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Toxoplasma gondii rhoptry discharge factor 3 is essential for invasion and microtubule-associated vesicle biogenesis [1]

['Rouaa Ben Chaabene', 'Department Of Microbiology', 'Molecular Medicine', 'Faculty Of Medicine', 'University Of Geneva', 'Geneva', 'Matthew Martinez', 'Department Of Biochemistry', 'Biophysics', 'Perelman School Of Medicine']

Date: 2024-08

Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They contain proteins and membranous materials injected directly into the host cells, participating in parasitophorous vacuole formation. Toxoplasma gondii tachyzoites harbor 8 to 12 rhoptries, 2 of which are docked to an apical vesicle (AV), a central element associated with a rhoptry secretory apparatus prior to injection into the host cell. This parasite is also equipped with 5 to 6 microtubule-associated vesicles, presumably serving as AV replenishment for iterative rhoptry discharge. Here, we characterized a rhoptry protein, rhoptry discharge factor 3 (RDF3), crucial for rhoptry discharge and invasion. RDF3 enters the secretory pathway, localizing near the AV and associated with the rhoptry bulb. Upon invasion, RDF3 dynamically delocalizes, suggesting a critical role at the time of rhoptry discharge. Cryo-electron tomography analysis of RDF3-depleted parasites reveals irregularity in microtubule-associated vesicles morphology, presumably impacting on their preparedness to function as an AV. Our findings suggest that RDF3 is priming the microtubule-associated vesicles for rhoptry discharge by a mechanism distinct from the rhoptry secretory apparatus contribution.

Host cell invasion is a vital step for the survival and dissemination of this obligate intracellular parasite. Active entry into host cells is a multistep process that occurs in as fast as ~30 s and involves the sequential, regulated secretion of 2 sets of specialized apical secretory organelles called micronemes and rhoptries. Microneme exocytosis is instrumental for parasite egress from infected cells, motility, and invasion, whereas discharge of rhoptries occurs at the time of invasion [ 2 – 4 ]. Rhoptries are club-shaped organelles that secrete RON (rhoptry neck) and ROP (rhoptry bulb) proteins implicated in invasion and subversion of host cellular functions, respectively. Rhoptries are formed de novo during endodyogeny, at a late stage of the division cycle [ 5 ]. T. gondii tachyzoite possesses 8 to 12 rhoptries, and disruption of the cargo receptor, sortilin-like receptor (SORTLR), impairs their biogenesis [ 6 ]. The armadillo repeats only protein (ARO) is crucial for the clustering and the apical anchoring of these organelles [ 7 ], while the coccidian-specific CORVET/HOPS Associated Protein (CSCHAP) is implicated in the apical positioning only [ 8 ]. Conditional depletion of any of these proteins leads to a severe block in invasion resulting from the absence of rhoptry discharge. The 3D structure and organization of the elements surrounding the rhoptries at the conoid have recently been resolved by cryo-electron tomography (cryo-ET) [ 9 – 12 ]. Two rhoptries enter the conoid [ 13 , 14 ] and are aligned along a pair of intraconoidal microtubules (ICMTs) with their necks reaching an apical vesicle (AV) located underneath the plasma membrane at the tip of the parasite. The AV is docked to an elaborate structure called “rhoptry secretion apparatus” (RSA) inserted in the parasite plasma membrane [ 10 , 15 ]. Five to 6 microtubule-associated vesicles (MVs) are aligned along the ICMTs and thought to be involved in replenishing the AV and enabling successive rounds of rhoptry discharge [ 13 , 16 ]. Concordantly, MVs and ICMTs are only present in parasites that harbor more than 2 rhoptries. All these elements are presumably functionally linked to rhoptry discharge in T. gondii [ 9 , 17 ]. Remarkably, several proteins involved in rhoptry discharge, such as the complex of non-discharge (Nd) proteins and cysteine repeats modular proteins (CRMPs) [ 9 , 18 ], are conserved outside Apicomplexa, in other Alveolate species (e.g., Ciliates) that contain organelles evolutionarily related to rhoptries [ 19 , 20 ]. Two proteins, initially designated as MIC15 and MIC14, have been identified to play a role in rhoptry discharge and were subsequently renamed RDF1 and RDF2, respectively [ 21 ]. These proteins exhibit a scattered punctate distribution that does not resemble any known subcellular compartment and have been identified as components of the CRMPs complex [ 18 , 22 ]. Additionally, a rhoptry apical surface protein 2 (RASP2) covers the end of the rhoptry neck and is crucial for rhoptry discharge [ 23 ].

