The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.     

The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex

Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.     

Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii

Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.     

The moving junction of apicomplexan parasites: a key structure for invasion

Most Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.     

Erythrocyte invasion: vocabulary and grammar of the Plasmodium rhoptry

Malaria is a dangerous infectious disease caused by obligate intracellular protozoan Plasmodium parasites. In the vertebrate host, erythrocyte recognition and establishment of a nascent parasitophorous vacuole are essential processes, and are largely achieved using molecules located in the microorganelles of the invasive-stage parasites. Recent proteome analyses of the phylogenetically related Toxoplasma parasite have provided protein catalogs for these microorganelles, which can now be used to identify orthologous proteins in the Plasmodium genome. Of importance is the formation of a complex between the proteins secreted from the rhoptry neck portion (RONs) and micronemes (AMA1), which localize at the moving junction during parasite invagination into the host cell. In this article I review the largely unexplored paradigm of the malaria merozoite rhoptry, focusing on the high molecular weight rhoptry protein complex (the RhopH complex), and speculate on its grammar during invasion.     

The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii

The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.     

Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium sp., are obligate intracellular protozoa. They enter into a host cell by attaching to and then creating an invagination in the host cell plasma membrane. Contact between parasite and host plasma membranes occurs in the form of a ring-shaped moving junction that begins at the anterior end of the parasite and then migrates posteriorly. The resulting invagination of host plasma membrane creates a parasitophorous vacuole that completely envelops the now intracellular parasite. At the start of this process, apical membrane antigen 1 (AMA1) is released onto the parasite surface from specialized secretory organelles called micronemes. The T. gondii version of this protein, TgAMA1, has been shown to be essential for invasion but its exact role has not previously been determined. We identify here a trio of proteins that associate with TgAMA1, at least one of which associates with TgAMA1 at the moving junction. Surprisingly, these new proteins derive not from micronemes, but from the anterior secretory organelles known as rhoptries and specifically, for at least two, from the neck portion of these club-shaped structures. Homologues for these AMA1-associated proteins are found throughout the Apicomplexa strongly suggesting that this moving junction apparatus is a conserved feature of this important class of parasites. Differences between the contributing proteins in different species may, in part, be the result of selective pressure from the different niches occupied by these parasites.     

Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites.     

Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.     

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill‐defined due to the lack of functional studies. In this study, we show that the rhoptry‐associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post‐invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra‐erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite–host cell interface are not simply by‐products of the invasion process but contribute to the growth of Plasmodium in vivo.     

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.     

Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.     

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so-called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium , the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 ( li ver- s pecific p rotein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1-deficient liver-stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.     

Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

Rhoptries: an arsenal of secreted virulence factors

Apicomplexan parasites use actin-based motility coupled with regulated protein secretion from apical organelles to actively invade host cells. Crucial in this process are rhoptries, club-shaped secretory organelles that discharge their contents during parasite invasion into host cells. A proteomic analysis of the rhoptries in Toxoplasma gondii demonstrated that this organelle contains a number of novel rhoptry proteins (ROPs) including serine-threonine kinases and protein phosphatases. A subset of rhoptry proteins called RONs have been shown to target the moving junction, which plays a key role in invasion and parasitophorous vacuole formation. Other ROP proteins have various destinations in the host cell including the host cell nucleus and the parasitophorous vacuole, probably reflecting their distinct targets and roles. Forward genetic analysis recently revealed that secretory ROP kinases dramatically influence host gene expression and are the major parasite virulence factors. Thus, ROP proteins are functionally analogous (though not homologous) to effectors released by type III and IV secretion systems, which are factors that play an important role in bacterial virulence. Deciphering the role of ROP effectors may allow specific disruption of these factors, thus offering new options for preventing disease.     

Molecular dissection of host cell invasion by the apicomplexans: the glideosome

Gliding motility is an essential and fascinating apicomplexan-typical adaptation to an intracellular lifestyle. Apicomplexan parasites rely on gliding motility for their migration across biological barriers and for host cell invasion and egress. This unusual substratedependent mode of locomotion involves the concerted action of secretory adhesins, a myosin motor, factors regulating actin dynamics and proteases. During invasion, complexes of soluble and transmembrane micronemes proteins (MICs) and rhoptry neck proteins (RONs) are discharged to the apical pole of the parasite, some protein acts as adhesins and bind to host cell receptors whereas others are involved in the moving junction formation. These complexes redistribute towards the posterior pole of the parasite via a physical connection to the parasite actomyosin system and are eventually released from the parasite surface by the action of parasite proteases.     

RON5 Is Critical for Organization and Function of the Toxoplasma Moving Junction Complex

Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.     

Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole

Most Apicomplexa reside and multiply in the cytoplasm of their host cell, within a parasitophorous vacuole (PV) originating from both parasite and host cell components. Trafficking of parasite-encoded proteins destined to membrane compartments beyond the confine of the parasite plasma membrane is a process that offers a rich territory to explore novel mechanisms of protein–membrane interactions. Here, we focus on the PVs formed by the asexual stages of two pathogens of medical importance, Plasmodium and Toxoplasma . We compare the PVs of both parasites, with a particular emphasis on their evolutionary divergent compartmentalization within the host cell. We also discuss the existence of peculiar export mechanisms and/or sorting determinants that are potentially involved in the post-secretory targeting of parasite proteins to the PV subcompartments.     

An Inhibitory Antibody Blocks Interactions between Components of the Malarial Invasion Machinery

Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1.     

Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite

Rhoptries are specialized secretory organelles characteristic of single cell organisms belonging to the clade Apicomplexa. These organelles play a key role in the invasion process of host cells by accumulating and subsequently secreting an unknown number of proteins mediating host cell entry. Despite their essential role, little is known about their biogenesis, components and targeting determinants. Here, we report on a conserved apicomplexan protein termed Armadillo Repeats-Only (ARO) protein that we localized to the cytosolic face of Plasmodium falciparum and Toxoplasma gondii rhoptries. We show that the first 20 N-terminal amino acids are sufficient for rhoptry membrane targeting. This protein relies on both - myristoylation and palmitoylation motifs - for membrane attachment. Although these lipid modifications are essential, they are not sufficient to direct ARO to the rhoptry membranes. Mutational analysis revealed additional residues within the first 20 amino acids of ARO that play an important role for rhoptry membrane attachment: the positively charged residues R9 and K14. Interestingly, the exchange of R9 with a negative charge entirely abolishes membrane attachment, whereas the exchange of K14 (and to a lesser extent K16) alters only its membrane specificity. Additionally, 17 proteins predicted to be myristoylated and palmitoylated in the first 20 N-terminal amino acids were identified in the genome of the malaria parasite. While most of the corresponding GFP fusion proteins were trafficked to the parasite plasma membrane, two were sorted to the apical organelles. Interestingly, these proteins have a similar motif identified for ARO.