Introduction Toxoplasma gondii is an obligate intracellular parasite that belongs to the phylum Apicomplexa. All members of this phylum exhibit a similar process for invading into host cells . The process begins with gliding motility that utilizes a reversible attachment to migrate over the surface of the host cell, perhaps to find a susceptible site for entry. The parasites then establish an intimate association, involving reorientation to put the apical secretory structures in contact with the host membrane . This reorientation is coincident with a transient spike in host plasma membrane conductance, consistent with a break in the bilayer . Next, the specialized secretory organelles in the parasite's apex, called micronemes and rhoptries, secrete their contents [1,4]. A moving junction (MJ), where the host and parasite plasma membranes are in intimate contact, is then formed. The MJ is associated with a visible constriction around the invading parasite that migrates from anterior to posterior end during invasion. This results in the creation of a parasitophorous vacuolar membrane (PVM) derived from invagination of the host plasma membrane [3–6]. The entire process is highly orchestrated and driven by the parasite's actin and myosin machinery [7–11]. Among other functions, the MJ apparently serves to exclude most host membrane proteins from entering the PVM . Transmission electron micrographs of invading Plasmodium merozoites and Toxoplasma tachyzoites have shown the MJ to be associated with electron-dense structures reminiscent of tight junctions, but the composition of these is not known [5,6]. Numerous proteins involved in invasion have been identified, but only one of these, MCP-1 of P. falciparum, has been shown to be associated with the MJ [12,13]. This protein has no signal peptide or transmembrane domain and is presumed to interact with the MJ complex from the inside of the parasite; hence the key surface molecules that form this complex have remained a mystery. Apical Membrane Antigen 1 (AMA1) is among the proteins directly implicated in host cell invasion by several members of the phylum, including Toxoplasma, Plasmodium, and Babesia [14–16]. AMA1 is unique to the Apicomplexa and has been localized to the micronemes of developing intracellular parasites and to the apical surface of extracellular parasites just prior to invasion [17–19]. During invasion, AMA1 migrates across the tachyzoite surface from where it is proteolytically shed in a soluble form [18–21]. These observations have led to the hypothesis that AMA1 is one of the parasite adhesins that transiently engage host receptors during the invasion process . This model is supported by the observation that anti-AMA1 antibodies block invasion at a stage following the initial attachment to the host plasma membrane in Toxoplasma , Plasmodium  and Babesia . As predicted from these data, TgAMA1 is an essential gene, and conditional knockout parasites are significantly impaired in their ability to invade . TgAMA1 and its homologues from other Apicomplexa have the structural characteristics of type-I transmembrane proteins. The ectoplasmic region contains 16 invariant cysteine residues that are present in all AMA1 sequences, indicating a conserved overall topology among the homologues [24,25]. AMA1 from P. vivax has been crystallized, and analysis of the structure suggests a receptor-binding role in invasion, requiring inter-domain interaction . To better understand the precise role of TgAMA1, we sought to identify the parasite proteins with which it associates. We describe here three such proteins, at least two of which, RON2 and RON4, surprisingly, derive from the rhoptry necks, not the micronemes, from which AMA1 is released. At least one, RON4, is secreted upon attachment to host cells, where it then precisely and predominantly localizes to the MJ as it migrates down the length of the invading parasite. In wild-type parasites, only a small amount of TgAMA1 is associated with RON4 at the MJ, but in engineered parasites with reduced amounts of TgAMA1 this proportion rises and a clear concentration of TgAMA1 is evident at the MJ. This crucial complex appears to be conserved across the Apicomplexa. Results Identification of AMA1-Associating Proteins To determine the proteins associating with TgAMA1, immunoprecipitation from detergent extracts of extracellular Toxoplasma tachyzoites was performed using an independent monoclonal antibody (mAb) recognizing one of two distinct TgAMA1 epitopes. The mAb B3.90  detects an epitope in the ectodomain whereas mAb CL22  was raised to a dodecapeptide at the C-terminus of TgAMA1. Each of these antibodies immunoprecipitated TgAMA1 (~70kDa) and near identical profiles of TgAMA1-associating proteins (AAPs; Figure 1A). These AAPs, with apparent sizes of ~145, 130, 110, and 45 kDa, were stably associated with TgAMA1 at high salt concentrations (500 mM NaCl), suggesting a highly specific interaction (unpublished data). The specificity of the AAP association was further