(A) Schematic representation of the mutants. Mut1 (green). Amino acids highlighted in green were mutated to alanine. Δ1 (purple); 11 amino acids highlighted in purple were deleted. Δ2 (pink); 20 amino acids highlighted in pink were deleted. (B) Western blot using anti-Myc antibodies showing the expression of the complemented mutated strains compared to the parental line DiCre and RDF3-depleted parasites. Catalase (anti-CAT) is used as a loading control. (C) Immunofluorescence of the 3 intracellular mutant parasites using anti-Myc antibody (green) and anti-ARO antibody (magenta). Scale bar = 2 μm. (D) A representative image of a plaque assay of different parasite strains (±Rapa) showing that complementation using RDF3-Δ1 and RDF3-Δ2 do not rescue the parasite lytic cycle (no plaque), whereas small plaques were observed for RDF3-mut1. (E) Red/green invasion assay (±Rapa) showing that complementation with RDF3 mutants does not rescue the invasion of the parasite. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. (F) Phospho-STAT6 assays assessing the ability of the parasite to secrete the rhoptry protein ROP16 into the host cell that phosphorylates host STAT6 in the nucleus. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. (G) Immunofluorescence of the 3 extracellular mutant parasites (±Rapa) in the presence of cytochalasin D (+CytD). Anti-Myc antibody (green) was used to visualize RDF3 protein and anti-ARO antibody (magenta). Scale bar = 2 μm. (H) Immunofluorescence of RDF3-Δ1 parasites fixed at different time points post-invasion. Anti-Ty antibody (green) is used to stain RDF3. Anti-ARO antibody is staining the rhoptry organelle. Counter-staining of DNA with DAPI (blue). Scale bar = 2 μm. Source data are provided as S1 Data . RDF3, rhoptry discharge factor 3.

(A) 2D slice through a tomogram of an RDF3 knockdown T. gondii, highlighting the components of the rhoptry secretion system. (B) Violin plot of the AV dimensions in wild-type, RDF3 control, and RDF3 knockdown parasites. A two-sided T test was performed to determine statistical significance between the knockdown and the control and wild-type together. N = 6 wild-type, 4 RDF3 control, and 10 RDF3 knockdown parasites. (C) 2D slice through 2 tomograms (top and bottom) of RDF3 knockdown parasites, highlighting extra conoidal vesicles forming an array, comparable to the MVs. (D) Plot of the number of MVs (left half) and extra conoidal vesicles (right half) in wild-type, RDF3 control, and RDF3 knockdown parasites. A two-sided Mann–Whitney U test was performed to determine statistical significance between the RDF3 knockdown and the control and wild-type together. N = 6 wild-type, 4 RDF3 control, and 20 RDF3 knockdown parasites. (E) Violin plot of the MV widths in wild-type, RDF3 control, and RDF3 knockdown parasites, and extra conoidal vesical widths in the knockdown parasites. A two-sided T test was performed to determine statistical significance between MVs or extra vesicles from the knockdown and MVs from the control and wild-type together. N = 6 wild-type, 4 RDF3 control, and 10 RDF3 knockdown parasites. (F) Side view (top) and top-down view (bottom) through a tomogram of an RDF3 knockdown parasite that has 2 AVs and 2 RSAs. (G) Different slices through one tomogram of an RDF3 knockdown parasite displaying a normal AV and RSA (top) and another AV-like vesicle with a partially formed RSA (bottom). Panels A, E–G are without (right) and with (left) color overlay. Scale bars are 100 nm in panels A and E, and 50 nm in panels F and G. Statistical significance was written as “ns” if p > 0.1. Source data are provided as S1 Data . AV, apical vesicle; MV, microtubule-associated vesicle; RDF3, rhoptry discharge factor 3; RSA, rhoptry secretion apparatus.

