An in vivo fitness gene of Toxoplasma, MIC11, is essential for PLP1-mediated egress from host cells

Domain: biology
Published: 2026-04-04
Editorial outcome: major_revision

Abstract
After invasion and replication, intracellular pathogens must egress from infected host cells. Toxoplasma gondii facilitates this process by permeabilizing host cells through induced secretion of perforin-like protein 1 (PLP1). However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identify the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen identifies MIC11 as the top in vivo fitness-conferring gene. Deletion of MIC11 results in severe defects in membrane rupture and egress. Scanning mutagenesis identifies functional motifs in MIC11, and mechanistic analyses support an association between MIC11 and PLP1, suggesting that MIC11 is involved in PLP1-dependent membrane disruption. Moreover, the merozoite-specific paralogue MIC22 functionally complements MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii. Collectively, the discovery of MIC11 advances our understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

Introduction
Intracellular bacteria and protozoa utilize various strategies to exit infected cells, a process known as egress1. Egress is a critical step for the obligate intracellular apicomplexan parasites, such as Toxoplasma gondii and Plasmodium falciparum, which cause human disease worldwide and represent a major global health problem2. In apicomplexans, this process relies on a dynamic interplay between host and parasite factors and typically leads to host cell lysis, resulting in detrimental effects for the host.

In T. gondii, egress is initiated by a signaling cascade that triggers microneme exocytosis, ultimately leading to disruption of the vacuolar membrane surrounding parasites3,4. Recent studies have markedly advanced our understanding of the key regulators. Guanylate cyclase (GC)5,6 and protein kinase G (PKG)7 drive a signaling cascade that branches into two major pathways: calcium mobilization, which activates calcium-dependent protein kinases 1 and 3 (CDPK1 and CDPK3)8-11 to phosphorylate many substrate proteins involved in microneme exocytosis, and lipid signaling involving diacylglycerol kinase 1 (DGK1) and acylated pleckstrin-homology domain-containing protein (APH)12, which also facilitates microneme secretion. In addition to responding to extrinsic signals, the parasite generates intrinsic signals via the dense granule protein diacylglycerol kinase 2 (DGK2) to control natural egress13.

Upon activation of these signaling pathways, micronemes, small secretory organelles at the apical end of the parasite, release a variety of microneme proteins (MICs) essential for invasion and egress14,15. While rhoptry secretion is not required for egress16, and a dense granule phospholipase LCAT contributes to the process17,18, microneme exocytosis is considered the primary driver of vacuolar membrane disruption. Although T. gondii is estimated to contain over 40 MICs19, many of them remain functionally uncharacterized.

Among the characterized MICs, perforin-like protein 1 (PLP1), a microneme protein widely conserved among the apicomplexans, is essential for egress in T. gondii20. Parasites lacking PLP1 exhibit significantly impaired egress from host cells and a complete loss of virulence in mice, highlighting the critical role of efficient egress in the in vivo fitness of T. gondii. Discharged PLP1 likely forms pores on the parasitophorous vacuole membrane (PVM), a membranous vacuole where parasites proliferate, leading to membrane permeabilization and disruption21. A current model supported by genetic, biochemical, and structural studies suggests that PLP1 alone is sufficient for membrane disruption20,22-25. Although a micronemal metalloprotease toxolysin 4 (TLN4) is implicated in egress, its contribution is limited26. PLP1 is therefore currently considered the only micronemal protein responsible for membrane disruption. However, the precise molecular mechanisms underlying this process remain poorly understood, raising the possibility that additional unidentified factors may contribute.

A genome-wide in vitro screen in human fibroblasts identified many fitness-conferring genes during the lytic cycle27, but paradoxically found PLP1 to be dispensable. This was an unexpected outcome given its established role in egress. This discrepancy likely results from the screening protocol, which bypasses egress by mechanically lysing host cells, or from delayed egress of PLP1-deficient parasites. In contrast, published in vivo CRISPR screens, free from such interventions, consistently identified PLP1 as crucial for parasite fitness28,29, suggesting their utility in uncovering egress factors. As these show, CRISPR/Cas9-based genetic screens have been instrumental in identifying essential genes involved in diverse biological processes in T. gondii 30,31.

Here, we performed a CRISPR-based in vivo screen targeting genes encoding proteins of the apical complex and pellicle, leading to identification of several previously unrecognized genes essential for T. gondii virulence. Among them, we identified microneme protein 11 (MIC11) as a key egress factor that likely cooperates with PLP1 to mediate membrane disruption and parasite egress from host cells. Our findings challenge the long-standing egress model of T. gondii and provide revised mechanistic insights into the molecular basis of parasite-induced membrane rupture during egress.

Results
CRISPR screen identifies MIC11 as the top-hit among in vivo fitness-conferring genes

The apical complex (including the conoid and apical secretory organelles) and the pellicle (inner membrane complex and parasite plasma membrane) are critical subcellular compartments of the apicomplexan parasites participating in motility, invasion, and egress32,33. This motivated us to search for genes essential for the parasite in vivo fitness by CRISPR-based screening targeting proteins localized to the apical complex and pellicle.

We employed a previously established in vivo CRISPR screen system using the type I RH strain and inbred C57BL/6 mice28 (Fig. 1a). We designed a new guide RNA (gRNA) library targeting selected genes encoding the apical complex or pellicle-localized proteins19 (Fig. 1b). RH∆hxgprt parasites were transfected with the gRNA library and subjected to pyrimethamine selection for four passages in cell culture. Parasite mutant pools from the fourth passage were subsequently inoculated into the footpads of C57BL/6 mice at a dose of 107 parasites per mouse. Seven days after infection, parasites were retrieved from the spleens, expanded in cell culture for one passage, and parasite genomic DNA was extracted for sequencing of genome-integrated gRNAs.

Fitness scores were calculated as the average log fold change of gRNA abundances between the input library and the fourth passage (in vitro), or the fourth passage and the spleen (in vivo). Fitness scores represent the relative abundance of each mutant within the parasite population, thereby reflecting the contribution of the targeted gene to parasite survival. Our in vitro fitness scores were highly correlated with published genome-wide in vitro fitness scores in human foreskin fibroblasts (HFFs)27 (Supplementary Fig. 1a). Our in vivo fitness scores were highly reproducible between independent infections in mice (Supplementary Fig.1b) and correlated well with previously reported genome-wide fitness data in mice (Supplementary Fig.1c). Together, these results support the robustness and reproducibility of our screening approach.

Comparing in vitro and in vivo fitness scores identified genes that contribute to parasite fitness during acute infection (Fig. 1c). The distance of each gene from the regression line was computed to identify top-ranking candidates (Supplementary Data 1). Known virulence factors such as ROP18 and GRA23 ranked among the top hits, further validating the robustness of the approach. Some microneme‐associated candidates were highly ranked, such as MIC11, SPATR, TGGT1_272380, and TGGT1_221180. MIC11 is also one of the top hits of another in vivo CRISPR screen29. SPATR was reported to be a component of the MIC complex essential for rhoptry discharge and contributes to virulence34-36. TGGT1_272380 was recently shown to be localized to the endoplasmic reticulum and dispensable for virulence37; thus, it was excluded from further analysis. Conoid‐associated candidates, such as PRP (TGGT1_291880) and CF6 (TGGT1_245640)38, were ranked highly. Consistently, pellicle‐associated candidates, such as PAP2 (TGGT1_247360), ULK kinase (TGGT1_235750), TGGT1_200460, and RAB6 (TGGT1_310460), were also ranked highly. Of these, TGGT1_200460 was recently shown to be essential for virulence29.

We selected nine genes displaying impaired in vivo fitness scores for validation, generating knockout parasites and inoculating them to the mouse footpad (Fig. 1d and Supplementary Fig.1d–e). Five knockouts (∆MIC11, ∆PAP2, ∆SPATR, ∆ULK, and ∆TGGT1_200460) were avirulent in mice. Four knockouts (∆RAB6, ∆PRP, ∆CF6, and ∆TGGT1_221180) showed attenuated virulence. These results demonstrate the high fidelity of the in vivo screen in identifying essential virulence factors. In this study, we further describe the characterization of the top-hit MIC11 in the following sections.

MIC11 is a microneme protein processed in an ASP3-dependent manner

MIC11 is a 16 kDa microneme protein conserved among coccidian parasites39, and its role remains unknown. MIC11 consists of a signal peptide and two additional peptides, α-chain and β-chain, separated by an internal γ-propeptide. During maturation, the γ-propeptide is removed, and the α- and β-chains are linked by a single disulfide bond to form mature MIC1139 (Supplementary Fig.2a). We confirmed that MIC11 localized to micronemes and its secretion was stimulated by calcium-mobilizing treatments (Supplementary Fig.2b–c). Under non-reducing conditions, the anti-MIC11 antibody detected a ~16 kDa band consistent with the mature species, whereas under reducing conditions two bands of ~10–15 kDa and ~20–25 kDa were observed, likely representing dissociated forms (Supplementary Fig.2d).