demonstrated by immunoblotting the material that co-precipitated with anti-TgAMA1 and probing for the highly abundant MIC2, GRA7, ROP1, or ROP2/3/4 proteins. None of these proteins were detected (unpublished data). Further evidence for the specificity of the AAPs for TgAMA1 was obtained using a Toxoplasma line in which the endogenous TgAMA1 locus has been disrupted and several tetracycline-repressible myc-tagged copies have been introduced (ΔAMA1/AMA1-myc; ). In the absence of added drug, this parasite line expresses ~10% of the constitutive TgAMA1 levels, which is a sufficient level for essentially wild-type levels of invasion and growth. Tetracycline treatment of these parasites reduces TgAMA1 levels to ~0.25% of wild-type amounts. Although such parasites have no apparent defect in motility or initial attachment to the host cell, they are almost completely blocked in their ability to invade . A specific role for TgAMA1 in invasion is further indicated by the observation that if tetracycline is added to cultures in which the parasites have already entered a host cell, there is no effect on further growth within that cell or on egress, but subsequent invasion is blocked. Immunoprecipitation of the AAPs from the ΔAMA1/AMA1-myc line grown in the absence of tetracycline resulted in recovery of the same profile of AAP bands (Figure 1B). As expected from its lower level of expression, markedly less TgAMA1 was recovered on a per-parasite basis, relative to wild-type parasites. For the four AAPs, however, nearly identical amounts were obtained (Figure 1 and unpublished data). As a result, the stoichiometry appeared to go from an excess of TgAMA1 over the AAPs in wild-type parasites to something closer to equal levels in the engineered parasites. This point is discussed further below. Under tetracycline-repressed conditions, immunoprecipitation with anti-TgAMA1 antibodies yielded much reduced TgAMA1 and the four AAPs (Figure 1B), although all were still detectable by Coomassie staining (Figure 1B) and/or immunoblot (Figure 1C). A variable number of bands below ~50 kDa were also observed in these gels, but apart from AAP45, they were not reproducibly detected in wild-type parasites (Figure 1A) and their abundance was not consistently reduced in the TgAMA1 knock-down conditions [Figure 1B, +Atc (anhydrotetracycline)]. Thus, they were not further pursued. Mass Spectrometry-Based Proteomic Analysis of TgAMA1 and the AAPs To determine the identity of the four AAPs, gel slices from the immunoprecipitation experiments were excised, digested with trypsin, and the eluted peptides were subjected to liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-MS/MS) analysis (Table 1). This analysis also confirmed that the ~70 kDa band was TgAMA1. Included among the identified peptides for this protein was one with an N-terminal sequence not derived from tryptic cleavage (TgAMA1 amino acids 40–54, Table 1). This likely derives from the pro-peptide cleavage of TgAMA1 described previously  and indicates cleavage of 17 amino acids to the C-terminal side of the predicted site for removal of the signal peptide. This N-terminal sequence for mature TgAMA1 was confirmed by Edman sequencing of the same immunoprecipitated TgAMA1 (unpublished data). Mass spectrometric analysis of the three largest AAPs revealed multiple peptides corresponding to Toxoplasma TwinScan predicted sequences Ts0698, Ts5626, and Ts4705 for AAP145, AAP130, and AAP110, respectively. Whether analyzed as a total gel slice representing the sizes from ~100 kDa to ~150 kDa, or as individually excised bands, these three predicted proteins and TgAMA1 were the only proteins for which multiple peptides were identified that met the confidence criteria described in the Materials and Methods (Table 1). Ts0698 corresponds to a protein of ~157 kDa, with a predicted signal sequence and three transmembrane domains. Proteomic analysis of the Toxoplasma rhoptries also recently identified this protein , and using antibodies to recombinant protein, it was shown to derive from the constricted, anterior portion of the rhoptries termed rhoptry necks. Ts0698 was one of four such neck proteins identified and was named RON2. The complete open reading frame was determined by cDNA sequencing; this confirmed the TwinScan prediction of a protein of ~157 kDa. Allowing for removal of a signal peptide, the predicted size (~153 kDa) is slightly larger than that predicted from the mobility on SDS-PAGE (~145 kDa). This leaves open the possibility that it may be processed by proteolytic cleavage to its mature size, a common property of many rhoptry proteins  although the discrepancy could also be a result of anomalous migration. There were no apparent functional motifs beyond the signal peptide and three putative hydrophobic transmembrane domains. RON2 is paralogous to two other Toxoplasma-predicted proteins, Ts3110 and Ts0430, but none of the peptides detected here exactly match these other two predicted proteins, and so the identity of p145 could be unambiguously