Given the apical localization of RDF3 and its crucial role in rhoptry secretion, we hypothesized that knockdown of RDF3 would result in a significant structural defect of the rhoptry secretion machinery. Upon imaging of RDF3 knockdown extracellular parasites, surprisingly there were no obvious structural changes around the rhoptries. We observed normal rhoptry docking to the AV and a similarly shaped AV compared to both the wild-type parasites and control parasites without the induction of RDF3 knockdown (Figs 8A, 8B and S8A ). Examination of the vesicles associated with the ICMTs showed that RDF3 knockdown parasites had an abundant number of vesicles in the vicinity of the ICMTs ( Fig 8C ). Some vesicles are clearly associated with the ICMTs and were identified as MVs ( Fig 8C , blue) while others were found more distant and were named extra vesicles (EVs) ( Fig 8C , brown). Quantification of the MVs number and size showed no significant differences between control and RDF3-depleted parasites ( Fig 8D ). When the same analysis was performed on EVs, their number varied significantly between control and RDF3-depleted parasites and their size appeared more variable even if not significantly different from the control (Figs 8D, 8E , S8B and S1 – S4 Movies ). Moreover, 4 out of 45 knockdown parasites displayed 2 AVs and 2 RSAs ( Fig 8F ). The phenomenon of 2 AVs has also been noted in the ICMAP2-iKD mutant [ 17 ], which resulted in disrupted MVs arrangement. The presence of a second AV might arise from premature anchoring of an MV to the cell apex to replenish the AV [ 17 ]. We additionally observed one parasite with a tentative second AV associated to an incomplete RSA ( Fig 8G ), potentially in the process of RSA biogenesis. Interestingly, there is still a chain of MVs near the ICMTs, but they display variable sizes compared to the control parasites ( Fig 8E ). Taken together, RDF3 appears to play a role in the proper trafficking, number, and morphology of the MVs.

First, to determine if RDF3 localization is dependent on these proteins, we endogenously tagged RDF3 in parasites strains where proteins are C-terminally fused to mAID-3HA cassette at the endogenous loci. The signal of RDF3 at the apex did not colocalize with Nd6, but it partially colocalized with RASP2 and CSCHAP signals in extracellular ( Fig 7B–7D , upper lanes) as well as in intracellular parasites ( S7B Fig ). Depletion of Nd6, RASP2, or CSCHAP did not affect the localization of RDF3 neither at the rhoptry bulb nor at the apex ( Fig 7B–7D , lower lanes). Reciprocally, RASP2 and CSCHAP were epitope tagged in the RDF3 inducible-knockdown parasites and confirmed that their apical localizations were not affected in the absence of RDF3 ( S7C Fig ). However, analysis of CSCHAP and RASP2 dynamics during invasion revealed that unlike RDF3, the signals of the 2 proteins persist even after parasite internalization ( S7D Fig ). Collectively, these findings suggest that while RDF3, RASP2, and CSCHAP exhibit some colocalization and play roles in rhoptry secretion, they do not rely on each other for targeting to the apical tip of the parasite.

To explore potential RDF3-interacting partners, we initially employed proximity labeling by introducing a second copy of RDF3 fused with the biotin-labeling protein, MiniTurbo [ 32 ] at the C-terminus. However, IFA indicated that the addition of this C-terminal tag disrupted the proper localization of RDF3 ( S7A Fig ). Consequently, we shifted our focus to investigating RDF3’s localization relative to proteins known to localize at the apex of the parasite. We also monitored how RDF3 localization was affected when these proteins were depleted. Nd6, RASP2, and CSCHAP are recognized to localize near the AV and play crucial roles in rhoptry discharge, albeit their precise functions remain unclear.

Next, we introduced a second copy of RDF3 under its own promoter (RDF3-4Myc) in the inducible knock-down mutants of ARO and SORTLR ( Fig 6D and 6E ). In the absence of ARO, rhoptries become detached from the apical complex and disperse in the cytosol (Figs 6D , S6E and S6F ) [ 7 ]. In ARO-depleted parasites treated with CytD, RDF3 dispersed in the cytosol, but its apical localization remained unaffected. Similarly, in SORTLR-depleted parasites treated with CytD, the localization of RDF3 at the rhoptry bulb was lost, while its localization at the tip remained unchanged, albeit with decreased intensity (Figs 6E and S6H ).

(A) Schematic representation of the apical complex of T. gondii highlighting the main structures and molecular players down-regulated in this study. (B) Immunofluorescence to assess RDF3 localization in ICMAP2-depleted parasites in extracellular parasites in presence of cytochalasin D (+CytD). Counter-staining of DNA with DAPI (blue). Scale bar = 2 μm. (C) Immunofluorescence to assess RDF3 localization in RNG2-depleted parasites in extracellular parasites in presence of cytochalasin D (+CytD). Counter-staining of DNA with DAPI (blue). Scale bar = 2 μm. (D) Immunofluorescence to assess RDF3 localization in absence of ARO in extracellular parasites in presence of cytochalasin D (+CytD). Counter-staining of DNA with DAPI (blue). Scale bar = 2 μm. (E) Immunofluorescence to assess RDF3 localization in absence of SORTLR in extracellular parasites in presence of cytochalasin D (+CytD). Counter-staining of DNA with DAPI (blue). Scale bar = 2 μm. Source data are provided as S1 Data . ICMAP2, intraconoidal microtubules associated protein 2; RDF3, rhoptry discharge factor 3.