Most secretory proteins of T. gondii are proteolytically processed either during trafficking to secretory organelles or after exocytosis. ASP3, an aspartyl protease, has been shown to process several rhoptry proteins and MICs, and is essential for parasite invasion and egress40. Previous proteomics data suggested that MIC11 might be processed in an ASP3-dependent manner40. To test this, we used the anhydrotetracycline (ATc)-inducible ASP3 knockdown strain tagged with Ty epitope tag (ASP3-iKD). Western blot analysis confirmed that ATc treatment for 48 h depleted ASP3 protein expression. In agreement with a previous report40, MIC6, a known substrate of ASP3, accumulated as the immature form, whereas MIC4 was not affected. Under this condition, MIC11 also exhibited reduced levels of the mature form (Supplementary Fig.2e–f), indicating ASP3-dependent processing at two predicted sites in accordance with the previous study40 (Supplementary Fig.2a). Taken together, these results demonstrate that MIC11 is a soluble microneme protein processed in an ASP3-dependent manner.

MIC11 is essential for parasite egress and vacuolar permeabilization

Plaque assays were performed to assess the impact of MIC11 deletion on the lytic cycle and ∆MIC11 produced smaller plaques compared to WT parasites. The complemented ∆MIC11::MIC11-Spot-HA parasites, C-terminally tagged with Spot and HA epitope tags, restored normal plaque formation (Fig. 2a and Supplementary Fig.3a–b), confirming that MIC11 is required for the lytic cycle of T. gondii. Further investigation revealed that MIC11 deletion did not significantly affect invasion or intracellular proliferation (Supplementary Fig.3c–d). In contrast, abnormal spherical structures where multiple parasites were entrapped, reminiscent of those observed in the ΔPLP1 parasite-infected cells20. These spheres were not observed in WT or the complemented strains (Fig. 2b). Considering that PLP1 is necessary for egress, we performed induced egress assays following stimulation with the Ca2+ ionophore ionomycin to assess the role of MIC11 in egress. Most WT parasites egressed from the infected cells within 5 min, while ∆MIC11 parasites remained inside vacuoles indicating that ionophore-induced egress was severely impaired in ∆MIC11 parasites. ∆MIC11::MIC11-Spot-HA parasites recovered the egress phenotype (Fig.2c–d). In addition to Ca2+ ionophore, other stimuli such as the phosphodiesterase inhibitor zaprinast and low pH also trigger parasite egress 9,22,41. ∆MIC11 parasites also exhibited severe defects in zaprinast- and low pH-induced egress assays (Supplementary Fig. 3e–f). After intraperitoneal injection, ∆MIC11 parasites lost virulence in mice, and ∆MIC11::MIC11-Spot-HA parasites restored virulence (Fig. 2e).

To unravel the mode of action of MIC11, we examined which steps are affected by its deficiency. Microneme secretion and gliding motility of ∆MIC11 parasites were normal (Fig. 2f–g and Supplementary Fig. 3g). To evaluate the PVM integrity, we generated transgenic parasites expressing GRA1-mCherry. Under unstimulated conditions, the PVM is intact and GRA1-mCherry resides in the PV. Parasites are then immobilized with the actin polymerization inhibitor cytochalasin D (CytD). Upon Ca2+ ionophore treatment, microneme secretion and subsequent PLP1 discharge trigger PV rupture, followed by diffusion of GRA1-mCherry into the host cytosol20 (Fig. 2h). We next monitored the permeabilization of the PVM after stimulation with ionomycin by using time-lapse imaging. WT parasites caused PV rupture after ionophore stimulation within 5 min. In contrast, no PV rupture was observed in ∆MIC11 vacuoles within 10 min (Fig. 2i and Supplementary Fig. 3h). These findings demonstrate that impaired membrane permeabilization accounts for the defective egress in ∆MIC11 parasites.

Scanning mutagenesis reveals functional regions in MIC11

MIC11 lacks homology to known proteins and has no apparent conserved domains other than a signal peptide. AlphaFold3 was used to model the mature MIC11 consisted of α- and β-chains. We then selected the model for which the complex was consistent with disulfide bond formation, which exhibited a four-helix bundle due to each chain forming an antiparallel pair of helices (Fig. 3a).

To determine which regions of MIC11 are required for egress, we performed scanning mutagenesis by replacing each set of five amino acid residues with five consecutive alanines42. In addition, we generated point mutations C90A and C194A to disrupt the disulfide bond between these residues39. Transfection of these constructs into ∆MIC11 parasites yielded mutant lines mut_01–mut_35, C90A, and C194A (Fig. 3b). Ionophore-induced egress assays were performed to assess the phenotype of each mutant parasite. The results mapped the critical and dispensable regions in MIC11 across the α-chain, β-chain, and γ-propeptide (Fig. 3c). Essential regions were concentrated at the anterior and central parts of the α-chain, whereas they were distributed throughout the β-chain. As expected, C90A and C194A mutations failed to restore the egress phenotype. Among the γ-propeptide mutants, mut_23 showed markedly impaired egress.

Localization of MIC11 mutants was validated by immunofluorescence assays (Supplementary Fig. 4). Some mutants exhibited altered localization in addition to micronemes. Among the γ-propeptide mutants, MIC11 in mut_23 (IRPHG137–141 to AAAAA) accumulated in the PV and was absent from micronemes (Fig. 3d–e). Since the γ-propeptide is removed during maturation and its role remains unknown, we hypothesized that it contains a determinant for microneme trafficking of MIC11. Dissection by two alanine substitutions showed that IR_AA and PH_AA mutants failed to target MIC11 to micronemes, whereas GF_AA mutant localized normally (Fig. 3e–f). These results demonstrate that the IRPH motif in the γ-propeptide is essential for proper trafficking of MIC11 to micronemes.

MIC11 likely cooperates with PLP1 to facilitate parasite egress

To understand how MIC11 contributes to the PVM disruption, we performed co-immunoprecipitation mass spectrometry (IP-MS) analysis. Comparison of protein abundance between immunoprecipitations from ∆MIC11::MIC11-Spot-HA parasites over the parental ∆MIC11 parasites revealed several MICs, including PLP1, MIC5, MIC10, MIC20, and MIC21 (Fig. 4a and Supplementary Data 2). It is well established that many MICs form functional complexes15. Among these hits, only PLP1 has been previously implicated in parasite egress, whereas MIC5, MIC10, MIC20, and MIC21 have not been implicated in this process43-46. Consistent with this, our in vivo CRISPR screen indicated that PLP1 was essential for parasite fitness, while the other four MICs were dispensable, at least upon single deletion28 (Fig. 4b). Therefore, we generated ∆PLP1 parasites and a quadruple knockout strain lacking MIC5, MIC10, MIC20, and MIC21 (referred to as QKO) (Supplementary Fig. 5a–c). As reported previously20, ∆PLP1 parasites exhibited severely impaired egress and virulence, while QKO parasites exhibited egress and virulence comparable to WT parasites (Fig. 4c–4d).

To perform reciprocal IP-MS, we generated endogenously tagged PLP1-HA parasites, along with rhoptry protein 9 (ROP9)-HA parasites as a control (Supplementary Fig. 5de). IP-MS of PLP1-HA parasites resulted in the detection of MIC11 within the precipitated fractions (Fig. 4e and Supplementary Data 2). To further examine the potential associations between MIC11 and PLP1, we performed reciprocal co-immunoprecipitation assays. In these experiments, MIC11 and PLP1 were reciprocally co-immunoprecipitated (Fig. 4f). These observations are consistent with the formation of a complex containing MIC11 and PLP1, supporting their essential roles during parasite egress.

To further investigate the functional relationship between MIC11 and PLP1, we generated double knockout parasites lacking MIC11 and PLP1 (∆MIC11∆PLP1, referred to as DKO). Complementation with either MIC11-HA or PLP1-Ty alone did not restore egress, whereas double complementation (DKO::MIC11-HA::PLP1-Ty) fully rescued the egress phenotype (Fig. 4g–4i). These findings suggest that both MIC11 and PLP1 are required for parasite egress and cannot compensate for each other.

MIC11 is not required for PLP1 localization or secretion

We confirmed co-localization of PLP1 with MIC11 by immunofluorescence analysis (Fig. 5a). To further investigate the role of MIC11, we examined the impact of gene deletions on reciprocal protein localization. MIC11 localization was not affected by PLP1 deletion (Fig. 5b), and conversely, PLP1 localization was not affected by MIC11 deletion (Fig. 5c). Next, we assessed the secretion and processing of PLP1 and found that they appeared normal in the absence of MIC11 (Fig. 5d). These results indicate that ∆MIC11 parasites fail to permeabilize the membrane despite normal secretion of PLP1.