assigned to RON2. Ts5626 corresponds to a protein that TwinScan predicts should be ~78 kDa with a signal peptide and no predicted transmembrane domains. Proteomic analysis of the Toxoplasma rhoptries also identified this protein and, using antibodies to recombinant protein showed it to also be a rhoptry neck protein . It was, therefore, dubbed RON4. The complete open reading frame was determined by cDNA sequencing. This indicated that the Ts5626 gene, in fact, corresponds to a predicted protein considerably larger (~107 kDa) than the TwinScan algorithm predicted (the TwinScan prediction miscalled some of the exon/intron boundaries, which is not uncommon). Allowing for removal of a signal peptide, the predicted size (~105 kDa) is still much smaller than that suggested by its mobility on SDS-PAGE (~130 kDa). The retarded migration in SDS-PAGE analysis may be explained by the 44 amino acid repeat sequence and the charges within this repeat . There is a paralogous protein identified in the T. gondii database, Ts2928, but none of the peptides detected here exactly match that protein and so the identification of AAP130 as RON4 was unambiguous. No functional motifs, other than a signal peptide, were observed. Ts4705 predicts a protein of ~179 kDa. This protein was also identified in the rhoptry proteome analysis although neither its size nor its localization were definitively determined . It therefore received no gene name and its true origin remains to be confirmed although all but one of 15 novel proteins that were definitively characterized in the Bradley et al. study proved to be from the rhoptry bulb or neck. It is therefore highly likely that Ts4705 corresponds to a rhoptry protein and given its association with RON2 and RON4 it most likely derives from the rhoptry neck. The discrepancy between the apparent (~110 kDa) and predicted (~179 kDa) sizes for this protein could indicate post-translational processing, as mentioned above for RON2. The identification of Ts4705 peptides in AAP45 is consistent with this possibility (see below) but some of the discrepancy could also be due to abnormal mobility or errors in the TwinScan prediction. In the excised gel band corresponding to AAP45, two significant peptide hits were identified corresponding to Toxoplasma actin (TgACT1), as well as seven peptides from the predicted C-terminus of Ts4705. Immunoblots with antibodies specific to TgACT1 confirmed that actin was present in the TgAMA1-immmunoselected material (unpublished data). Importantly, however and unlike the Coomassie staining of AAP45, there was no difference in the anti-actin signal using TgAMA1-immunoprecipitated material from untreated or tetracycline-treated ΔAMA1/AMA1-myc parasites and comparable signals were also seen in preparations obtained by immunoprecipitation using antibodies to an irrelevant control protein, ROP1 (unpublished data). These results strongly argue against a specific association of TgACT1 with TgAMA1. Consistent with this, TgACT1 has been proposed to interact with aldolase  and Toxofilin  yet no peptides corresponding to either of these two proteins were detected by MS, and neither were detected on immunoblots of material immunoprecipitated with anti-TgAMA1 (unpublished data). The Ts4705 peptides identified in p45 were all derived from the C-terminal region, with no overlap to peptides identified from p110 (Table 1). This is consistent with a proteolytic processing of the Ts4705 predicted polypeptide into p110 and p45 both of which are associated, directly or indirectly, with TgAMA1. Functional Analysis of TgAMA1 and RON Protein Interactions The finding that the micronemal protein TgAMA1 associates with at least two rhoptry neck proteins was unexpected and begged the question of whether these proteins associate with TgAMA1 in intact parasites or only when given the artificial opportunity of parasite lysis. To determine whether there is an in vivo interaction between the RONs and TgAMA1, therefore, we used chemical cross-linking of live, cultured parasites followed by immunoprecipitation from parasite extracts denatured so as to disrupt most non-covalent interactions (boiled in the presence of detergent). The cross-linker used was the membrane-impermeable, thiol-cleavable 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) and the parasites were from a culture in which invasion was synchronized with a high potassium block  that was released simultaneously with the addition of the cross-linker. Under these conditions, any proteins that are exposed to the medium and are within twelve angstroms of each other should be cross-linked, including those that are put out onto the surface during invasion. Following two washes and quenching of any remaining cross-linker, the fibroblast monolayer and attached parasites were lysed and boiled in 1% SDS and then equilibrated into RIPA buffer. Immunoprecipitation with anti-TgAMA1 was performed, as before, followed by DTT reduction and analysis by SDS-PAGE. Immunoblotting revealed RON4 and RON2 