Citation: Bargieri D, Lagal V, Andenmatten N, Tardieux I, Meissner M, Ménard R (2014) Host Cell Invasion by Apicomplexan Parasites: The Junction Conundrum. PLoS Pathog 10(9): e1004273. https://doi.org/10.1371/journal.ppat.1004273
Editor: Chetan E. Chitnis, International Centre for Genetic Engineering and Biotechnology, India
Published: September 18, 2014
Copyright: © 2014 Bargieri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funds from the Pasteur Institute, the French National Research Agency, the EviMalar, the Wellcome Trust, and the Fondation pour la Recherche Médicale (FRM team support, Barriers and Pathogens). DB was a recipient of a Manlio Cantarini Fellowship, NA is supported by an EviMalaR PhD fellowship (European FP7/2007-2013, grant number 242095), VL is supported by an EC Marie Curie integration grant (PIRG05-GA-2009-249158), and MM is funded by a Wellcome Trust Senior Fellowship (087582/Z/08/Z). The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust (085349). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Apicomplexans form a large phylum of parasitic protists, some of which cause severe diseases in humans. Most notorious is Plasmodium, the agent of malaria, which kills around a million people each year, mostly young children in Africa. Most successful is Toxoplasma, which parasitizes nearly a third of the human population, making those people at risk of life-threatening complications, primarily encephalitis or pneumonia, in case of immunosuppression. Other apicomplexans of human importance include Cryptosporidium, Isospora, and Sarcocystis, which are opportunistic pathogens that cause severe diarrhea often associated with AIDS. Several apicomplexan parasites cause heavy losses in livestock, particularly Theileria and Babesia in cattle and Eimeria in poultry.
Most apicomplexans are obligate intracellular parasites. Their extracellular stages, called zoites, display several conserved features: they are elongated and polarized cells, their shape is maintained by a set of microtubules running longitudinally, and their anterior pole contains secretory vesicles, called micronemes and rhoptries, which secrete their content at the anterior tip of the parasite. Most zoites also share two unique properties among eukaryotic cells. They move on substrate by a gliding type of motility, i.e., without overt deformation of the cell shape, at speeds of several microns per second. They also typically invade host cells by forming a ring-like junction with the host cell membrane. Zoites slide through the junction into an invagination of the host cell surface that becomes the parasitophorous vacuole (PV) after pinching off from the host cell plasma membrane, in a process that takes less than a minute. Once inside the PV niche, the zoite can multiply into multiple new zoites that eventually egress the infected cell to infect new host cells.
Much work has been performed since the late 1970s to understand the cellular and molecular bases of host cell invasion by apicomplexans, using various zoites as models. The overall invasion process encompasses several steps, including loose followed by intimate attachment, reorientation relative to the host cell surface, and organelle discharge with junction formation. The ultimate step, sliding through the junction inside the PV and called here internalization, is commonly viewed as powered by the zoite submembrane actin-myosin motor. The junction is thought to act as a traction point for the motor, to bridge the cortical cytoskeletons in the two cells, and to be made of parasite proteins conserved in the apicomplexan phylum. In this review, we confront these established notions with genetic data recently obtained in Plasmodium and Toxoplasma parasites.
The Junction: From “Moving” to Stationary
The first observation of a junction between an apicomplexan zoite and its host cell was made using Plasmodium merozoites and their target cells, erythrocytes . Electron microscopy showed that the merozoite, after initial random binding, reorients so that its apical tip faces the erythrocyte surface, and then induces a circumferential zone of close apposition of the zoite and erythrocyte membranes over ∼250 nm and the thickening of the inner leaflet of the erythrocyte membrane . This junctional area was described as “actively moving down the body of the merozoite,” since the poorly motile merozoite was not thought to be capable of actively moving inside the cell, and was thus termed “moving junction” .
Studies in the 1980s focused on the highly motile Eimeria sporozoites. They showed that several activities at the zoite surface were dependent on parasite actin, including the posterior translocation (capping) of various surface ligands and beads . Videomicroscopic studies revealed that host cell invasion by Eimeria sporozoites was continuous with extracellular gliding . This led to the proposal that the zoite actin-based system would power both gliding motility and host cell invasion by capping substrate-binding ligands or the junction, respectively, which implied that the zoite actively moved inside the host cell , . After myosins were identified in Toxoplasma  and in Plasmodium , it was assumed that an actin-myosin motor powered the zoite motile processes.
The role of the host cell during zoite invasion has been studied mainly with Toxoplasma tachyzoites, which can be made to invade host cells at high frequency and synchronicity. The host cell was initially described as displaying no detectable actin reorganization and playing no active role during tachyzoite invasion , . More recent work found that Toxoplasma tachyzoites induced, specifically at the junction, host actin polymerization and recruitment of the Arp2/3 complex, an actin-nucleating factor, which is important for tachyzoite entry . Videomicroscopic studies showed a stationary ring of host F-actin at the parasite constriction, in agreement with the junction acting as an anchor for zoite traction inside the cell. In addition to de novo actin polymerization at the junction, tachyzoite invasion also requires disorganization of the host cortical actin meshwork. This activity is in part dependent on Toxofilin , a Toxoplasma protein that sequesters actin monomers in vitro  and promotes actin turnover at the leading edge of the cell . Localized actin disassembly might thus release G-actin necessary to feed actin reassembly at the junction, regulated by recruited Arp2/3 complex, to anchor the junction to the host cortical cytoskeleton.