A putative MIC11 paralogue is expressed in the feline-restricted stages of T. gondii

Sexual reproduction of T. gondii occurs exclusively during the infection of the definitive feline host47. The parasites of feline-restricted stages need to egress from the intestinal cells, as do tachyzoites in the intermediate hosts. However, PLP1 and MIC11 are not expressed during the feline-restricted stages48. A previously proposed model suggested that PLP2, a PLP1 paralogue, is involved in egress in the feline gut49. This motivated us to search for a homologue of MIC11 expressed during the feline-restricted stages. We identified one gene, TGME49_295662, encoding an uncharacterized hypothetical protein that shares 25% amino acid sequence similarity with MIC11 (Fig. 6a). TGME49_295662 possesses a signal peptide, α-chain, β-chain, and γ-propeptide-like sequences. Within the γ-propeptide-like region, an IAPH sequence resembles the IRPH microneme trafficking determinant in MIC11 (Fig. 6a). The structure of mature TGME49_295662 was modeled in an equivalent manner as MIC11. In spite of the low sequence identity (25%), the two models were found to have the same overall topology, with TGME49_295662 containing an additional N-terminal helix in the β-chain (Fig. 6b). The Root Mean Square Deviation (RMSD) was 2.47 Å over 56 residues for the α-chains and 2.49 Å over 48 residues for the β-chain. In contrast to MIC11, TGME49_295662 is highly expressed in merozoites48 (Fig. 6c).

To characterize the putative paralogue of MIC11 in tachyzoites, we expressed the open reading frame (ORF) of TGGT1_295662 tagged with Spot and HA epitopes under the control of the SAG1 promoter in ∆MIC11 parasites (Fig. 6d). Immunofluorescence analysis revealed that TGGT1_295662-Spot-HA localized to micronemes in tachyzoites (Fig. 6e). We then compared the biochemical properties of MIC11 and TGGT1_295662 by subjecting ∆MIC11::MIC11-Spot-HA and ∆MIC11::295662-Spot-HA parasites to western blotting under non-reducing and reducing conditions. TGGT1_295662-Spot-HA showed a pattern like MIC11-Spot-HA (Fig. 6f), suggesting that the mature form of TGGT1_295662 consists of α-chain and β-chain, connected by a disulfide bond between C93 and C202 (Fig. 6b). Based on these findings, TGGT1_295662 was named microneme protein 22 (MIC22). Given the similarity of MIC11 and MIC22, we next examined whether MIC22 could functionally compensate for MIC11 deficiency. Indeed, the ∆MIC11::MIC22-Spot-HA strain fully restored both egress and virulence (Fig. 6g–6i). Collectively, these results suggest that MIC11–PLP1-mediated egress in tachyzoites may be replaced by MIC22–PLP2-mediated egress in feline-restricted stages.

Methods
Ethics statement

All animal experiments were conducted under the approval of the Animal Research Committee of the Research Institute for Microbial Diseases in The University of Osaka. C57BL/6NCrSlc (C57BL/6N) mice were purchased from SLC. Female mice aged 8–12 weeks were used in all experiments.

Toxoplasma strains

All T. gondii strains were maintained in Vero cells by serial passage using RPMI (Nacalai Tesque) supplemented with 2% heat-inactivated fetal bovine serum (FBS; JRH Bioscience), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Nacalai Tesque) in incubators at 37℃ and 5% CO2. The parental strains used in this study were RHΔhxgprt28 and RHΔhxgprtΔku8068. ASP3-iKD was described previously40. Depletion of ASP3 was induced by addition of 1 µg/mL of anhydrotetracycline (Takara).

Host cell culture

Vero cells were maintained in RPMI (Nacalai Tesque) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Nacalai Tesque) in incubators at 37℃ and 5% CO2. HFFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Nacalai Tesque) in incubators at 37℃ and 5% CO2.

Antibodies

The following primary antibodies were used in the immunofluorescence assay and immunoblotting. Mouse anti-HA (MBL, M180-7), mouse anti-HA.11 (BioLegend, 901514), mouse anti-Ty (BB2), mouse anti-GRA1 (TG17-43), mouse anti-MIC2 (T34A11)69, rabbit anti-MIC2 (this study), rabbit anti-MIC11 (this study), rabbit anti-MIC670, rabbit anti-MIC460, rabbit anti-PLP1 (N-terminus)23, rabbit anti-PLP120, rabbit anti-PLP1 (this study), rabbit anti-GRA1728, rabbit anti-ROP1871, mouse anti-SAG172, rabbit anti-GAP4572. We raised custom antibodies against synthetic peptides in rabbits as follows. MIC11 (RGEGRLDKEEAVDDESD), PLP1 (PTSKTMSSLKLAPVK), MIC2 (TKVVMEEEKETLV). The epitope identification, peptide synthesis, rabbit immunization, and serum collection were commercially conducted by Cosmo Bio. The following secondary antibodies were used in the immunofluorescence assay or immunoblotting. Donkey anti-mouse CF488A (Biotium, 20014), donkey anti-rabbit CF488A (Biotium, 20015), donkey anti-mouse CF594 (Biotium, 20115), donkey anti-rabbit CF594 (Biotium, 20152), goat anti-rabbit Alexa Fluor 647 (Invitrogen, A21244), goat anti-mouse-HRP (Invitrogen, G21040), goat anti-rabbit-HRP (Invitrogen, G21234). Dilutions of antibodies used in each assay are listed in Supplementary Data 3.

Parasite transfection

Parental parasites were filtered through 5-µm filter (PALL) and resuspended in transfection solution containing 2 mM ATP, 5 mM glutathione, DNA, and cytomix (10mM KPO4, 120mM KCl, 0.15 mM CaCl2, 5mM MgCl2, 25 mM HEPES, 2 mM EDTA). Parasites were electroporated in a 2-mm cuvette at 1500 V and 25 µF by GENE PULSER II (Bio-Rad). Stable transgenic parasites were selected with the appropriate antibiotics or sorted by cell sorter, cloned in 96-well plates by limiting dilution, and verified by genotyping, mRNA expression, or protein expression. The primers used for generating transgenic parasites are listed in Supplementary Data 3.

Generation of parasite transgenic lines

For generating knockout parasites, the specific gRNAs targeting each gene's 5′ and 3′ ends were cloned into the pU6-Universal vector (Addgene #52694). The floxed HXGPRT construct was PCR amplified with 60 bp homology arms to each gene's 5′ and 3′ UTRs. Parasites were transfected with two gRNAs and the PCR-amplified HXGPRT repair template. Transfected parasites were selected with 25 µg/ml mycophenolic acid (Sigma) and 50 µg/ml xanthine (Wako).

For generating quadruple knockout parasites (∆MIC5∆MIC10∆MIC20∆MIC21, QKO), parasites were transfected with eight gRNAs and the PCR-amplified HXGPRT, DHFR-TS, Venus, and mCherry repair templates targeting each gene. Transfected parasites were selected with 25 µg/ml mycophenolic acid, 50 µg/ml xanthine, and 3 µM pyrimethamine (Sigma) for serial passage. Stable resistant parasites were sorted for Venus and mCherry double positive parasites by BD FACS Aria III.

To generate endogenously tagged PLP1 and ROP9 strains, the 3HA-HXGPRT cassette was PCR amplified with 40 bp homology arms to the C-terminus of the respective loci. The resulting repair templates were co-transfected into parasites with gRNA vectors targeting the C-terminus of PLP1 or ROP9. Transfected parasites were selected with 25 µg/ml mycophenolic acid and 50 µg/ml xanthine.

To generate GRA1-mCherry expressing parasites, pUPRT-pTub-GRA1-mCherry plasmid was co-transfected with a gRNA vector targeting UPRT locus. Transfected parasites were selected with 10 µM fluorodeoxyuridine (Wako).

To complement PLP1 deletion, PLP1 cDNA was amplified from RH strain cDNA and cloned into the pUPRT vector. The pUPRT-PLP1-Ty construct was co-transfected with a gRNA vector targeting the UPRT locus. Transfected parasites were selected with 10 µM fluorodeoxyuridine.

To generate ∆MIC11∆hxgprt parasites, the loxP-flanked HXGPRT cassette inserted at the endogenous MIC11 locus was excised by transient transfection with Cre recombinase. Transfected parasites were then selected in medium containing 80 µg/ml 6-thioxanthine (Wako).

To complement MIC11 knockout parasites, wild-type MIC11 cDNA was amplified from the cDNA of the RH strain. MIC22 ORF was amplified from the genomic DNA of the RH strain. Each fragment was cloned into pHXGPRT vector. ∆MIC11∆hxgprt parasites were transfected with the pHXGPRT-pSAG1-MIC11/MIC22-Spot-HA plasmid and selected with 25 µg/ml mycophenolic acid and 50 µg/ml xanthine.