To dissect RDF3 localization relative to the perturbation of components of conoid, we tagged the protein in 2 parasite lines that conditionally regulate the expression of genes orchestrating this structure ( Fig 6A ). First, RDF3 was tagged in the intraconoidal microtubules associated protein 2 (ICMAP2)-mAID-3HA strain. Depletion of ICMAP2 leads to the dissociation of the 2 ICMTs, their detachment from the conoid and the dispersion of the MVs and rhoptries [ 17 ]. Under this condition, RDF3 localization is not affected in intracellular parasites ( S6A Fig ) or in extracellular parasites treated with CytD (Figs 6B and S6B ). Subsequently, RDF3 was tagged in the Myc-RNG2-mAID-3H strain. RNG2 is known to have a dual localization at the base of the conoid and the apical polar ring. Depletion of RNG2 results in the detachment of the conoid, ICMTs, MVs, and leads to a disorganization of the rhoptries [ 31 ]. In the absence of a molecular marker for the AV, the impact of RNG2 depletion on its fate remains unclear. Nevertheless, under this condition, RDF3 maintains normal localization at the tip, whereas the staining of the rhoptry bulb becomes disorganized, indicating that the bulb-associated localization of RDF3 follows the disorganization of the rhoptries (Figs 6C , S6C and S6D ).

Rhoptry secretion relies on an arsenal of proteins located at the RSA at the tip of the parasite [ 3 , 4 , 15 ]. Some components of the apparatus might be perennial while others might show a dynamic localization when rhoptry discharge occurs. Therefore, we sought to dissect the dynamics of RDF3 localization by performing a time course infection using RDF3-2Ty expressing parasites. The invasion was synchronized by preincubation on ice and then started by incubating the infected culture at 37°C. Samples were fixed at different time points and a drastic change in RDF3 localization was observed over the course of invasion ( Fig 5A ). The percentage of intracellular parasites at each time point was quantified ( S5A Fig ) together with the localization of RDF3. In extracellular parasites treated with CytD (block of invasion), RDF3 is found at the bulb and at the tip in 90% of parasites. RDF3 signal is absent in 40% of parasites at 2 min post-invasion, coinciding with only 50% of parasites being internalized ( Fig 5A and 5B ). This likely accounts for the observed signal retention in the bulb and tip in 60% of parasites by IFA and the detectable signal by western blot (Figs 5B , S5A and S5B ). In contrast, RDF3 was undetectable in internalized parasites at 15 min to 1 h post-invasion by IFA and WB (Figs 5A, 5B and S5B ). After 4 h, the signal of RDF3 reappeared as a punctate staining observed around the rhoptry in 60% of the parasites, whereas in almost 35% the staining was observed at the bulb ( Fig 5A and 5B ). At that time, the parasite has not yet replicated and therefore the punctuated RDF3 staining is likely distinct from de novo rhoptry organelle biogenesis seen during daughter cell formation. Six hours post-invasion, a staining around the bulb is observed in almost 70% of the parasites with a punctate signal still observed in around 25% of the parasites ( Fig 5A and 5B ). Finally, when the parasite starts to replicate (>6 h post-invasion), a staining is observed at the tip and at the rhoptry bulb ( Fig 5A and 5B ). To refine the localization of RDF3 at a higher resolution, we employed ultrastructure Expansion Microscopy (U-ExM) (36). U-ExM analysis of extracellular RDF3-3Ty-U1 parasites revealed that RDF3 indeed localizes to the rhoptry bulb. However, its signal appears punctate and more dispersed compared to ROP5 staining ( Fig 5C ). The absence of RDF3 signal at the rhoptry tip using this technique raises questions about its localization. These findings, coupled with RDF3’s expression profile differing from luminal rhoptry proteins, prompt further investigation into whether RDF3 resides inside the rhoptry lumen or on the organelle surface ( S5C Fig ). To elucidate RDF3’s topology of RDF3, we performed proteinase K protection assays ( Fig 5D ). We subjected parasites to digitonin and proteinase K treatment, followed by western blot. Under these conditions RDF3, like the luminal rhoptry protein ROP1, was not proteolytically degraded, suggesting that it is not exposed to the cytosol. In contrast the rhoptry surface protein ARO was degraded ( Fig 5D ). Also, immuno-electron microscopy (EM) was performed on RDF3-2Ty strains, but the results did not yield any additional data to resolve RDF3 localization ( S5D Fig ). The gold particles were found scattered randomly throughout the cytoplasm, nucleus, and occasionally on the bulb of rhoptries.