The Parasite Motor
The motor is located in the space (∼20 nm) between the zoite plasma membrane and the inner membrane complex (IMC), a continuous layer of flattened vesicles apposed onto the microtubule structure typical of alveolates (Figure 1A) . The development in the 1990s of gene targeting techniques in Toxoplasma and Plasmodium has allowed the identification of some of the main players of the apicomplexan motor. The first proposed motor-substrate link was the thrombospondin-related anonymous protein (TRAP), a transmembrane protein of Plasmodium sporozoites conserved in the phylum that was found to be essential for sporozoite gliding . The first (and, to date, only) myosin shown to operate during apicomplexan gliding is MyoA, a single-headed unconventional myosin of the apicomplexan-specific XIV class, which was shown to be crucial for gliding of Toxoplasma tachyzoites . The orientation of the motor was determined by two main findings: the association of the cytoplasmic tails of Plasmodium TRAP and its MIC2 ortholog in Toxoplasma with actin  and the localization of the MyoA light chain (also called myosin A tail domain interacting protein, MTIP) in the IMC .
A. The parasite motor (glideosome) is located in the space between the parasite plasma membrane (PPM) and the inner membrane complex (IMC) apposed to the microtubules. Gliding motility is mediated by the binding of the ectodomain of transmembrane TRAP-family proteins to a solid substrate, while the cytoplasmic tail of the protein is linked to the parasite motor. The integrity of the glideosome is maintained by the gliding-associated protein 45 (GAP45), which is anchored to the PPM at one end and to the IMC, via GAPs 40 and 50, at the other end. The link between the GAPs, and ultimately the IMC, to actin is provided by Myosin A (MyoA) and the MyoA Light Chain 1 (MLC1). The movement of the cell is the consequence of the capping, by myosin-actin activity, of the TRAP-family protein. B. The model of invasion seen as the junction structured by the AMA1-RON complex. The figure on the left shows a Toxoplasma tachyzoite invading a host cell. The arrow indicates the direction of movement. Immunostaining of surface MIC2 (sMIC2) stains the part of the zoite cell still extracellular (blue), while the rest of the cell, already internalized, is not stained. Immunostaining of total RON4 (tRON4, red) marks the junction as a ring at the point of constriction, indicated by the circle, and the rhoptries at the apical pole of the zoite cell. After a first step of adhesion to the host cell plasma membrane (HCPM) mediated by parasite surface adhesins and host cell surface receptors, the binding of the transmembrane protein AMA1 to RON2, inserted at the host cell membrane and complexed with RONs 4 and 5, forms the junction. The link to the parasite motor is as in (A), while host actin recruited at the junction provides the link to the host cell cytoskeleton. The movement of the zoite towards the interior of the newly formed parasitophorous vacuole membrane (PVM) is thus a consequence of the capping of AMA1, which would be anchored at the junction by binding to RON2. C. Models of zoite invasion in which the functions of AMA1 and RONs are dissociated. Color codes and acronyms are as in (A) and (B). After a first step of adhesion mediated by parasite surface adhesins and host cell surface receptors, AMA1 binding to a host cell receptor provides a strong attachment between the zoite and host cell membranes, possibly leading to reorientation of the zoite to allow junction formation. Three different hypotheses could then explain junction formation: 1. A still-unknown transmembrane parasite protein binds to the motor and to RON2, taking the place previously assigned to AMA1. 2. Unknown proteins structure the junction and connect the parasite motor to the host cell cortical actin, in which case the role of the RONs at the junction is not structural. 3. Unknown proteins structure the junction without a role of the parasite motor during invasion.
Actin polymerization in apicomplexans has been studied in some detail. In these parasites, which contain a limited repertoire of actin-binding factors , actin forms inherently short and unstable filaments , and normal zoite gliding requires rapid actin dynamics . In the absence of Arp2/3 complex in these parasites, F-actin nucleation is promoted by formins , , which were detected as an apical ring at the junction, like actin itself , in the invading Plasmodium merozoite . Actin depolymerization is controlled by actin-depolymerizing factor (ADF)  while profilin sequesters monomeric actin , . How actin filaments are connected to the cytoplasmic tails of the TRAP/MIC2 proteins remains unclear. The link was originally thought to occur via aldolase , a glycolytic enzyme that binds the cytoplasmic tails of the TRAP/MIC2 proteins and is known to bundle actin filaments in mammalian cells. However, genetic data have now clearly shown that aldolase does not play such a mechanical, bridging role during gliding motility and invasion ,  but is important for providing energy via its glycolytic activity . Finally, structural components of the motor complex have also been identified, primarily in Toxoplasma, called gliding-associated protein 45 (GAP45) , , GAP50 , and GAP40 . Although the individual contributions of the GAP proteins during gliding motility and invasion remain uncertain, they are thought to maintain the cohesion and integrity of the pellicle during zoite gliding and invasion, especially via GAP45 that spans the entire space between the plasma membrane and the IMC and anchors the motor complex at the IMC (Figure 1A) , .
The motor is still typically viewed as linear, i.e., as “linear arrangements of transmembrane proteins transducing the force generated by the actin-myosin motor and posteriorly capped,” as originally proposed by King . However, recent studies using biophysical approaches to measure the force Plasmodium sporozoites exert on the surface during gliding indicate that zoite movement is not continuous, as predicted by the linear motor, but follows a stick-and-slip pattern . This involves, in addition to backward capping of adhesion proteins, the formation/disengagement of adhesion sites at the front and rear ends of the zoite. Interestingly, TRAP appears to have a key role in the release of adhesion sites, not in retrograde capping (in stick but not in slip), while actin is important for both processes. Moreover, sporozoites lacking the motor-binding TRAP-like protein (TLP), which glide less efficiently by more frequently detaching from the substrate, were complemented by the addition of actin stabilizing drugs . These data illustrate the complex bases of apicomplexan gliding, which may be more akin to crawling of mammalian cells than previously anticipated.