Scanning mutagenesis of MIC11

Mutated MIC11 cDNAs were commercially synthesized (FASMAC) and cloned into an expression vector in which MIC11 was expressed under the SAG1 promoter, while a Venus-P2A-HXGPRT selectable marker cassette was driven by the tubulin promoter. ∆MIC11∆hxgprt parasites were transfected with empty vector, wild-type MIC11, or each mutant construct, and selected with 25 µg/ml mycophenolic acid and 50 µg/ml xanthine. After drug selection, stable polyclonal populations were obtained. Flow cytometry analysis (Attune NxT, Thermo Fisher) confirmed that 70–90% of parasites were Venus-positive in all lines, indicating efficient transfection and selection. Each mutant population was then subjected to ionophore-induced egress assays.

Construction of the apical–pellicle gRNA library

The gRNA sequences of the apical–pellicle library were selected from the genome-wide gRNA library27 based on hyperLOPIT data19. The selected gRNA libraries were cloned into the modified pU6-Universal vector as previously described28. The construction of the gRNA library was performed by VectorBuilder.

In vitro and in vivo CRISPR screens

In vitro and in vivo CRISPR screens were conducted as previously described 28. Briefly, the gRNA library was linearized with NotI (Takara) and transfected into approximately 1–2×108 RHΔhxgprt parasites, divided between four cuvettes. Transfected parasites were grown in 4×150-mm dishes of confluent Vero cells. Medium was replaced with fresh one containing 10 µg/ml DNase I and 3 µM pyrimethamine 24 h post-transfection. Every 3 days until passage 3, parasites were scraped, mechanically lysed, and passaged onto new dishes. After 2 days (passage 4), parasites were syringe-lysed, filtered, and counted for either genomic DNA preparation or mouse infection.

For genomic DNA preparation, 1×108 parasites were pelleted and stored at -80 °C. For mouse infection, parasites were resuspended in PBS at 2.5×108 parasites/ml, and 1×107 parasites in 40 µl PBS were injected into the footpad of each anesthetized C57BL/6 mouse. Seven days post infection, mice were euthanized, and spleens were collected, homogenized by a plunger, and passed through a cell strainer to prepare cell suspensions. These suspensions were added to 2×150-mm Vero cell dishes per spleen. Parasites typically lysed out within 2–4 days. At least 1×108 parasites were pelleted and stored at -80 °C for genomic DNA extraction.

Parasite genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen). The gRNAs were amplified and barcoded with KOD FX Neo (TOYOBO) using Primer 1 and Primer 2. The resulting libraries were sequenced on a DNBSEQ-G400RS (MGI) using Primer 3 and Primer 4. All primers are listed in Supplementary Data 3.

Data analysis of the CRISPR screen

Following demultiplexing, raw read counts for each gRNA were obtained after alignment to the library. To account for differences in sequencing depth, counts were normalized by dividing each gRNA count by the total number of aligned reads per sample, yielding relative abundances. These normalized values were used to calculate log2 fold-changes between samples. For in vitro analysis, log2 fold-change values were computed between the passage 4 sample and the input library for each gRNA. For in vivo analysis, log2 fold-change values were computed between each in vivo sample and the passage 4 sample for each gRNA. The median fitness score across mouse replicates was used as the in vivo fitness score. P values were calculated using the Wilcoxon rank-sum test comparing passage 4 vs in vivo samples, and adjusted p values were calculated using the Benjamini-Hochberg method. The distance of each gene from the regression line was calculated as below.

All analyses were conducted by R (v4.1.1) using the stats package (v3.6.2) and visualized by ggplot2 (v3.4.0).

Assessment of in vivo virulence in mice

Mice were infected with 103 tachyzoites in 200 µl (intra-peritoneum) or 40 µl (footpad) of PBS. The mouse health condition and survival were monitored daily until 30 days post infection.

Immunofluorescence assay

HFFs were grown on coverslips, infected with parasites for 24–30 h, and fixed in 3.7% paraformaldehyde (PFA) for 10 min at room temperature. Cells were permeabilized with PBS containing 0.1% Triton-X or 0.002% digitonin for 10 min and then blocked with PBS containing 3% BSA for 1 h at room temperature. Then, the coverslips were incubated with the primary antibodies for 1 h, followed by incubation with appropriate secondary antibodies and DAPI for 30 min. The coverslips were mounted using PermaFluor (Thermo Scientific). Images were acquired by confocal laser microscopy (Olympus FV3000 IX83). Images were acquired using an Olympus FV3000 IX83 confocal microscope equipped with oil-immersion objective lenses (UPLXAPO60XO, NA 1.42, and UPLXAPO100XO, NA 1.45). Fluorophores were excited with 405, 488, 561, and 640 nm laser lines, and emissions were collected using spectral detection windows set to 440–480 nm (DAPI), 500–540 nm (CF488A), 581–631 nm (CF594), and 654–692 nm (Alexa Fluor 647).

Western blotting

Parasites were lysed in lysis buffer (1% NP-40, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing a protease inhibitor cocktail (Nacalai Tesque). The parasite lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked in 5% skim milk in 0.05% Tween-20 in PBS for 1 h at room temperature, followed by incubation with primary antibodies for 1 h at room temperature. Membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. HRP was detected using the Immobilon Forte Western HRP substrate (Millipore) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), visualized by ImageQuant LAS 4000mini (GE HealthCare).

Quantitative RT-PCR

Total RNA was extracted by RNeasy kit (Qiagen), and cDNA was synthesized by Verso reverse transcription (Thermo Fisher Scientific). Quantitative RT-PCR was performed with CFX Connect real-time PCR system (Bio-Rad) and Go-Taq real-time PCR system (Promega). The data was analyzed by the ∆∆CT method and normalized to ACT1 in each sample. The primers used are listed in Supplementary Data 3.

Microneme secretion assay

Freshly egressed parasites (1×108) were pelleted and resuspended in serum-free DMEM with 2% ethanol for 30 min at 37°C. To suppress microneme secretion, parasites were also treated with 100 µM BAPTA-AM. After incubation, parasites were pelleted, and the supernatants were transferred to new collection tubes and re-centrifuged. The final supernatants (ESA) were resuspended in SDS loading buffer. The pellet lysates and ESA were subjected to immunoblot.

Plaque assay

A total of 400 parasites were infected into a well of a six-well plate of HFFs and grown for 7 days in DMEM supplemented with 5% heat-inactivated FBS. Parasites were fixed with 3.7% PFA, stained with crystal violet, washed, and dried overnight. Plaque images were visualized using a stereo microscope (SZX16, Evident) equipped with a 1× objective and a digital camera (AdvanCam-HD2s, AdvanVision). Images were acquired using the camera's default software, and plaque areas were measured manually using ImageJ.

Invasion assay

Parasites (1×106) were inoculated on coverslips with HFF monolayer in a 24-well plate. The plate was centrifuged for 5 min at 250 G, incubated for 1 h at 37°C, and fixed with 3.7% PFA. Extracellular parasites were stained with anti-SAG1 antibody without permeabilization. After washing three times, the cells were permeabilized with 0.1% Triton-X, and all parasites were stained with anti-GAP45 antibody. At least 100 parasites were counted and determined as extracellular or intracellular parasites.

Replication assay

A total of 4×104 parasites were inoculated on coverslips with HFF monolayer in a 24-well plate. Twenty-four hours post-infection, the monolayers were fixed with 3.7% PFA and processed for immunofluorescence assay with anti-GAP45 antibody to stain parasites. The number of parasites per vacuole was counted for at least 100 vacuoles.

Induced egress assay

Freshly egressed parasites (1×105) were inoculated on coverslips with HFF monolayer and grown for 24–30 hours at 37°C. Egress was induced under the following conditions:

Ionophore-induced egress assay: monolayers were washed twice with serum-free DMEM and incubated with 2 µM ionomycin (Nacalai Tesque) in serum-free DMEM for 5 min at 37°C.

Zaprinast-induced egress assay: monolayers were washed twice with serum-free DMEM and incubated with 500 µM zaprinast (Tocris) in serum-free DMEM for 5 min at 37°C.

Low-pH induced egress assay: monolayers were washed twice with intracellular (IC) buffer (5 mM NaCl, 142 mM KCl, 1 mM MgCl2, 2 mM EGTA, 5.6 mM glucose, 25 mM HEPES, pH adjusted to 7.4 with KOH). The buffer was then replaced with SIC buffer (pH adjusted to 5.4 or 7.4 with HCl) with or without 0.002% digitonin, and coverslips were incubated for 3 min at 37°C. Cells were fixed with 3.7% PFA and processed for immunofluorescence assay using anti-SAG1 and anti-GRA17 antibodies to stain parasites and PVs, respectively. At least 100 vacuoles were manually scored as occupied or egressed.