(A) Microneme secretion of extracellular parasites stimulated with BIPPO to assess the release of MIC2 in culture supernatant. ESA excreted–secreted antigens. GRA1 is used as a control for constitutive secretion from dense granules. Samples derived from the same experiment and gels were processed in parallel. Image representative of 3 biologically independent experiments. (B) E-vacuole assays assessing the ability of the parasites to secrete the rhoptry protein ROP1 into the host cell. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. (C) Phospho-STAT6 assays assessing the ability of the parasite to secrete the rhoptry protein ROP16 into the host cell that phosphorylates host STAT6 in the nucleus. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. (D) Quantification of secreted RON4 at the tip of CytD-treated parasites. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. (E) ssTEM analysis confirmed the normal morphology and positioning of rhoptries in absence of RDF3 in intracellular parasites. Shown is gallery of 8 consecutive sections (i–viii) at 70 nm thickness through the one tachyzoite highlighting the normal rhoptries (r) morphology and bundled positioning at the apical tip of the parasite, together with one rhoptry neck docked (arrow in panel iv) inside the conoid (c). Shown also are the apicoplast (a) and mitochondrion (m). Scale bar: 0.5 μm. Source data are provided as S1 Data . RDF3, rhoptry discharge factor 3; ssTEM, serial section transmission electron microscopy.

The concerted action of microneme secretion and rhoptry discharge is essential for parasite invasion. We therefore examined whether these organelles were properly secreted in RDF3-depleted parasites. To assess microneme secretion integrity in RDF3-depleted parasites, we induced secretion with BIPPO and quantified processed MIC2, a secreted microneme protein, in the supernatant via western blot. A comparable amount of secreted MIC2 was found in the supernatant of the control strain (DiCre) and RDF3-depleted parasites, indicating no defect in microneme secretion ( Fig 4A ). We then examined the ability of the parasite to discharge rhoptry content in the absence of RDF3 by 3 different methods assessing the discharge of membranous materials and lipids (e-vacuoles assay), the injection of ROP16 (STAT6-phosphorylation assay), and the secretion of RON4 at the contact point between the parasite and the host cell (RON4-dot). RDF3-depleted parasites exhibited a significant impairment in rhoptry discharge, as evidenced by the 3 assays ( Fig 4B–4D ), which explains the observed invasion blockade. Unlike ASP3 depletion, which results in aberrant rhoptry morphology and positioning, rhoptries in RDF3-depleted parasites maintained proper morphology and positioning at the apical tip of intracellular parasites, as observed via serial section transmission electron microscopy (ssTEM) ( Fig 4E ). Moreover, rhoptry proteins are properly expressed and targeted to the organelles in absence of RDF3, as showed with RON9 and ROP5 that exhibit a normal localization, expression and processing assessed by IFA ( S4A Fig ) and western blot ( S4B Fig ).

(A) Plaque assay of different parasite strains (±Rapa) showing that depletion of RDF3 impairs the parasite lytic cycle (no plaque), a phenotype that is fully rescued by complementation with RDF3 wild-type copy (RDF3-4Myc). Image representative of 3 biologically independent experiments. (B) Quantification of plaque assays for DiCre, RDF3-3Ty-U1, and complemented RDF3 strain (±Rapa). (Mean ± SD; n = 3 biologically independent experiments.) Statistical significance was assessed by a two-way ANOVA significance with Tukey’s multiple comparison. (C) Intracellular replication assay. Graph representing the number of parasites per vacuole observed at 36 h post-invasion. (Mean ± SD; n = 3 biologically independent experiments.) (D) Induced egress assay. Graph representing the percentage of ruptured vacuoles following treatment with the egress inducers BIPPO for DiCre and RDF3-U1-3Ty parasites (±Rapa, 48 h). (Mean ± SD; n = 3 biologically independent experiments.) (E) Red/green invasion assay (±Rapa 48 h and 72 h) showing that depletion of RDF3 impairs the invasion of the parasite. ASP3-depleted parasites were used as a negative control. (Mean ± SD; n = 3 biologically independent experiments.) A parametric unpaired t test was used to assess significance; the two-tailed p-values are written on the graphs. Source data are provided as S1 Data . RDF3, rhoptry discharge factor 3.