What Is in the Junction?
Numerous proteins in Plasmodium merozoites have been described as being released from apical organelles, important for invasion and found at the junction. This is the case of several members of the micronemal erythrocyte binding like (EBL) family of proteins, such as Plasmodium falciparum erythrocyte-binding antigen 175 (EBA175)  and P. knowlesi Duffy binding protein (DBP) . This is also the case of several rhoptry proteins, including the P. falciparum reticulocyte-binding like (RBL) homologue PfRh1 , PfRh2a , and PfRh5 , and of the rhoptry-associated leucine zipper-like protein 1 (RALP1) . However, function is difficult to assess in the Plasmodium merozoite, typically relying on antibody inhibition and negative transfection experiments. In Toxoplasma, one member of the micronemal MIC family of proteins, MIC8, was shown to be specifically crucial for junction formation . Importantly, none of these proteins is conserved across the phylum and most are stage-specific. If these proteins are indeed part of the junction, the latter might then be at least in part zoite-specific. If instead the junction is composed of a molecular core conserved across apicomplexans, then these stage-specific proteins may constitute adaptations to particular zoite-host cell combinations.
Since the junction is viewed as a traction point for the motor, other candidates for junction components were the transmembrane proteins involved in gliding motility, i.e., the TRAP family of proteins including TRAP, TLP, and TRAP-related protein (TREP) in the Plasmodium sporozoite and MIC2 in the Toxoplasma tachyzoite . However, there is no evidence that any of these proteins participates specifically at the junction during host cell invasion. In the Toxoplasma tachyzoite, inactivation of MIC2 impairs but does not preclude motility or invasion  and MIC2 is not specifically enriched at the junction during tachyzoite internalization. The hypothesis that TRAP/MIC2 might play a role as junction components was also favored by the presence in their extracellular domains of one or more A domains of von Willebrand factor, which are homologous to integrin I domains and thus potential ligands of host cell surface receptors . However, the A domains of the TRAP family member CS and TRAP-related protein (CTRP), expressed by the Plasmodium motile ookinete stage that does not invade host cells, were shown to be important for gliding motility . Together, these data favor the view that the TRAP family of proteins is involved in gliding motility, but not specifically for host cell entry.
In 2005, two papers identified a set of parasite proteins in Toxoplasma tachyzoites, called rhoptry neck proteins (RON), which specifically marked the constriction around invading zoites in a ring-like manner (Figure 1B) , . Later, a ring-like staining of RON proteins was also shown in invading Plasmodium merozoites . It was additionally found that in tachyzoite extracts, several RON proteins formed a complex with apical membrane antigen 1 (AMA1) , a transmembrane protein first identified in Plasmodium merozoites  and a leading malaria vaccine candidate. In humans, evidence suggests that AMA1 is an important target of naturally acquired protective antibodies preventing merozoite invasion of erythrocytes . AMA1 vaccines have demonstrated protective efficacy in rodent and simian models against blood-stage challenge with the homologous strain , although human vaccine trials using AMA1 have shown poor efficacy so far .
Both AMA1 and RON proteins are conserved in the apicomplexan phylum and the AMA1-RON complex has now been detected in extracts from Toxoplasma tachyzoites and sporozoites , Plasmodium merozoites of various species , and Neospora . Several independent lines of evidence have first favored the view that the AMA1-RON complex might constitute the building block of the junction. (i) The ectodomain of AMA1 binds RON2 , , which inserts into the host cell membrane  and is thought to bind to the cell cortical cytoskeleton via other RON proteins, a view recently strengthened by the observation that Toxoplasma RON4 may bind host cell tubulin . (ii) The crystal structure of the AMA1–RON2 interaction in Toxoplasma  and Plasmodium  reveals a conserved RON2 loop that inserts deep into a hydrophobic groove in AMA1, suggesting that it might withstand mechanical forces and act as the traction point for the zoite motor. (iii) Antibodies or peptides that inhibit the AMA1–RON2 interaction reduce host cell invasion by Toxoplasma tachyzoites and Plasmodium merozoites –. These results clearly pointed to the view that cross-membrane AMA1-RON2 complexes shaped the junction for zoite internalization ,  (Figure 1B). Consequently, the conserved AMA1–RON2 interaction has sparked much interest as a broad target for intervention against apicomplexan parasites , .
AMA1-Dependent Attachment and AMA1-Independent Internalization
The development of conditional mutagenesis techniques in Toxoplasma ,  and Plasmodium  has allowed the addressing of the functions of AMA1 and the RON complex (Table 1). All attempts to directly inactivate either RON4 or RON2 in Toxoplasma and Plasmodium have failed so far. P. berghei RON4 conditional sporozoites, obtained by Flp/FRT-mediated recombination, are unable to invade hepatocytes . P. berghei RON2 conditional sporozoites, obtained by a promoter swap strategy, are unable to invade mosquito salivary gland cells . Toxoplasma gondii RON5 and RON2 conditional tachyzoites, generated with a Tet-repressible promoter, are drastically impaired in invasion , . Therefore, the RON proteins appear to play crucial roles during host cell invasion by all zoites.