Gliding motility assay

Freshly egressed parasites (2×106) were resuspended in serum-free DMEM containing 2% ethanol and added to poly-L-Lysine coated coverslips in a 24-well plate. Following incubation for 30 min at 37°C, the coverslips are fixed with 3.7% PFA and processed for immunofluorescence assay using anti-SAG1 antibody to stain parasites and trails. Parasite trail lengths were manually measured using ImageJ.

Parasite-induced PVM permeabilization assay

For time-lapse imaging, parasites (5×105) were inoculated on HFF monolayers in poly-lysine coated 35mm glass-bottom dishes (Matsunami) and grown for 24–30 hours at 37°C. HFF monolayers were pre-treated with 2 µM Cytochalasin D in DMEM for 5 min followed by the addition of an equal volume of 2 µM Cytochalasin D and 4µM ionomycin in DMEM. Time-lapse images were acquired every 10 sec for up to 10 min by a confocal laser microscopy (Olympus FV1200).

For quantification, infected HFF monolayers were pre-treated with 2 µM Cytochalasin D in DMEM for 5 min followed by the addition of an equal volume of 2 µM Cytochalasin D and 4µM ionomycin in DMEM for additional 5 min. Samples were then fixed and subjected to immunofluorescence assay using an anti-GRA1 antibody to stain PV. At least 100 vacuoles were manually scored as permeabilized or intact.

Co-immunoprecipitation followed by mass spectrometry (IP-MS)

Parasites were fixed with 0.1% PFA for 10 min, quenched with 100 mM glycine for 4 min, washed with HEPES-buffered saline, and lysed in HEPES-RIPA buffer (20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 0.25% sodium deoxycholate, 0.05% SDS, 1% NP-40) supplemented with protease inhibitor cocktail cOmplete EDTA-free (Roche) and Benzonase (Merck).

After centrifugation, the supernatants were processed as follows. For MIC11 immunoprecipitation, lysates of ∆MIC11::MIC11-Spot-HA parasites and ∆MIC11 parasites (control) were incubated with Spot-Trap magnetic agarose beads (Protein tech, etma). For PLP1 immunoprecipitation, lysates of RH::PLP1-HA parasites and RH::ROP9-HA parasites (control) were incubated with anti-HA antibody (#3724, Cell Signaling Technology) and Sera-Mag SpeedBeads Protein A/G (Cytiva). All incubations were performed for 3 h at 4°C. The beads were washed three times with HEPES-RIPA buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng of trypsin/Lys-C mix (Promega) at 37°C overnight. The digested peptides were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–100 min followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range from 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against T. gondii GT1 protein data (ToxoDB release 55) supplemented using Proteome Discoverer version 2.5 (Thermo Fisher Scientific, USA) with Sequest HT search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Label-free precursor ion quantification was performed using the precursor ions quantifier node, and normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Co-immunoprecipitation assay

Parasite lysates were prepared as described in the IP-MS section but lysed in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) supplemented with a protease inhibitor cocktail (Nacalai Tesque). After centrifugation, the supernatants were incubated with anti-HA antibody (sc-7392, Santa Cruz) for 2 h at 4°C with gentle rotation. Subsequently, the samples were incubated with Protein G Sepharose 4 Fast Flow (GE Healthcare) for 1 h at 4°C. The beads were washed three times with wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.2% Triton X-100, 0.2% Deoxycholate). Proteins bound to the beads were eluted by boiling at 95°C for 10 min in SDS-sample buffer and then subjected to immunoblotting.

AlphaFold prediction of MIC11 and MIC22 structure

The structures of MIC11 and MIC22 were predicted using the AlphaFold server (https://alphafoldserver.com/) 73 with the α- and β-chain sequences as input. The RMSD was computed using cealign in PyMOL (version 3.1.0). The resulting structural model was visualized using UCSF ChimeraX (version 1.10.1) 74.

Homologue search and sequential alignment

VEuPathDB75 was searched for MIC11 homologues using BLAST. MAFFT76 was used to align and visualize MIC11 and MIC22.

Statistics and Reproducibility

Statistical analyses of CRISPR screen were performed using R (4.1.1, https://www.r-project.org/). Statistical analyses of proteomics were performed using Proteome Discoverer (version 2.5) and GraphPad Prism10. Statistical analyses of other experiments were performed using GraphPad Prism10 and details of the analyses are reported in each legend. Quantification of image-based assay, such as plaque assay, egress assay, and motility assay, was manually performed. All fluorescence microscopy experiments were conducted in at least two biological repeats.

