IP3), Phosphoinositide phospholipase C (PI-PLC), Protein kinase G (PKG), Guanylate cyclase (GC) Acylated plekstrin homology domain containing protein (APH), Double C2 containing protein (DOC2.1), Parafusins (PRP1/PGM2), Centrin 2 (Cen2), Dynein light chain 8a (DLC8a), Ring2 (RNG2), Apical polar ring 1 (APR1), Kinesin A and a representative V-T SNARE complex.
Figure 3. 3D reconstruction showing micronemes in conoid.
A-D. Orthogonal views of apical part of the T. gondii tachyzoite from imaged volume acquired with focused ion beam SEM (FIBSEM) microscope and (B, D) corresponding 3D
reconstruction, highlighted the distribution of the apical micronemes (blue) around or inside the conoid (transparent black) together with rhoptry’s necks (transparent yellow), dense granules (transparent brown) and tachyzoite plasma membrane (transparent cyan) for ΔKU80 (A,B) and TFP2-KO (C, D) strain (KO does not affect micronemes) (Hammoudi et al., 2018).
Scale bar: 1μm.
Table 1. Conservation of microneme related genes.
Table showing conservation of featured genes in apicomplexa and chromerida. Presence of ortholog/s indicated with a black dot, absence with white.
Toxoplasma (To), Neospora (N), Hammondia (H), Sarcocystis (S), Eimeria (E), Cryptosporidium (Cr), Babesia (B), Theileria (Th), Plasmodium falciparum (Pf), Plasmodium berghei (Pb), Gregarinicae (G) and Chromera (Ch). Ortholog list obtained from literature, if unavailable through EuPathDB.org – transform by orthology feature.