In contrast, all AMA1 knock-down (AMA1KD) or knock-out (AMA1KO) zoites constructed are still invasive. AMA1KD P. berghei sporozoites invade hepatocytes 3-fold better than the wild type (WT) in vitro and in vivo , and AMA1KO Toxoplasma tachyzoites and P. berghei merozoites are internalized into host cells indistinguishably from the WT, i.e. systematically form normal RON rings and are internalized at normal speed , . Nonetheless, AMA1KO Toxoplasma tachyzoites and P. berghei merozoites display reduced invasion efficiency , , , along with a major impairment in host cell attachment , . A recent report suggested that the adhesion defect of AMA1KO tachyzoites might be secondary to a failure to form a normal junction leading to parasite detachment from the cell, based on immunofluorescence (IF) assays of rhoptry secretion . However, what causes rhoptry secretion is still unknown and no direct evidence was provided that AMA1KO tachyzoites formed an abnormal junction before detaching. AMA1KO tachyzoites observed by real-time imaging did not display abortive invasions and their attachment defect (upright instead of flattened positioning relative to the cell surface) concerned the entire population, irrespective of invasion , . In Plasmodium, AMA1KO merozoites were 15-fold less adhesive to erythrocytes than controls after only 3 minutes incubation, which cannot be accounted for by failed invasions given that merozoite invasion efficiency is less than 5% . A primary role of AMA1 in zoite binding to host cells is also in agreement with earlier work showing that AMA1 mediates attachment of Toxoplasma tachyzoites  and Plasmodium merozoites  to their respective host cells, and that Plasmodium AMA1 binds erythrocytes – and to the erythrocyte membrane receptor Kx .
Recent work revealed that the Toxoplasma genome encodes paralogs of AMA1 and RON2 in specific combinations. In the tachyzoite, RON2 interacts with AMA1 or AMA2 and RON2L1 with AMA4 , while in the sporozoite RON2L2 interacts with AMA3 in a manner mutually exclusive with tachyzoite paralogs . Therefore, the hypothesis was raised that AMA1 paralogs might account for the residual invasive capacity of AMA1KO tachyzoites , . In agreement with this, the latter were found, during parasite selection, to up-regulate AMA2 as well as the RON2L1–AMA4 pair . However, evidence that these paralogs act at and/or structure the junction is lacking, and the weaker affinity of RON2 for AMA2 compared with AMA1  is at odds with the fully efficient internalization of AMA1KO  (supposedly mediated by AMA2). In Plasmodium, the compensation theory appears particularly unlikely. The AMA1KD Flp/FRT sporozoites undergo AMA1 excision after parasite selection and yet are 100% invasive . Additionally, Plasmodium expresses no AMA1 or RON2 paralog. The protein most closely related to AMA1 is the trans-membrane protein MAEBL, with which it shares the presence of a cysteine-rich domain but differs by an unrelated cytoplasmic tail and the absence of RON2-binding ability. MAEBL was shown by gene targeting in both P. berghei  and P. falciparum  sporozoites to function as a stage-specific adhesion; it mediates oocyst sporozoite binding to the mosquito salivary glands, but not internalization into hepatocytes. This further suggests that AMA1, its paralogs in Toxoplasma, and MAEBL in Plasmodium form a family of stage-specific, host cell–binding proteins.
If AMA1 primarily mediates zoite intimate binding to host cell surfaces, irrespective of RON2 interaction and junction assembly, what could be the role of the conserved and therefore important AMA1–RON2 interaction? AMA1 might still bind to RON2, possibly to help further stabilize the zoite prior to internalization, although direct evidence for such a step is still lacking. Alternatively, AMA1–RON2 interactions might serve to process AMA1 at the junction during internalization of the AMA1-covered zoite. For example, interaction with RON2 might serve to disengage the AMA1–host cell receptor interaction and help the zoite slide free inside the PV, separated from the vacuole membrane. In agreement with this, RON2 binding induces conformational changes in AMA1 , which might impact AMA1 processing by the substilisin-like protease SUB2  or intramembrane rhomboids . Likewise, RON2L2 binding alters AMA3, including allosterically in its membrane-proximal domain . Such AMA processing function of the AMA–RON2 interactions would be dispensable for internalization and yet block invasion if perturbed, reconciling the inhibition and genetic data.
The contribution of the apparently essential RON proteins is also unclear. They might be structural components of the junction, by linking it to the host cell cytoskeleton (Figure 1C, hypothesis 1), or might not be part of the force-transducing link (Figure 1C, hypothesis 2). Perhaps favoring the latter, the RON proteins are present in apicomplexans that are not known to form a junction, like Theileria , which raises the possibility that another zoite–host cell interaction might structure the junction, possibly involving host cell receptor(s). In any hypothesis, the junction constitutes a traction point for zoite internalization into the host cell.
The Toxoplasma tachyzoite is ideal to study zoite invasion, not just due to the frequency of observable invasion events but also the genetic tractability of the parasite. The use of a transcriptional regulation system based on artificial Tet-transactivators (TATi) allowed the generation of knock-down mutants and the functional dissection of individual components of the motor (Table 1). As already said, knocking down MIC2  or MyoA  does not result in a complete block in host cell invasion. Even knocking down GAP45, while leading to the detachment of the IMC from the plasma membrane (PM) and the release of the motor complex in the cytosol, does not abolish host cell entry . In contrast, as mentioned above, a knock-down for MIC8 does not affect gliding motility but completely blocks host cell invasion due to an inability to form a junction . These partial phenotypes were typically explained by the leakiness of the Tet-inducible system, but were also a hint that the motor might not be essential for host cell invasion.