specifically associating with significant amounts of TgAMA1 but only if cross-linked before the precipitation (Figure 2A). The specificity of the cross-linking was demonstrated by showing that the control rhoptry protein ROP1 was not co-immunoprecipitated by any of the heterologous antisera and neither did anti-ROP1 antibodies co-precipitate detectable amounts of any of the other three proteins (Figure 2B). Reciprocal experiments using immunoprecipitation with anti-RON4 yielded TgAMA1 (Figure 2C). Some TgAMA1 is detected in the absence of DTSSP, but cross-linking significantly increased the amount of TgAMA1 co-precipitating with RON4. Interestingly, RON2 was pulled down with anti-RON4 regardless of the presence of cross-linker suggesting a covalent or other strong association between these two proteins (Figure 2C). No co-precipitation of TgAMA1 and RON2, or RON4 was seen using heat-denatured lysates from extracellular parasites, with or without prior cross-linking (not shown). These results indicate that an association between TgAMA1 and RONs occurs during the process of invasion and is surface-exposed. That is, the association is not simply an artifact of parasite lysis although this may increase the amount of association that is seen ( Figure 1). To explore the association further, tachyzoites that were intracellular and ones that were entering and exiting fibroblast monolayers were analyzed by indirect immunofluorescence (IIF) to identify where in the cell TgAMA1, RON4 and RON2 might be associating. Unfortunately, the antisera available for RON2 gave an IIF signal only using methanol-fixation, and not the formaldehyde-fixation conditions needed to preserve good morphology for IIF. While this did allow identification of RON2 as a rhoptry neck protein , no signal was observed on invading parasites indicating that either this protein is released into the medium during invasion or, more likely, the structures it is within are not stabilized with this fixative (unpublished data). Antibodies to RON4, however, worked well using formaldehyde-fixation and so co-localization with TgAMA1 could be assessed. Using intracellular parasites, and as predicted from the literature, anti-RON4 and anti-TgAMA1 antibodies gave the closely apposed but clearly distinct IIF patterns typical of rhoptry necks and micronemes, respectively (Figure 3). RON4 was also detected in the parasitophorous vacuole (Figure 3, arrows) as previously reported . No TgAMA1 is seen in the vacuolar space. These results confirmed that TgAMA1 and the RON2/4 proteins are initially in distinct compartments within the parasite. Analysis of AAP Localization in Invading Parasites The above results predicted that the association of TgAMA1 and RON proteins happens only upon initiation of invasion when the micronemes and rhoptries discharge. To examine this, we used IIF and the invasion-synchronization method described above. At the very start of invasion, antibodies to RON4 can be seen staining the apical tip of parasites that are in intimate contact with the host cell and a very small, circumferential ring (Figure 4A–4D). RON4 is not detected on the surface of unattached parasites (unpublished data). Antibodies to surface antigen 1, SAG1, show bright staining under non-permeabilized conditions and this can be used to identify the portion of an invading parasite outside of the host cell up to the point of contact with the host plasma membrane . Subsequent permeabilization and staining for RON4 shows this protein localizes precisely with the limit of SAG1 staining (Figure 4A–4D). As the parasite enters the host cell, this ring of RON4 staining migrates down the length of the parasite, coincident with the constriction that demarcates the MJ (arrows) between host and parasite (Figure 4E–4H). Fully invaded parasites show a focal staining at the posterior end of the parasite where the PVM appears to contact the host plasma membrane (Figure 4I–4L). There is also some apical staining within the parasite, the intensity of which varies depending on the degree of saponin permeabilization (Figure 4I–4L and unpublished data). The RON4 staining of the MJ, including the final posterior focus is fully apparent in the absence of detergent permeabilization (Figure 4M–4T) consistent with the observation that RON4 is outside the portion of the MJ that keeps antibodies out of the nascent parasitophorous vacuole (PV). The focal surface staining at the site of invasion is only observed within about six min after release from the high potassium block to initiate invasion; after that, and likely when the PV has dissociated from the host plasma membrane , this posterior focal staining is no longer detected (unpublished data). The surface accessibility of RON4 to antibodies suggested that anti-RON4 antiserum might be also inhibitory for invasion, similar to the findings for some antibodies against TgAMA1 and PfAMA1 [2,18,33]. This was tested by pre-incubation of parasites with anti-RON4 antisera and/or inclusion of the antisera upon adding