Data availability

The CRISPR screen data have been deposited in the NCBI GEO under accession number GSE290078 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE290078]. The MS proteomics data have been deposited in the ProteomeXchange Consortium via the jPOST partner repository with the dataset identifiers PXD062810 [https://proteomecentral.proteomexchange.org/?pxid=PXD062810] and PXD068211 [https://proteomecentral.proteomexchange.org/?pxid=PXD068211]. Source Data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Tachibana et al perform an CRISPR-based in vivo screen of Toxoplasma proteins localized to the apical complex and the\npellicle. They identify MIC11 as a novel egress protein interacting with PLP1 essential for in vitro and in vivo virulence. This\nis a comprehensive study which includes extensive characterization of MIC11, for example by identifying functional sites in a\nscanning mutagenesis experiment. The results challenge the longstanding idea that PLP1 is the only microneme protein\nrequired for parasite egress. While this is an interesting and novel finding, the study would benefit from increased scientific\nrigor, mainly by supporting their claims with statistical analysis and by providing sufficient detail in the materials and\nmethods section. In addition, a few experiments and conclusions require further validation and additional analysis.\nMajor concerns:\nStatistical rigor: Throughout the manuscript many statistical tests have been omitted or have not been reported (Fig 1D, Fig\n2D,E, Fig S3A, C-E, Fig 3A, Fig 4A, C, D, G, Fig 6C, H, I, Fig S6C). The authors should perform the appropriate statistical\ntests for all quantitative data presented in this manuscript and report the results in line with journal instructions (e.g. provide\nR2 value and p value for correlation calculations, always provide sample size and (corrected) p value along with the\nstatistical test used; when used specify what the error bars represent in the figure legends (e.g. Fig S3D)\nThe materials and method section is insufficient. The authors should update this section to allow for replication of their\nexperimental work and data analysis.\nThe authors should provide the working concentrations of their antibodies for all assays.\nThe authors never explicitly explain what the fitness score is in the main text or the Methods section\nL506-507 (“Images were acquired by confocal laser microscopy”). The authors should for example provide information about\nthe objectives and filters used.\nThe authors should explicitly state the methods used for normalization of their data. For example: L484-485 “The abundance\nof each gRNA was calculated and normalized to the total number of aligned reads.”\nThe number of parasites used for the experiments are missing at several parts in the manuscript (invasion assay, induced\negress assay, gliding motility assay, live cell imaging assay)\nThe plaque assay section lacks information about how the images were obtained.\nWhenever results were manually annotated the authors should explicitly indicate that - Otherwise provide access to the code\nthat was used to (for example plaque assay, intracellular vs egressed vacuoles, …)\nFor the gliding motility assay no information on potential quantification and analysis was provided.\nData presentation: Many of the results and quantification were obtained manually, e.g. gliding motility assay, localization of\nproteins in mutagenesis experiment. Thus, the authors should include larger more representative images to include more\nvacuoles and highlight particular areas of interest by cropping and enlarging the image.\nThe data presented for the mutagenesis screen require additional validation and analysis:\nThe authors are lacking sufficient evidence to claim that: “These results demonstrated that the IRPH sequence in the γ-\npropeptide is essential for trafficking to micronemes” (L213-214). They should either change the statement to apply it only to\nMIC11 or provide further evidence that 1) it is a conserved sequence and more microneme proteins have this sequence; 2)\nmutate the sequence in other microneme proteins to show whether these are also mislocalised. For example, MIC22 protein\ndoes not have an IRPH sequence (Fig 6B), contradicting the authors statement that IRPH is a universal microneme\nlocalization signal.\nThe mutants that were generated for the scanning mutagenesis have not been verified by PCR. It is not possible to affirm\nthat the mislocalisation is due to mutations of specific sequences or integration into a wrong locus. The authors should\nvalidate their mutants by PCR.\nIn Fig S4 many other parasite lines seem to mislocalize; for example: mut_10, mut_11 and mut_05. The authors should 1)\ncalculate the pixel correlation between the anti HA and MIC2 stainings within vacuoles for all mutants to be able to quantify\nthe localization. 2) provide larger representative images with more vacuoles and then highlight individual vacuoles through\ncrops.\nMany of these mislocalisation-causing mutations do not fall on the y propetide but still show strong egress phenotypes (Fig\n3A). The authors should comment on this. At an earlier point in the manuscript, the authors state that the scanning\nmutagenesis was applied to identify “ which regions of MIC11 are required for membrane permeabilization”. this point is\nnever addressed in the manuscript, since the authors focus on the γ-propeptide. The authors should comment on this.\nThe authors identify PLP1 as a direct interaction partner for MIC11 (Fig. 4). The authors could provide a connection between\nFig 3 and 4 by investigating which mutations hinder the interaction between PLP1 and MIC11. It would be interesting to see\nwhether the mutations that affect egress also have an impact on the interaction with PLP1. At the very least, the authors\nshould model the interaction between the two proteins using Alphafold and comment on this finding in connection with the\nmutagenesis results. In addition, it could be valuable to perform the CoIP experiments at different stages of infection (for\nexample extracellular vs intracellular) to assess whether the interaction is temporally regulated.", "reviewer_2": "In this study, the authors investigated the role of secretion from micronemes in the permeabilization of the host cell during\negress of Toxoplasma gondii. Using an in vivo CRISPR screen targeting proteins predicted to localize to the apical complex\nand pellicle membrane, they identified several genes essential for parasite virulence. Among these, they focused on MIC11,\nexamining its role in parasite egress and vacuolar permeabilization. Scanning mutagenesis revealed key functional motifs in\nMIC11 and identified residues critical for its trafficking to the micronemes. Authors investigate the interaction between MIC11\nand PLP1 to promote egress. Notably, complementation with the merozoite-specific paralog MIC22 rescued the ∆MIC11\nphenotype, suggesting a conserved role for MIC22 in feline-restricted sexual stages.\nThe rationale for identifying novel egress factors to better understand host cell permeabilization is well-supported.\nCritique major\n• The approach used, an in vivo CRISPR screen, has been previously used, and as acknowledged by the authors, several\ntop hits identified here were also reported previously. In addition, MIC11, the focus of this work, has been previously\nrecognized, raising the question of what new insights this screen adds beyond prior findings. The discussion would benefit\nfrom placing the results in the context of previous findings, with a clearer emphasis on the new insights this study presents.\n• The experimental setup for Co-IP analysis to identify MIC11 interactors included fixation with 0.1% PFA. PFA is known to\ncross-link molecules and normally would need to be quenched with glycine. However, the method detailed in the manuscript\ndid not include such step. The residual PFA could still cross-link proteins during lysis and could alter the analysis. Second,\nPFA can capture proximity interactors, as the linker is between 2-3 angstrom. It remains uncertain whether all the\nidentified hits represent direct interactions.\n• The scanning mutagenesis approach generated extensive data, analyzing multiple motifs of MIC11. While the authors\nappropriately focused on the γ-propeptide’s role in localization, further analysis was centered on mut_23, which showed the\nmost pronounced mislocalization. However, other mutants such as mut_06 and mut_27 also showed altered localization,\nand mut_27 being essential for egress. A clear rationale for not looking into these mutants is missing.\n• Concerning the scanning mutagenesis experiment analyzing egress of the mutants, are these clonal populations? If not,\nhow is the efficiency of the transfection and expression of the respective mutant accounted for in the egress analysis?\n• The scanning mutagenesis represents an impressive effort. However, the functional significance of these findings remains\nunclear. The result that Mut 23 affects MIC11 localization is interesting, but the authors do not explain why this matters or\nfollow through with validation of the interaction (or lack of) with PLP1.\n• The authors verified the interaction between PLP1 and MIC11 in Fig 4A. To validate their potential direct interactions,\nrecombinant PLP1 and MIC11 were purified, and IP was performed. To strengthen the evidence for a specific interaction, the\nauthors should include an anti-FLAG IP as a reciprocal control. Additionally, incorporating a negative control, such as rGFP-\nFLAG, would help rule out nonspecific binding. An equivalent assay with the mutant protein (mut_23 or other) could be\nincluded to further validate the interaction. This experiment would address if any of the motifs are important for this\ninteraction giving some insights to the study.\n• Figure 4G does not clearly support a direct interaction between PLP1 and MIC11. Instead, the results indicate that both\nproteins are important for egress and can not compensate for each other.\n• The rationale for studying the interaction using recombinant proteins is unclear. Why the authors did not use in-parasite\napproaches, like dual tagging of the genes followed with reciprocal IPs from parasite lysates or using antibodies against\nPLP1 for reciprocal IPs. It is not the same to study the interaction between two recombinant proteins in vitro as it is to study\ntheir interaction inside the cell. The recombinant proteins may not replicate the true cellular environment. Post-translational\nmodifications or folding might be different. The experiment may miss effects of cofactors, crowding, membrane context, or\nadditional interactors. A case in point would be the proposed cleavage of MIC11 that does not happen with the\nrecombinantly generated protein.\n• Figure 5: Based on IFA and microneme secretion assays, the authors conclude that ∆MIC11 does not affect PLP1\nlocalization or secretion. To further test their hypothesis that MIC11 facilitates PLP1's activity at PVM, it would be interesting\nto examine PLP1 localization in a DKO::PLP1-Ty mutant treated with cytochalasin D, with and without ionomycin. If MIC11\nfunctions to promote PLP1’s activity at the PVM, one might expect PLP1 to accumulate in the PV, instead of the PVM upon\nionomycin treatment in the presence of cytochalasin D.\n• Figure 5B: PLP1-myc signal looks slightly reduced in ∆MIC11, please include loading and secretion controls for\nquantification to validate the claim that ∆MIC11 does not affect PLP1 secretion.