TGGT1_242820 is a fitness-conferring gene [ 24 ], conserved only in a few members of the coccidian subgroup of Apicomplexa, Hammondia and Neospora species that are closely related to T. gondii ( Fig 1A ). The product of TGGT1_242820 is a hypothetical protein of 22 kDa that possesses a predicted signal peptide clustered with the rhoptries by LOPIT [ 24 ] ( Fig 1A and 1B ). This small protein exhibits alternative stretches of acidic and basic amino acid residues but no identifiable domains ( Fig 2A ). Three regions, C31-Q41, the hydrophobic region G113-A123 and the stretch of charged amino acids at the C-terminus are highly conserved among the putative apicomplexan orthologues ( Fig 2A ). Based on the functional evidence described below, the gene was named RDF3. CRISPR-Cas9 mediated carboxy-terminal 2Ty-epitope tagging at the endogenous locus ( S2A Fig ) refined the rhoptry localization of RDF3 ( Fig 2B ). Intracellular RDF3-2Ty parasites demonstrate a dual localization pattern, with most of the signal colocalizing with ROP5, a marker of the rhoptry bulb. Moreover, an additional RDF3 signal was observed above the RON9 signal, which denotes the neck of the rhoptries ( Fig 2B ). This dual localization was not observed in intracellular RDF3-3Ty-U1 parasites wherein RDF3 was only visible in the bulb of the rhoptry ( Fig 1B ). This difference in the RDF3 signal observed between both strains might be explained by the fact that the integration of the LoxP sequences within the 3′ UTR in RDF3-3Ty-U1 parasites leads to a slight decrease in the level of protein expression as observed by western blot (Figs 2C and S2B ). This phenomenon has been reported for other proteins [ 27 ]. Surprisingly, even though no staining was observed at the rhoptry tip in RDF3-3Ty-U1 intracellular strains, extracellular parasites treated with Cytochalasin D (+ CytD) in presence of host cells, showed an accumulation of the protein at the tip comparable to RDF3-2Ty ( Fig 2D ). CytD is an inhibitor of actin polymerization, which blocks parasite motility and invasion without impairing rhoptry and microneme secretion [ 28 , 29 ]. This allows us to observe parasites attached to the host cell that have discharged their rhoptries. This result suggests an accumulation of RDF3 at the tip of the rhoptries at the time of invasion likely close to the AV. Moreover, RDF3’s dual localization remains consistent whether the parasite has a retracted (+BIPPO/+CytD) or extruded conoid ( S2C Fig ).

In the recent years, advancements in genome- and proteome-wide experimental data sets have enhanced our understanding of apicomplexan parasite biology. To uncover novel rhoptry proteins critical in T. gondii, we integrated these data sets and prioritized genes meeting the following criteria: (1) encoding uncharacterized or hypothetical proteins predicted to localize to rhoptries based on Localization of Organelle Proteins by Isotope Tagging (LOPIT); (2) encoding proteins with predicted transmembrane domains or signal peptides; and (3) showing a negative fitness score from a global CRISPR/Cas9 screen (≤ −2) [ 24 ]. According to these criteria, 4 candidate genes were chosen ( Fig 1A ). Inducible-knockdown parasites were then created by epitope tagging and fusion with the DiCre-mediated conditional U1 gene silencing system at the 3′-untranslated region (UTR) of the gene loci ( S1A Fig ). This approach uses a parasite line expressing a split Cre recombinase (DiCre) [ 25 ]. Treatment with rapamycin (Rapa) triggers Cre dimerization, resulting in excision of the sequence between the LoxP sites and substitution of the exogenous 3′ UTR of the gene of interest with a repeat of 4 U1 recognition sequences, leading to RNA degradation. The CRISPR-Cas9-mediated tagging was conducted at the endogenous locus for all 4 candidates using the 3xTy-U1 cassette to investigate their localization and function. Correct integration, replacement of the 3′ UTR, and rapamycin-induced DiCre-mediated excision were analyzed by genomic PCR ( S1A Fig ). Indirect immunofluorescence assay (IFA) showed that all selected proteins are localizing to the rhoptries as they partially co-localize with ARO, a marker of the entire rhoptry surface ( Fig 1B ). Two of them (TGGT1_242820 and TGGT1_276210) are mostly found associated with the bulbous part of the rhoptries while others (TGGT1_305590 and TGGT1_315470) are more apical. Effective reduction of protein expression was observed upon addition of Rapa, as confirmed by western blot analysis, with protein levels significantly decreased after 72 h of treatment ( S1B Fig ). Plaque assays were conducted to evaluate the collective effect of protein depletion on the parasite lytic cycle. While 3 parasite lines (TGGT1_305590-3Ty-U1, TGGT1_276210-3Ty-U1, and TGGT1_315470-3Ty-U1) exhibited similar lysis plaque formation compared to the parental strain, depletion of TGTT1_242840 resulted in a significant reduction in parasite fitness.