parasites to the human foreskin fibroblasts (HFFs). No significant reduction in invasion efficiency was observed (unpublished data); hence, binding of antibodies to RON4 either does not inhibit the invasion process or the titer of the antibodies used was insufficient to exert an effect. IIF of invading wild-type parasites using mAb CL22 and mAb B3.90 to detect TgAMA1 confirmed the reported surface localization of TgAMA1 on invading parasites and showed a marked concentration on the posterior region up to the MJ (Figure 5A and 5B). Co-staining with anti-RON4 showed overlap of the two signals at the MJ (Figure 5C and 5D) although the majority of TgAMA1 was not coincident with RON4. Nevertheless, rotation of the three dimensional rendering makes clear the overlap of TgAMA1 throughout the RON4 ring at the MJ (Figure 5E–H). While these results show some co-localization of RON4 and TgAMA1 at the MJ, the majority of TgAMA1 is clearly posterior to this position. The increase in the stoichiometry of RON4-association with TgAMA1 observed with the ΔAMA1/AMA1-myc parasites suggested that their association might be more apparent in IIF using these parasites. This line is fully invasion-competent, yet it has, on average, only ~10% of the wild-type amount of TgAMA1 . Hence, the overall TgAMA1 staining should be less, but that which is observed may be concentrated in regions on the surface most critical for invasion. Repeating the IIF on invading ΔAMA1/AMA1-myc parasites confirmed the reduced level of TgAMA1 staining relative to wild-type but revealed that approximately one in twenty of these parasites have a level that is substantially lower than the average (i.e., 3.2 and a D-CN >0.25, and the individual spectra examined for ion coverage and overall quality. Nucleic acid techniques. The generation of complete coding region cDNAs for RON2 and RON4 were as described previously, as was the use of recombinant versions of these proteins to produce antibodies in mice . Constructs for the disruption of RON4 were generated using HXGPRT selection and the vector pMini-GFP.ht . For the knockout construct, a PCR fragment from genomic bases −3941 to −1939, relative to the start codon, were digested with Kpn and Apa1, and ligated into pMini-GFP.ht similarly cut. Into the resulting construct, a PCR fragment of genomic bases 12,053–14,276 (again relative to the ATG start codon) digested with Not1 and SpeI was cloned. The primers used to expand the flanking regions were 5′-CGGGGTACCCCAATCAAAATCCGCAATAGCC-3′, and 5′-CGGGTGCACCAGGTGACCCGTCCATAC −3′ for the upstream flank and 5′-GGACTAGTTTGCCTTGTTTCGCCTTAC-3′, and 5′-AAGCGGCCGCTGTTTCCCTTTGAACTCTGCCAC-3′ for the downstream flank. The resulting vector pKORON4 was linearized with NotI, and 50 μg of DNA was electroporated into RHΔHXGPRT strain parasites by standard methods, and selection of HXGPRT parasites was performed as previously described . Four independent transformations were carried out. Ten days after transformation and MPA/xanthine selection, DNA was isolated from selected populations and PCR used to screen for the presence of knockout parasites. An HXGPRT-internal primer (5′-GTGGCGATTCTCATCGACTT-3′) and a primer representing the region upstream of RON4 (5′- CTTCTTCGGTTCCTCGTTAG-3′) were used for PCR detection of integration into the upstream flanking region. Microscopy. Analysis of tachyzoite-invasion was performed following potassium buffer shift to synchronize invasion essentially as described above. Specifically, 106 parasites in Endo buffer were added onto HFF monolayers grown on 20-mm cover-slips in 24-well plates and incubated for 15 min at 37 °C to settle and contact the monolayer. Invasion was initiated by exchanging the buffer to HBSS, supplemented with 1% FBS at 37 °C followed by incubation for either 1 min to capture partially invaded parasites, or 20 min for a predominantly fully invaded population. Tachyzoites were washed in PBS, and fixed with 3.5% formaldehyde in 150 mM phosphate buffer [pH 7.2], washed and processed for indirect immunofluorescence as described previously . Alternatively, fixation was carried out using 100% methanol at −20 °C for 2 min. Specific antibody staining was developed with appropriate Alexa488- or Alexa594-secondary antibodies (Molecular Probes, Eugene Oregon, United States). Phase and fluorescence images were captured at 100× on an Olympus BX60 and a Hamamatsu Orca100 CCD, and were pseudo-colored and merged using Image pro-plus 2.0 software (Mediacybernetics, Silver Spring, Maryland, United States). Where indicated, serial Z-stacks images were collected at 100× on a motorized Zeiss (Thornwood, New York, United States) Axiovert 200M equipped for DIC light microscopy. In these cases, fluorescence images were captured with a Hamamatsu Orca2 CCD camera (Hamamatsu, Hamamatsu City, Japan) and were deconvolved by using an iterative algorithm and calculated point spread function, pseudo-colored, and merged using Openlab 4.02 and Velocity 3.01 software (Improvision, Lexington, Massachusetts, United States).