The current adaptation of a conditional recombination system based on dimerizable Cre has allowed the construction of a series of tachyzoite mutants completely lacking individual components of the motor complex, including MyoA, GAP45, MLC1, and Act1 , . All of these mutants are affected in invasion efficiency but retain some invasive capacity (Table 1). Strikingly, tachyzoites devoid of MyoA, MLC1, or Act1, which is a single copy gene in Toxoplasma, can invade host cells through a junction , demonstrating that at least junction formation is independent of connection to the motor. Moreover, GAP45KO tachyzoites, in which the IMC detaches from the parasite membrane and MyoA and MLC1 become cytosolic, remain motile and also invade . These genetic data suggest that tachyzoites can move and enter host cells without a functional motor. However, whether this motor-independent entry pathway is the normal pathway used by the WT, or an alternative pathway discernable only when the motor is not functional, remains to be seen.
How could tachyzoites with a deficient actin-myosin motor invade host cells? Until recently, actin polymerization and actin-myosin contraction were thought to underlie force generation during movement. However, this view is currently being challenged by new models, in which hydrodynamic forces generate changes in cell shape during motility . Indeed, there is mounting evidence that osmotic pressure and hydrodynamic fluids are critical for motility of amoeboid cells, while the actin-myosin system is critical for the formation and release of attachment sites and associated traction forces . A recent report demonstrates the poroelastic nature of the cytosol , where force can be generated by differences in hydrodynamic pressure that can be higher in one part of a cell than another, leading to tension. This pressure can be generated by actin-myosin activity or by the localized activation of osmogenic ion transporters in the plasma membrane . In agreement with such a model, Na+/H+ antiporters have been implicated in invasion and egress of host cells by Toxoplasma tachyzoites –, and monovalent ion concentrations have been involved in gliding motility and host cell invasion efficiencies in Toxoplasma , . Based on this, a gelsolation model for gliding motility and zoite internalization, in which the acto-myosin system of the parasite is required as a clutch for force transmission but not for the generation of the force itself, has recently been proposed .
Another possibility that cannot be excluded is that the force for parasite internalization might originate from the host cell. Theileria sporozoites and merozoites, which lack an IMC and subpellicular microtubules and are not motile, invade host cells in any orientation, without a junction, and by a mechanism of circumferential zippering of parasite and host cell membranes . Others, like Cryptosporidium sporozoites, are motile but rest on the host cell surface and induce the formation of host cell membrane folds that progressively encapsulate the “epicellular parasite” inside the PV . Interestingly, these membrane protrusions recruit a host cell Na+/glucose cotransporter and aquaporin 1, which generate localized water influx and are required for parasite invasion .
If zoite internalization is powered by the host cell or by hydrodynamic forces, then the junction would no longer be connected to the parasite motor. In this case, the junction might serve as a membrane “seal,” possibly regulating protein processing upon entry into the PV, facilitating membrane dynamics, fluidity and curvature, and/or ensuring correct formation of the PV (Figure 1C, hypothesis 3).
New mutagenesis data question the current view that apicomplexan zoites invade host cells by a unique pathway involving their motor and AMA1–RON2 interactions as traction points at the junction. AMA1 appears not to be involved in junction function and the RON proteins are the only parasite proteins known to functionally associate with the junction. Whether the RON complex transmits force at the junction remains uncertain, though the evidence of a link between the RON proteins and the host cell cytoskeleton points to this direction. The conservation of the RON proteins in apicomplexans and of their essential role in invasion suggests a conserved molecular kit for junction formation. Still, there is no definitive evidence for such a conserved apicomplexan junction core, and at least part of its structure might be stage-specific. Whether the zoite provides all pieces of the junction, or whether the host cell also provides receptors, possibly located in specific microdomains, is also unclear.
Moreover, it now appears that the force required for gliding motility and host cell entry might, at least in Toxoplasma, be generated in a motor-independent manner. Whether this holds true for other apicomplexan genera remains to be seen. In any event, apicomplexan invasion of host cells appears more complex than previously thought. Dissecting the process and its possible versatility will require establishing novel experimental approaches, including biophysical, and investigating unconventional force-generation means in zoites, as well as in host cells, that might facilitate, or even replace, the activity of the parasite motor.
- 1. Aikawa M, Miller LH, Johnson J, Rabbege J (1978) Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite. J Cell Biol 77: 72–82.
- 2. King CA (1988) Cell motility of sporozoan protozoa. Parasitol Today 4: 315–319.
- 3. Russell DG, Sinden RE (1981) The role of the cytoskeleton in the motility of coccidian sporozoites. J Cell Sci 50: 345–359.
- 4. Russell DG (1983) Host cell invasion by Apicomplexa: an expression of the parasite's contractile system? Parasitology 87 (Pt 2): 199–209.
- 5. Schwartzman JD, Pfefferkorn ER (1983) Immunofluorescent localization of myosin at the anterior pole of the coccidian, Toxoplasma gondii. J Protozool 30: 657–661.
- 6. Pinder JC, Fowler RE, Dluzewski AR, Bannister LH, Lavin FM, et al. (1998) Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion. J Cell Sci 111 (Pt 13): 1831–1839.
- 7. Dobrowolski JM, Sibley LD (1996) Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84: 933–939.
- 8. Morisaki JH, Heuser JE, Sibley LD (1995) Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J Cell Sci 108 (Pt 6): 2457–2464.
- 9. Gonzalez V, Combe A, David V, Malmquist NA, Delorme V, et al. (2009) Host cell entry by apicomplexa parasites requires actin polymerization in the host cell. Cell Host Microbe 5: 259–272.