Discussion

Rhoptries represent unique organelles essential for apicomplexan parasites such as T. gondii, assisting establishment of infection and subverting host cellular functions. The process of rhoptry discharge, crucial for successful invasion, involves intricate temporal and spatial coordination. It requires the secretion of their contents into the host cell while navigating through multiple physical barriers, including the rhoptry membrane, parasite plasma membrane, and host cell plasma membrane. This complex event involves a conserved macromolecular machine, the RSA, which forms a rosette at the parasite apex and docks an AV [9,10,33]. In the coccidian subgroup where there are more than 2 rhoptries, the secretion machinery expands to include 2 ICMTs and a series of up to 6 MVs. This expansion is believed to facilitate iterative rhoptry discharge [9,13]. The rosette, found in ciliates as well, facilitates the fusion of the rhoptry membrane with the parasite plasma membrane and is crucial for the exocytosis of secretory organelles. In contrast, some molecular players of rhoptry secretion such as CSCHAP, RASP2, RDF1, and RDF2 are unique to Apicomplexa and might be tailored to act in the fusion event with the host plasma membrane leading to the delivery of rhoptry contents into the host cytosol [8,21,23]. Moreover, the AV is also unique to Apicomplexa and likely instrumental for this last step, which is mechanistically and molecularly not understood to date. Importantly, the composition of the AV and how the rhoptries are docked to it remain unknown.

Here, we characterized RDF3, a small protein conserved in a subset of apicomplexans possessing multiple rhoptries. Unlike the broadly conserved ARO or ASP3, whose depletion results in mispositioning or morphological alteration of the rhoptries, respectively, [7,30,34], depletion of RDF3 blocks rhoptry secretion without affecting organelle positioning or morphology. Although little is known about what triggers rhoptry discharge, it is tightly linked to microneme secretion [21,29] and importantly microneme secretion occurs normally in RDF3-depleted parasites.

RDF3 is a highly charged protein with multiple stretches of acidic and basic residues that may play a role in its interaction with other proteins or membranes, facilitating rhoptry discharge at the time of invasion. Though RDF3 has no homolog in Plasmodium spp., the Plasmodium Apical Sushi protein (PfASP), also known as PfRON1, contains similar repetitions of negatively charged residues at the C-terminal domain [35]. However, this protein is not fully characterized, and its precise role is not known.

