Plasmodium falciparum polypeptides interacting with human red cell membranes show high affinity binding to Band-3

P. falciparum proteins were labelled with [³⁵S]methionine and harvested at various asexual stages. A number of parasite proteins bound to uninfected red cell membranes (ghosts). Some of these proteins differentially partitioned when ghosts were extracted with detergent. Several of these proteins bound very strongly to immobilised whole ghost proteins or immobilised purified Band-3 in a stage-specific manner, but not to a sham-coupled matrix or to immobilised Band-3 extract from cells rendered refractory to invasion. Such specific binding of parasite proteins to immobilised Band-3 supports recent conjecture as to its role as a host receptor during parasite invasion. However, our results demonstrate the complex and multifactorial nature of the interaction between parasite and host proteins during invasion and development.     

Human erythrocyte Band-3 has an altered N terminus in malaria-resistant Melanesion ovalocytosis

There is a high prevalence of the erythrocyte polymorphism ovalocytosis associated with reduced susceptibility to malaria in Papua New Guinea. The major erythrocyte integral membrane protein, Band-3, showed markedly increased phosphorylation in whole cells or isolated ghosts from ovalocytic individuals. The cytoplasmic domain of the ovalocyte Band-3 was found to be approx. 3 kDa largen than the normocytic protein. The N-terminal sequence of the ovalocytic Band-3 was different from the reported sequence for human Band-3, suggesting that the increased size results from an N-terminal extension. Since this is the region of Band-3 which is phosphorylated and interacts with the red cell cytoskeleton, it is likely that this alteration in ovalocytic Band-3 is the underlying cause of the diverse alterations in ovalocytic cells including increased phosphorylation, increased membraned rigidity, decreased agglutinability by blood group antibodies and refractoriness to invasion by malarial parasites.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.     

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.     

Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell''s periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing disease in fetuses and immunocompromised individuals (23). The parasite infects a wide range of nucleated cells of most warm-blooded animals. The mechanisms underlying this wide tropism are not known but could be due to either the parasite infecting cells using a ubiquitously expressed host receptor and associated machinery, inserting its own receptor into the host cell''s plasma membrane, or using multiple host cell receptors/machinery (5).Toxoplasma invasion is a multistep, complex process consisting of parasite contact to host cells, intimate attachment, parasite motility, and then penetration (5). Host cell contact is a loose, low-affinity interaction that is mediated by parasite surface antigens. An unknown signal then triggers the release of proteins from a specialized secretory organelle called micronemes whose contents include proteins that function as adhesins. This is then followed by parasite gliding motility on the host cell surface. At some point, proteins from a second secretory organelle, named rhoptries, are exocytosed. Among these rhoptry proteins, several (RON2, RON4, RON5, and RON8) are part of a preformed complex that binds the previously secreted AMA1 microneme protein (1, 2, 20, 33). Together, these proteins form the moving junction complex, which defines the parasite entry site on the host cell plasma membrane. Parasite penetration occurs by the parasite propelling itself forward, via acto-myosin-dependent motility, into the host plasma membrane (35). This causes an invagination of the plasma membrane resulting in the formation of the parasitophorous vacuole (PV), which is the compartment that the parasite resides in throughout its time in the host cell. However, host plasma membrane-associated proteins are selectively incorporated into the developing PV such that glycosylphosphatidylinositol (GPI)-linked proteins are included, while single-pass transmembrane proteins are excluded (7, 24).In contrast to parasite molecules that function during invasion, few host cell components involved in this process are known. A notable exception is the finding that host Arp2/3-dependent actin polymerization promotes Toxoplasma invasion (11). Nevertheless, how actin or other host molecules function during invasion remains to be determined. The host microtubule cytoskeleton has been widely studied for its role during receptor-mediated endocytosis, as well as in bacterial and viral infections, where microtubules act to facilitate cargo transport from the host cell periphery to the interior (8, 15, 27, 29, 40). Consistent with this role in cargo transport, host microtubules also promote trafficking of rhoptry proteins secreted into the host cell (12). However, whether this host cell structure functions during parasite invasion per se is unknown.Here, we tested the hypothesis that host microtubules are used by Toxoplasma tachyzoites to penetrate into its host cell. Using synchronized parasite invasion assays, we find that disruption of host microtubules significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are localized to the moving junction but, unlike their previously described role in pathogen invasion, host microtubules promote tachyzoite invasion by hastening the time that parasites initiate invasion.     

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.     

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background

The unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.

Methodology/Principal Findings

Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.

Conclusions/Significance

Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.     

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.     

A new release on life: emerging concepts in proteolysis and parasite invasion

Cell invasion by apicomplexan pathogens such as the malaria parasite and Toxoplasma is accompanied by extensive proteolysis of zoite surface proteins (ZSPs) required for attachment and penetration. Although there is still little known about the proteases involved, a conceptual framework is emerging for the roles of proteolysis in cell invasion. Primary processing of ZSPs, which includes the trimming of terminal peptides or segmentation into multiple fragments, is proposed to activate these adhesive ligands for tight binding to host receptors. Secondary processing, which occurs during penetration, results in the shedding of ZSPs by one of two mechanistically distinct ways, shaving or capping. Resident surface proteins are typically shaved from the surface whereas adhesive ligands mobilized from intracellular secretory vesicles are capped to the posterior end of the parasite before being shed during the final steps of penetration. Intriguingly, recent studies have revealed that ZSPs can be released either by being cleaved adjacent to the membrane anchor or actually within the membrane itself. Mounting evidence suggests that intramembrane cleavage is catalysed by one or more integral membrane serine proteases of the Rhomboid family and we propose that several malaria adhesive ligands may be potential substrates for these enzymes. We also discuss the evidence that the key reason for ZSP shedding during invasion is to break the connection between parasite surface ligands and host receptors. The sequential proteolytic events associated with invasion by pathogenic protozoa may represent vulnerable pathways for the future development of synergistic anti-protozoal therapies.     

Molecular interactions and signaling mechanisms during erythrocyte invasion by malaria parasites

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

Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.     

Reduced Parasite Motility and Micronemal Protein Secretion by a p38 MAPK Inhibitor Leads to a Severe Impairment of Cell Invasion by the Apicomplexan Parasite Eimeria tenella

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.     

Role of the parafusin orthologue,PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii

The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.     

Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells

Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells. Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite. Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro. The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively. These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.     

Toxoplasma Co-opts Host Cells It Does Not Invade

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.     

Time-Lapse Imaging of Red Blood Cell Invasion by the Rodent Malaria Parasite Plasmodium yoelii

In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum.     

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.