- 10. Delorme-Walker V, Abrivard M, Lagal V, Anderson K, Perazzi A, et al. (2012) Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J Cell Sci 125: 4333–4342.
- 11. Delorme V, Cayla X, Faure G, Garcia A, Tardieux I (2003) Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii Toxofilin. Mol Biol Cell 14: 1900–1912.
- 12. Morrissette NS, Murray JM, Roos DS (1997) Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J Cell Sci 110 (Pt 1): 35–42.
- 13. Sultan AA, Thathy V, Frevert U, Robson KJ, Crisanti A, et al. (1997) TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 90: 511–522.
- 14. Meissner M, Schluter D, Soldati D (2002) Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298: 837–840.
- 15. Jewett TJ, Sibley LD (2003) Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Mol Cell 11: 885–894.
- 16. Bergman LW, Kaiser K, Fujioka H, Coppens I, Daly TM, et al. (2003) Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites. J Cell Sci 116: 39–49.
- 17. Schuler H, Matuschewski K (2006) Regulation of apicomplexan microfilament dynamics by a minimal set of actin-binding proteins. Traffic 7: 1433–1439.
- 18. Skillman KM, Diraviyam K, Khan A, Tang K, Sept D, et al. (2011) Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites. PLoS Pathog 7: e1002280.
- 19. Mehta S, Sibley LD (2011) Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii. Mol Biol Cell 22: 1290–1299.
- 20. Baum J, Tonkin CJ, Paul AS, Rug M, Smith BJ, et al. (2008) A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion. Cell Host Microbe 3: 188–198.
- 21. Daher W, Plattner F, Carlier MF, Soldati-Favre D (2010) Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii. PLoS Pathog 6: e1001132.
- 22. Angrisano F, Riglar DT, Sturm A, Volz JC, Delves MJ, et al. (2012) Spatial localisation of actin filaments across developmental stages of the malaria parasite. PLoS ONE 7: e32188.
- 23. Mehta S, Sibley LD (2010) Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin. J Biol Chem 285: 6835–6847.
- 24. Skillman KM, Daher W, Ma CI, Soldati-Favre D, Sibley LD (2012) Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro. Biochemistry 51: 2486–2495.
- 25. Plattner F, Yarovinsky F, Romero S, Didry D, Carlier MF, et al. (2008) Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. Cell Host Microbe 3: 77–87.
- 26. Shen B, Sibley LD (2014) Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion. Proc Natl Acad Sci U S A 111: 3567–3572.
- 27. Starnes GL, Coincon M, Sygusch J, Sibley LD (2009) Aldolase is essential for energy production and bridging adhesin-actin cytoskeletal interactions during parasite invasion of host cells. Cell Host Microbe 5: 353–364.
- 28. Pomel S, Luk FC, Beckers CJ (2008) Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites. PLoS Pathog 4: e1000188.
- 29. Gaskins E, Gilk S, DeVore N, Mann T, Ward G, et al. (2004) Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J Cell Biol 165: 383–393.
- 30. Gilk SD, Gaskins E, Ward GE, Beckers CJ (2009) GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex. Eukaryot Cell 8: 190–196.
- 31. Frenal K, Polonais V, Marq JB, Stratmann R, Limenitakis J, et al. (2010) Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8: 343–357.
- 32. Egarter S, Andenmatten N, Jackson AJ, Whitelaw JA, Pall G, et al. (2014) The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion. PLoS ONE 9: e91819.
- 33. Munter S, Sabass B, Selhuber-Unkel C, Kudryashev M, Hegge S, et al. (2009) Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host Microbe 6: 551–562.
- 34. Hellmann JK, Perschmann N, Spatz JP, Frischknecht F (2013) Tunable substrates unveil chemical complementation of a genetic cell migration defect. Adv Healthc Mater 2: 1162–1169.
- 35. Gilberger TW, Thompson JK, Reed MB, Good RT, Cowman AF (2003) The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking. J Cell Biol 162: 317–327.
- 36. Singh AP, Ozwara H, Kocken CH, Puri SK, Thomas AW, et al. (2005) Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion. Mol Microbiol 55: 1925–1934.
- 37. Gunalan K, Gao X, Yap SS, Huang X, Preiser PR (2013) The role of the reticulocyte-binding-like protein homologues of Plasmodium in erythrocyte sensing and invasion. Cell Microbiol 15: 35–44.
- 38. Gunalan K, Gao X, Liew KJ, Preiser PR (2011) Differences in erythrocyte receptor specificity of different parts of the Plasmodium falciparum reticulocyte binding protein homologue 2a. Infect Immun 79: 3421–3430.
- 39. Baum J, Chen L, Healer J, Lopaticki S, Boyle M, et al. (2009) Reticulocyte-binding protein homologue 5 - an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum. Int J Parasitol 39: 371–380.
- 40. Ito D, Hasegawa T, Miura K, Yamasaki T, Arumugam TU, et al. (2013) RALP1 is a rhoptry neck erythrocyte-binding protein of Plasmodium falciparum merozoites and a potential blood-stage vaccine candidate antigen. Infect Immun 81: 4290–4298.
- 41. Kessler H, Herm-Gotz A, Hegge S, Rauch M, Soldati-Favre D, et al. (2008) Microneme protein 8—a new essential invasion factor in Toxoplasma gondii. J Cell Sci 121: 947–956.
- 42. Morahan BJ, Wang L, Coppel RL (2009) No TRAP, no invasion. Trends in Parasitology 25: 77–84.