RDF3 shows a dual localization at the rhoptry bulb and close to the AV, not yet reported for any rhoptry protein. The detection of RDF3 at the rhoptry tip appears to be dependent on its level of expression since the introduction of LoxP sequences at the 3′ UTR of the gene lowers RDF3 levels and hampers its detection at the tip in intracellular parasites. Additionally, in intracellular parasites, the signal of RDF3 close to the AV is very faint compared to extracellular parasites. This implies that the protein accumulates at the rhoptry tip in extracellular parasites ready to invade and might be triggered by the attachment of the parasite to a host cell. RDF3 is fully soluble in PBS, suggesting that the N-terminal predicted signal peptide is cleaved. RDF3 seems to be enclosed in the lumen of the rhoptry organelles or vesicles as it is largely protected from degradation either by proteolysis under selective permeabilization conditions or by auxin-mediated proteasomal degradation. However, determining if this applies to both localizations of the protein is challenging due to technical limitations. Interestingly, RDF3 protein becomes undetectable, upon internalization of the parasite (2 min post-invasion). It is unlikely that the protein is secreted into the host cell as parasites treated with CytD retain RDF3 signal. Thus, we hypothesize that RDF3 is likely subjected to degradation process following completion of invasion. The investigation of the AV and MVs as key players in rhoptry secretion has been hindered thus far by the lack of specific molecular markers. The Nd6 signal persists at the apex of the parasite for up to 5 min post-invasion, whereas the CRMPs signal at the apical tip of the parasite disappears [18]. No RDF3 signal was observed at the tip of the rhoptries even after 6 h post-invasion, indicating that replenishment at the tip occurs later, possibly concurrently with parasite multiplication. This dynamic localization can speak for a replenishment process as the one proposed for the MVs [16,17]. The dynamic of disappearance and replenishment have not been reported before for any proteins, and the precise mechanisms underlying the changes in localization of RDF3 and its temporal regulation during invasion warrant further investigation. U-ExM results showed that the staining of RDF3 was different from the luminal rhoptry bulb protein ROP5 and was visible as small punctate signal likely corresponding to vesicles decorating the rhoptry bulb. Collectively, the results suggest that RDF3 is not located inside the rhoptry bulb lumen but is likely present in vesicles that are delivered around the rhoptry bulb via vesicular trafficking. Subsequently, RDF3 may be replenished at the rhoptry tip during invasion. Colocalization between RDF3 and ICMAP2 firmly establishes that RDF3 localizes close to the AV. The apical localization of RDF3 is independent of the presence of the ICMTs, conoid, or rhoptries and remained invariably unaffected under perturbation of these structures. The bulbous localization of RDF3 is tightly associated with the organelle and always colocalizes with ROP5 under organelle positioning perturbations. Additionally, in SORTLR-depleted parasites, only the bulb localization disappeared confirming that this localization is dependent on the presence of the rhoptries. Intriguingly, RDF3 partially colocalizes with known rhoptry discharge factors CSCHAP and RASP2 without affecting their expression or trafficking. Similarly, RASP2 and CSCHAP do not influence RDF3 localization. Cryo-ET is a cutting-edge technique that has recently been used to dissect the structural arrangement of the RSA of Apicomplexa. Therefore, it was instrumental to use this technique to investigate if in the absence of RDF3, morphological changes had occurred in the apical complex structures. This analysis revealed that the core of the secretion machinery is not affected in RDF3-depleted parasites. However, given the presence of a smaller population of MVs and abundant extra vesicles in the conoid with the size and appearance of MVs, the absence of RDF3 likely perturbed the biogenesis and maintenance of the MVs. For example, the regulation of their trafficking may be disrupted, leading to the accumulation of numerous MV-like vesicles within the conoid. Moreover, it has been hypothesized that MVs serve as precursors to AVs, facilitating iterative rhoptry discharge [16,17]. More recently, a second AV and RSA have been observed in ICMAP2 knockdown parasites [17]. The mutant exhibits dispersed MVs that may be capable of becoming an AV if they prematurely reach the cell apex. In the RDF3 knockdown parasites, nearly 10% contained 2 AVs and 2 RSAs, indicating that the extra conoidal vesicles are indeed dysregulated MVs that prematurely became an AV with an RSA. Given the severity of the rhoptry discharge defect observed, which contrasts with the effect seen in ICMAP2-depleted parasites, RDF3 likely also plays a role in the function of the AV. To dissect the function of RDF3, we evaluated the localization and functional complementation of an RDF3 mutant with 3 strictly conserved residues among coccidian sequences downstream of the signal peptide. This RDF3-Mut1 affects RDF3 trafficking, with a punctate signal dispersed through the cytosol, yet the parasite was still able to form small plaques and invasion was restored to almost 30%. Additionally, in extracellular parasites RDF3-Mut1 accumulated at the apex, whereas no signal was observed at the bulb. This accumulation can explain the slight rescue of the rhoptry discharge in this mutant. RDF3-Δ2 led to a severe decrease in RDF3 protein abundance indicating that the C-terminal region is important for the stability of the protein. Deletion of the hydrophobic region (RDF3-Δ1) was the most striking since it resulted in a severe defect in rhoptry discharge and invasion, while the protein was properly targeted. This indicates that the hydrophobic stretch is essential for RDF3 function but is not required for the protein’s localization or trafficking. Intriguingly, the apical localization of the nonfunctional RDF3-Δ1 persisted after invasion, whereas the functional WT RDF3 disappeared upon internalization of the parasite. In contrast, RDF3-Δ1 localization at the bulb disappeared just like the WT RDF3. In light of these results, it is tempting to speculate that the hydrophobic stretch plays a crucial role in RDF3 function at the tip. Functional RDF3 at the apex may be utilized during invasion, while RDF3 associated with the bulb, whether functional or not, may undergo degradation following invasion.

In summary, RDF3 emerges as a pivotal factor in rhoptry discharge, affecting MV biogenesis and likely influencing AV functionality. Its localization near the rhoptry bulb suggests a role in replenishing the new AV derived from MVs, potentially reloading the machinery for subsequent rhoptry discharge. While the hydrophobic region of RDF3 appears unrelated to protein trafficking and localization, it likely mediates interactions with crucial yet unidentified partners to facilitate AV function during invasion. This study unveils a dynamic process essential for effective rhoptry secretion, warranting further exploration.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002745

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