\n• Figure 5D and 5E: PLP1 is between 140-180 kDa, however, the band that corresponds to the normal size is not present in\nthe western blot, even in the wild type parasites so this needs to be properly explained. There is a band at 250 that\napparently shows up and the authors claim that it is PLP1. A similar fainter band appears in the knockouts. The legend\nindicates that PLP1 has a non-specific band of the same size as the high MW PLP1 band size the authors observed. This\nexplanation does not adequately address the issue. First the authors need to try a different antibody. Second, they need to\ncut the band at 250 kDA and run mass spec to validate that it is indeed PLP1. Additionally, the blot needs loading controls\nand quantifications.\n• Complementation with MIC22 restores egress and virulence in the ∆MIC11 background, suggesting that MIC22 may\nperform a similar function during the feline-restricted stage. While the authors propose that PLP1–MIC11–mediated egress\nin tachyzoites is functionally replaced by PLP2–MIC22–mediated egress in the feline stage, this model would be\nstrengthened by additional experiments, such as assessing egress in a ∆PLP1∆MIC11::MIC22 mutant to directly test the\nfunctional relationship between these paralogs.\n• Fig S1D and Fig S6B: To validate the KOs, the authors performed qRT-PCR. However, it was not indicated how many\nbiological replicates were tested.\n• While the statistical analysis methods are described in the materials and methods section, figures and legends do not\nclearly show the application of these analyses. Statistical analysis should be included in the figure and legends. Legends in\ngeneral are incomplete. It is not possible to understand the experiments by reading the legends.\nMinor Comments:\n• Figure 2F: MIC2 signal from ∆MIC11 in ESA seems to be stronger when compared to WT. Is this an actual phenotype or is\nthis due to uneven loading? Please include a secretion control with GRA1 and calculate the ratio.\n• To validate their potential direct interactions, recombinant PLP1 and MIC11 were purified, and IP was performed. However,\nthe observation that rPLP1-FLAG can be pulled down in an anti-HA IP even without rMIC11-HA (Fig 4H, fainter band) raises\nsome questions about the specificity of the assay.\n• Figure S3A: plaque area should be presented as mm2 not mm3.\n• Figure 6F: What is the band between 25-30 kDa that is present in +2ME sample?\nConclusion:\nThis study presents a well-designed in vivo genetic screen that builds on prior work to identify and functionally characterize\nMIC11 as a contributor to Toxoplasma gondii egress. Although MIC11 was identified in earlier datasets, this study\ninvestigates its potential interaction with PLP1. The study presents an impressive number of experiments. However, some\ncritical controls are missing, which are important for interpreting the data. Most important, the conclusion that PLP1 and\nMIC11 interact is not convincingly supported by the presented evidence. Additionally, while the authors suggest MIC11 as a\npromising therapeutic target, this claim is not yet supported by compound inhibition data. The study raises interesting\nquestions about the broader biological and clinical relevance, especially concerning the feline stage and host specificity.", "reviewer_3": "This study reported by Tachibana et al. reveals an unexpected role for MIC11 in PLP1-mediated egress in Toxoplasma\ngondii. Using a targeted CRISPR/Cas9 screen, MIC11 was highly enriched for in vivo essentiality. Mutants lacking MIC11\ngrow poorly in vitro and are avirulent in vivo. Mechanistically, MIC11 was determined to regulate host cell egress by\ncooperating with PLP1 for PVM disruption. This model was supported by interactome analysis and genetic approaches.\nImpressively, the authors screened the full MIC11 sequence by mutational analysis to pinpoint domains required for host cell\negress. Additionally, a paralog of MIC11 expressed in merozoites was shown to rescue loss of MIC11 in tachyzoites,\nsuggesting similar mechanisms are used in cat stages. Since MIC11 orthologs are not present in Plasmodium, MIC11 may\nrepresent an adaptation to support PLP1-dependent egress in coccidians. Additional studies are needed to determine how\nMIC11 promotes PLP1 activity post secretion, but that does not dampen the key advances described in this report.\nSuggestions for revision:\nIt is interesting that MIC11 had a neutral in vitro fitness score in Fig 1C, yet MIC11 deletion reduced parasite growth in vitro\nFig. 2A. Please comment on this discrepancy in the results or discussion.\nIf PLP1 was screened in Fig. 1C, please label it. It seems like it should have been a hit as stated on line 69. I assume it was\nnot in the apical-pellicle library?\nLoss of MIC11 results in remarkably poor egress in response to calcium ionophore. Is this true for cGMP (zaprinast/BIPPO)\ninduced egress as well?\nThe main conclusion of Fig. 2 is that MIC11 is essential for parasite egress and PVM permeabilization. Please quantify the\ndata in Fig. 2I to properly support this claim.\nAn Alphafold structure model of MIC11 with chains colored by domains depicted in Fig 3A would help visualize the mutated\nregions and inform how they might disrupt MIC11 function.\nIt’s not clear when MIC11 and PLP1 interact. Do they reside in the same vesicles or do they only interact following\nsecretion? Do the mass spec data suggest what residues MIC11 and PLP1 may interact with?\nThe data presented in this study point to an interesting model whereby MIC11 acts as a regulator of post-secretion PLP1\nactivity for egress. Please include a model figure for the different mechanisms for how this might occur (e.g. membrane\ninsertion, oligomerization, pore formation, processing etc.).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We appreciate the reviewer’s overall evaluation of our manuscript and the recognition of the novelty\nand significance of our findings. Below, we provide a point-by-point response to each of the\nreviewer’s comments.\nMajor concerns:\nStatistical rigor: Throughout the manuscript many statistical tests have been omitted or have not been\nreported (Fig 1D, Fig 2D, E, Fig S3A, C-E, Fig 3A, Fig 4A, C, D, G, Fig 6C, H, I, Fig S6C). The\nauthors should perform the appropriate statistical tests for all quantitative data presented in this\nmanuscript and report the results in line with journal instructions (e.g. provide R2 value and p value\nfor correlation calculations, always provide sample size and (corrected) p value along with the\nstatistical test used; when used specify what the error bars represent in the figure legends (e.g. Fig\nS3D)\nIn response, we have carefully reviewed all figures and ensured that appropriate statistical tests have\nbeen performed for each quantitative dataset (Figs. 1D, S1E, 2D-F, S3A, S3C-H, 3C, 4C-D, 4G,\nS5B, 5D, and 6H-I). No statistical test was performed on Fig. 6C because this is public RNA-seq\ndata from biological duplicates, and we just want to show the expression pattern of two genes.\nWe now report the following in accordance with the journal’s guidelines such as sample size,\nstatistical test used, p-values, and clarification of what the error bars represent. These details have\nbeen added to each corresponding figure legends.\nThe materials and method section is insufficient. The authors should update this section to allow for\nreplication of their experimental work and data analysis.\nIn response, we have thoroughly revised and expanded the Materials and Methods section to provide\ncomprehensive details of our experimental procedures and data analysis workflows.\nThe authors should provide the working concentrations of their antibodies for all assays.\nWe have now included the working dilutions of all primary and secondary antibodies used in our\nassays in the supplemental data Table S3.\nThe authors never explicitly explain what the fitness score is in the main text or the Methods section.\nAs in other CRISPR-based screens, fitness scores represent the relative abundance of each mutant\nwithin the parasite population, thereby reflecting the contribution of the targeted gene to parasite\nsurvival. More specifically, fitness scores were calculated as the log2 fold-change of gRNA\nabundances between input and subsequent samples. We have now added this explanation to the Main\ntext (L100–102) and clarified the calculation procedure in the Methods section (L599–606).\nL506-507 (“Images were acquired by confocal laser microscopy”). The authors should for example\nprovide information about the objectives and filters used.\nWe added following information in the Methods section (L623–628).\n“Images were acquired using an Olympus FV3000 IX83 confocal microscope equipped with oil-\nimmersion objective lenses (UPLXAPO60XO, NA 1.42, and UPLXAPO100XO, NA 1.45).\nFluorophores were excited with 405, 488, 561, and 640 nm laser lines, and emissions were collected\nusing spectral detection windows set to 440–480 nm (DAPI), 500–540 nm (CF488A), 581–631 nm\n(CF594), and 654–692 nm (Alexa Fluor 647).”\nThe authors should explicitly state the methods used for normalization of their data. For example:\nL484-485 “The abundance of each gRNA was calculated and normalized to the total number of\naligned reads.”\nWe added following information in the Methods section (L599–602).\n“Following demultiplexing, raw read counts for each gRNA were obtained after alignment to the\nlibrary. To account for differences in sequencing depth, counts were normalized by dividing each\ngRNA count by the total number of aligned reads per sample, yielding relative abundances. These\nnormalized values were used to calculate log₂ fold-changes between samples.”\nThe number of parasites used for the experiments are missing at several parts in the manuscript\n(invasion assay, induced egress assay, gliding motility assay, live cell imaging assay)\nIn response, we have revised the Methods section to include the number of parasites used in each assay.\nThe plaque assay section lacks information about how the images were obtained.\nWe have now included additional details regarding image acquisition in the Methods section (L656–\n659). Specifically, plaque images were visualized using a stereo microscope (SZX16, Evident)\nequipped with a 1× objective and a digital camera (AdvanCam-HD2sA, AdvanVision). Images were\nacquired using the camera's default software, and plaque areas were measured using ImageJ.\nWhenever results were manually annotated the authors should explicitly indicate that - Otherwise\nprovide access to the code that was used to (for example plaque assay, intracellular vs egressed\nvacuoles, …)\nAll image-based phenotypic assays described in the manuscript, including the plaque assay and\negress assay, were manually annotated by the authors. We have now explicitly stated this in the\nMethods section (L756–757).\nFor the gliding motility assay no information on potential quantification and analysis was provided.\nWe repeated the gliding motility assay and quantified the trail lengths. Trail lengths were manually\nmeasured using ImageJ. The analysis revealed no significant differences between WT and ∆MIC11\nparasites (Fig. S3G).\nData presentation: Many of the results and quantification were obtained manually, e.g. gliding\nmotility assay, localization of proteins in mutagenesis experiment. Thus, the authors should include\nlarger more representative images to include more vacuoles and highlight particular areas of interest\nby cropping and enlarging the image.\nTo address the reviewer’s suggestion, we replaced the representative images of the gliding motility\nassay with lower-magnification images to provide a broader view (Fig. 2G). For the MIC11 mutant\npanel, given the large number of mutants, it was not feasible to include all images in the\nsupplementary figures due to space constraints. Instead, we have added representative low-\nmagnification images to the Source Data file.