- 43. Huynh MH, Carruthers VB (2006) Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog 2: e84.
- 44. Song G, Koksal AC, Lu C, Springer TA (2012) Shape change in the receptor for gliding motility in Plasmodium sporozoites. Proc Natl Acad Sci U S A 109: 21420–21425.
- 45. Ramakrishnan C, Dessens JT, Armson R, Pinto SB, Talman AM, et al. (2011) Vital functions of the malarial ookinete protein, CTRP, reside in the A domains. Int J Parasitol 41: 1029–1039.
- 46. Alexander DL, Mital J, Ward GE, Bradley P, Boothroyd JC (2005) Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog 1: e17.
- 47. Lebrun M, Michelin A, El Hajj H, Poncet J, Bradley PJ, et al. (2005) The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion. Cell Microbiol 7: 1823–1833.
- 48. Riglar DT, Richard D, Wilson DW, Boyle MJ, Dekiwadia C, et al. (2011) Super-resolution dissection of coordinated events during malaria parasite invasion of the human erythrocyte. Cell Host Microbe 9: 9–20.
- 49. Deans JA, Alderson T, Thomas AW, Mitchell GH, Lennox ES, et al. (1982) Rat monoclonal antibodies which inhibit the in vitro multiplication of Plasmodium knowlesi. Clin Exp Immunol 49: 297–309.
- 50. Thomas AW, Trape JF, Rogier C, Goncalves A, Rosario VE, et al. (1994) High prevalence of natural antibodies against Plasmodium falciparum 83-kilodalton apical membrane antigen (PF83/AMA-1) as detected by capture-enzyme-linked immunosorbent assay using full-length baculovirus recombinant PF83/AMA-1. Am J Trop Med Hyg 51: 730–740.
- 51. Remarque EJ, Faber BW, Kocken CH, Thomas AW (2008) Apical membrane antigen 1: a malaria vaccine candidate in review. Trends Parasitol 24: 74–84.
- 52. Laurens MB, Thera MA, Coulibaly D, Ouattara A, Kone AK, et al. (2013) Extended safety, immunogenicity and efficacy of a blood-stage malaria vaccine in malian children: 24-month follow-up of a randomized, double-blinded phase 2 trial. PLoS ONE 8: e79323.
- 53. Poukchanski A, Fritz HM, Tonkin ML, Treeck M, Boulanger MJ, et al. (2013) Toxoplasma gondii sporozoites invade host cells using two novel paralogues of RON2 and AMA1. PLoS ONE 8: e70637.
- 54. Narum DL, Nguyen V, Zhang Y, Glen J, Shimp RL, et al. (2008) Identification and characterization of the Plasmodium yoelii PyP140/RON4 protein, an orthologue of Toxoplasma gondii RON4, whose cysteine-rich domain does not protect against lethal parasite challenge infection. Infect Immun 76: 4876–4882.
- 55. Straub KW, Cheng SJ, Sohn CS, Bradley PJ (2009) Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements. Cell Microbiol 11: 590–603.
- 56. Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, et al. (2011) The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS Pathog 7: e1001276.
- 57. Tyler JS, Boothroyd JC (2011) The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex. PLoS Pathog 7: e1001282.
- 58. Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M (2009) Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog 5: e1000309.
- 59. Takemae H, Sugi T, Kobayashi K, Gong H, Ishiwa A, et al. (2013) Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular beta-tubulin. Sci Rep 3: 3199.
- 60. Tonkin ML, Roques M, Lamarque MH, Pugniere M, Douguet D, et al. (2011) Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science 333: 463–467.
- 61. Vulliez-Le Normand B, Tonkin ML, Lamarque MH, Langer S, Hoos S, et al. (2012) Structural and functional insights into the malaria parasite moving junction complex. PLoS Pathog 8: e1002755.
- 62. Collins CR, Withers-Martinez C, Hackett F, Blackman MJ (2009) An inhibitory antibody blocks interactions between components of the malarial invasion machinery. PLoS Pathog 5: e1000273.
- 63. Richard D, MacRaild CA, Riglar DT, Chan JA, Foley M, et al. (2010) Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites. J Biol Chem 285: 14815–14822.
- 64. Srinivasan P, Beatty WL, Diouf A, Herrera R, Ambroggio X, et al. (2011) Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion. Proc Natl Acad Sci U S A 108: 13275–13280.
- 65. Srinivasan P, Yasgar A, Luci DK, Beatty WL, Hu X, et al. (2013) Disrupting malaria parasite AMA1-RON2 interaction with a small molecule prevents erythrocyte invasion. Nat Commun 4: 2261.
- 66. Baum J, Cowman AF (2011) Biochemistry. Revealing a parasite's invasive trick. Science 333: 410–411.
- 67. Shen B, Sibley LD (2012) The moving junction, a key portal to host cell invasion by apicomplexan parasites. Curr Opin Microbiol 15: 449–455.
- 68. Macraild CA, Anders RF, Foley M, Norton RS (2011) Apical membrane antigen 1 as an anti-malarial drug target. Curr Top Med Chem 11: 2039–2047.
- 69. Miller LH, Ackerman HC, Su XZ, Wellems TE (2013) Malaria biology and disease pathogenesis: insights for new treatments. Nat Med 19: 156–167.
- 70. Andenmatten N, Egarter S, Jackson AJ, Jullien N, Herman JP, et al. (2013) Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat Methods 10: 125–127.
- 71. Combe A, Giovannini D, Carvalho TG, Spath S, Boisson B, et al. (2009) Clonal conditional mutagenesis in malaria parasites. Cell Host Microbe 5: 386–396.
- 72. Giovannini D, Spath S, Lacroix C, Perazzi A, Bargieri D, et al. (2011) Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe 10: 591–602.
- 73. Murata E, Tokunaga N, Tachibana M, Tsuboi T, Torii M, et al. (2012) The investigation of the mechanism how malaria sporozoites invade salivary glands. Molecular Approaches to Malaria Meeting 2012. Abstract Book. Lorne, Australia.
- 74. Beck JR, Chen AL, kim EW, Bradley PJ (2014) RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS Pathog 10: e1004025.
- 75. Lamarque MH, Roques M, Kong-Hap M, Tonkin ML, Rugarabamu G, et al. (2014) Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites. Nat Commun 5: 4098.
- 76. Bargieri DY, Andenmatten N, Lagal V, Thiberge S, Whitelaw JA, et al. (2013) Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nat Commun 4: 2552.
- 77. Yap A, Azevedo MF, Gilson PR, Weiss GE, O'Neill MT, et al. (2014) Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites. Cell Microbiol 16: 642–656.
- 78. Mital J, Meissner M, Soldati D, Ward GE (2005) Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol Biol Cell 16: 4341–4349.
- 79. Mitchell GH, Thomas AW, Margos G, Dluzewski AR, Bannister LH (2004) Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells. Infect Immun 72: 154–158.
- 80. Fraser TS, Kappe SH, Narum DL, VanBuskirk KM, Adams JH (2001) Erythrocyte-binding activity of Plasmodium yoelii apical membrane antigen-1 expressed on the surface of transfected COS-7 cells. Mol Biochem Parasitol 117: 49–59.
- 81. Urquiza M, Suarez JE, Cardenas C, Lopez R, Puentes A, et al. (2000) Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein. Vaccine 19: 508–513.
- 82. Valbuena J, Rodriguez L, Vera R, Puentes A, Curtidor H, et al. (2006) Synthetic peptides from Plasmodium falciparum apical membrane antigen 1 (AMA-1) specifically interacting with human hepatocytes. Biochimie 88: 1447–1455.
- 83. Kato K, Mayer DC, Singh S, Reid M, Miller LH (2005) Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx. Proc Natl Acad Sci U S A 102: 5552–5557.
- 84. Poukchanski A, Fritz HM, Tonkin ML, Treeck M, Boulanger MJ, et al. (2013) Toxoplasma gondii sporozoites invade host cells using two novel paralogues of RON2 and AMA1. PLoS ONE 8: e70637.
- 85. Kariu T, Yuda M, Yano K, Chinzei Y (2002) MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland. J Exp Med 195: 1317–1323.
- 86. Saenz FE, Balu B, Smith J, Mendonca SR, Adams JH (2008) The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands. PLoS ONE 3: e2287.
- 87. Olivieri A, Collins CR, Hackett F, Withers-Martinez C, Marshall J, et al. (2011) Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies. PLoS Pathog 7: e1002448.
- 88. Parussini F, Tang Q, Moin SM, Mital J, Urban S, et al. (2012) Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates host-cell invasion but is dispensable for replication. Proc Natl Acad Sci U S A 109: 7463–7468.
- 89. Shaw MK (2003) Cell invasion by Theileria sporozoites. Trends Parasitol 19: 2–6.
- 90. Mitchison TJ, Charras GT, Mahadevan L (2008) Implications of a poroelastic cytoplasm for the dynamics of animal cell shape. Semin Cell Dev Biol 19: 215–223.
- 91. Keren K, Yam PT, Kinkhabwala A, Mogilner A, Theriot JA (2009) Intracellular fluid flow in rapidly moving cells. Nat Cell Biol 11: 1219–1224.
- 92. Moeendarbary E, Valon L, Fritzsche M, Harris AR, Moulding DA, et al. (2013) The cytoplasm of living cells behaves as a poroelastic material. Nat Mater 12: 253–261.
- 93. Francia ME, Wicher S, Pace DA, Sullivan J, Moreno SN, et al. (2011) A Toxoplasma gondii protein with homology to intracellular type Na(+)/H(+) exchangers is important for osmoregulation and invasion. Exp Cell Res 317: 1382–1396.
- 94. Karasov AO, Boothroyd JC, Arrizabalaga G (2005) Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii. Int J Parasitol 35: 285–291.
- 95. Arrizabalaga G, Ruiz F, Moreno S, Boothroyd JC (2004) Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation. J Cell Biol 165: 653–662.
- 96. Endo T, Tokuda H, Yagita K, Koyama T (1987) Effects of extracellular potassium on acid release and motility initiation in Toxoplasma gondii. J Protozool 34: 291–295.
- 97. Endo T, Yagita K (1990) Effect of extracellular ions on motility and cell entry in Toxoplasma gondii. J Protozool 37: 133–138.
- 98. Valigurova A, Jirku M, Koudela B, Gelnar M, Modry D, et al. (2008) Cryptosporidia: epicellular parasites embraced by the host cell membrane. Int J Parasitol 38: 913–922.
- 99. Chen XM, O'Hara SP, Huang BQ, Splinter PL, Nelson JB, et al. (2005) Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion. Proc Natl Acad Sci U S A 102: 6338–6343.
- 100. Kappe S, Bruderer T, Gantt S, Fujioka H, Nussenzweig V, et al. (1999) Conservation of a gliding motility and cell invasion machinery in apicomplexan parasites. J Cell Biol 147: 937–943.