\nThe data presented for the mutagenesis screen require additional validation and analysis:\nThe authors are lacking sufficient evidence to claim that: “These results demonstrated that the IRPH\nsequence in the γ-propeptide is essential for trafficking to micronemes” (L213-214). They should\neither change the statement to apply it only to MIC11 or provide further evidence that 1) it is a\nconserved sequence and more microneme proteins have this sequence; 2) mutate the sequence in\nother microneme proteins to show whether these are also mislocalised. For example, MIC22 protein\ndoes not have an IRPH sequence (Fig 6B), contradicting the authors statement that IRPH is a\nuniversal microneme localization signal.\nWe agree that our original statement was misleading. The IRPH sequence is unique to MIC11 and is\nnot conserved across other microneme proteins. We have revised the sentence to read:\n“These results demonstrate that the IRPH motif in the γ-propeptide is essential for proper trafficking\nof MIC11 to micronemes.” (L247–248).\nAs shown in Figs. 6A, MIC22 possesses a related sequence, IAPH, in the γ-propeptide region.\nAlthough we speculate that this motif may also play a role in the trafficking of MIC22 to\nmicronemes, we did not mention this in the manuscript because we have not experimentally assessed\nits function in the present study.\nThe mutants that were generated for the scanning mutagenesis have not been verified by PCR. It is\nnot possible to affirm that the mislocalisation is due to mutations of specific sequences or integration\ninto a wrong locus. The authors should validate their mutants by PCR.\nWe have now included additional information regarding the scanning mutagenesis strategy in the\nmethods section (L560–568) and Fig.3B.\nThe mutants were generated by random integration of MIC11 expression constructs. As such, the\nexpression cassette might be inserted into different genomic loci in each mutant. Therefore, PCR\nvalidation of a specific integration site is not applicable in this context. Initially, we attempted\ntargeted knock-in of the MIC11expression constructs into the UPRT locus to ensure uniform\nexpression; however, this approach was unsuccessful for unknown reasons. We then adopted a\nrandom integration strategy, which consistently yielded stable MIC11 expression.\nIn Fig S4 many other parasite lines seem to mislocalize; for example: mut_10, mut_11 and mut_05.\nThe authors should 1) calculate the pixel correlation between the anti HA and MIC2 stainings within\nvacuoles for all mutants to be able to quantify the localization. 2) provide larger representative\nimages with more vacuoles and then highlight individual vacuoles through crops.\nAs requested, we quantified the localization of each MIC11 mutant by calculating the pixel\ncorrelation between MIC11 (HA) and MIC2 signals using the Coloc2 plugin in Fiji. Most mutants\ndisplayed Pearson correlation coefficients in the range of 0.7–0.9, consistent with normal microneme\nlocalization, whereas mut_23 exhibited a markedly reduced value (~0.4), consistent with its severe\nmislocalization phenotype (Rebuttal Figure 1).\nRegarding representative images, due to the large number of mutants analyzed, it was not feasible to\ninclude all in the supplementary figures. Instead, we have added representative low-magnification\nimages including multiple vacuoles to the Source Data file, where cropped enlargements of\nindividual vacuoles are also provided.\nRebuttal Figure 1. Correlation between HA and MIC2 signal.\nImmunofluorescence images of MIC11 mutants were analyzed for correlation between HA and\nMIC2 signals. Pearson’s correlation coefficients were calculated within vacuoles using the Coloc2\nplugin in Fiji. Most mutants exhibited high correlation (0.7–0.9), whereas mut_23 displayed a much\nlower correlation (0.4), consistent with its abnormal mislocalization phenotype.\nMany of these mislocalisation-causing mutations do not fall on the y propetide but still show strong\negress phenotypes (Fig 3A). The authors should comment on this. At an earlier point in the\nmanuscript, the authors state that the scanning mutagenesis was applied to identify “which regions of\nMIC11 are required for membrane permeabilization”. this point is never addressed in the manuscript,\nsince the authors focus on the γ-propeptide. The authors should comment on this.\nFirst, while several mutations within the α- and β-chains of MIC11 resulted in partial mis-\nlocalization and showed strong egress phenotypes, these mutants still exhibited localization to the\nmicronemes. In contrast, MIC11 of mut_23 was completely absent from the micronemes, which is\nwhy we chose to further characterize this mutant in detail. Following the reviewer’s suggestion, we\nhave now explicitly stated that some mutants exhibited altered localization in the main text (L241–\n242).\nSecond, we agree that our original sentence was misleading. The aim of the scanning mutagenesis\nwas to identify regions of MIC11 required for parasite egress, not for membrane permeabilization.\nWe therefore have revised the sentence accordingly to:\n“To determine which regions of MIC11 are required for egress, …” (L230).\nThe authors identify PLP1 as a direct interaction partner for MIC11 (Fig. 4). The authors could\nprovide a connection between Fig 3 and 4 by investigating which mutations hinder the interaction\nbetween PLP1 and MIC11. It would be interesting to see whether the mutations that affect egress\nalso have an impact on the interaction with PLP1. At the very least, the authors should model the\ninteraction between the two proteins using Alphafold and comment on this finding in connection\nwith the mutagenesis results.\nTo investigate the interaction between MIC11 and PLP1, we used AlphaFold3 to predict the complex\nstructure (please see Rebuttal Figure 2 below). However, the resulting model had low confidence,\nwith an inter-protein TM-score (ipTM) of 0.29. According to AlphaFold documentation, ipTM\nvalues below 0.6 suggest that the predicted protein–protein interface is unreliable. Therefore, this\npredicted model does not support a confident structural prediction of MIC11–PLP1 interaction.\nTherefore, we believe it would be premature at this stage to correlate our mutagenesis results with\nthe putative interaction surface between MIC11 and PLP1.\nWe agree with the reviewer that exploring how specific mutations affect the MIC11–PLP1\ninteraction is an exciting direction. We plan to pursue this in future studies.\nRebuttal Figure 2. MIC11–PLP1 interaction model predicted by AlphaFold.\nAlphaFold Server (https://alphafoldserver.com/) was used to model the interaction between MIC11\nand PLP1. The resulting model had low confidence with ipTM of 0.29, indicating this predicted\nmodel is unreliable.\nIn addition, it could be valuable to perform the CoIP experiments at different stages of infection (for\nexample extracellular vs intracellular) to assess whether the interaction is temporally regulated.\nWe appreciate the reviewer’s insightful suggestion. In the revised manuscript, we performed\nreciprocal co-immunoprecipitation using parasites expressing PLP1-3HA and observed co-\nprecipitation of MIC11, indicating an interaction between the two proteins. These experiments were\nconducted using syringe-lysed unstimulated tachyzoites, indicating that the MIC11–PLP1 interaction\nlikely occurs prior to microneme secretion.\nHowever, as current models propose that PLP1—and possibly MIC11—functions at the\nparasitophorous vacuole membrane (PVM) during egress, it remains possible that additional\nregulation or dynamic changes in their interaction take place after secretion. While we were unable\nto explore this further during the current revision, we agree that investigating the temporal regulation\nof this interaction across different stages of infection represents a promising future direction. This\npoint has been added to the Discussion (L459–461).\nReviewer #2 (Remarks to the Author):\nIn this study, the authors investigated the role of secretion from micronemes in the permeabilization\nof the host cell during egress of Toxoplasma gondii. Using an in vivo CRISPR screen targeting\nproteins predicted to localize to the apical complex and pellicle membrane, they identified several\ngenes essential for parasite virulence. Among these, they focused on MIC11, examining its role in\nparasite egress and vacuolar permeabilization. Scanning mutagenesis revealed key functional motifs\nin MIC11 and identified residues critical for its trafficking to the micronemes. Authors investigate\nthe interaction between MIC11 and PLP1 to promote egress. Notably, complementation with the\nmerozoite-specific paralog MIC22 rescued the ∆MIC11 phenotype, suggesting a conserved role for\nMIC22 in feline-restricted sexual stages.\nThe rationale for identifying novel egress factors to better understand host cell permeabilization is\nwell-supported.\nWe appreciate the reviewer’s overall evaluation of our manuscript and the recognition of the novelty\nand significance of our findings. Below, we provide a point-by-point response to each of the\nreviewer’s comments.\nCritique major\n• The approach used, an in vivo CRISPR screen, has been previously used, and as acknowledged by\nthe authors, several top hits identified here were also reported previously. In addition, MIC11, the\nfocus of this work, has been previously recognized, raising the question of what new insights this\nscreen adds beyond prior findings. The discussion would benefit from placing the results in the\ncontext of previous findings, with a clearer emphasis on the new insights this study presents.\nWe agree that in vivo CRISPR screening has become a competitive field, and substantial overlap\namong datasets has been observed. Indeed, we found that our screen and that of Giuliano et al. (Nat\nMicrobiol. 2024, PMID: 38977907) correlated well (Pearson’s r = 0.61; Fig. S1C), underscoring the\nreproducibility and robustness of this approach. We think such consistency across independent\nstudies strengthens confidence in the biological relevance of the identified hits (L105–106).\nWhile MIC11 was also listed among the hundreds of hits in previous screens, it was not functionally\nanalyzed or discussed. The key novelty of our study lies in providing the first functional analysis of\nMIC11, showing that it is indispensable for egress and essential for parasite-induced membrane\ndisruption. We believe this functional characterization provides a significant advance in\nunderstanding T. gondii biology.\n• The experimental setup for Co-IP analysis to identify MIC11 interactors included fixation with\n0.1% PFA. PFA is known to cross-link molecules and normally would need to be quenched with\nglycine. However, the method detailed in the manuscript did not include such step. The residual PFA\ncould still cross-link proteins during lysis and could alter the analysis. Second, PFA can capture\nproximity interactors, as the linker is between 2-3 angstrom. It remains uncertain whether all the\nidentified hits represent direct interactions.\nWe apologize for our incomplete description in the Methods section. In our co-immunoprecipitation\nexperiments, parasites were indeed fixed with 0.1% PFA, but this was followed by a quenching step\nusing 100 mM glycine for 4 minutes, and subsequent washes with HEPES-buffered saline. We have\nnow clarified this procedure in the revised Methods section as follows (L708–709):\n“Parasites were fixed with 0.1% PFA for 10 min, quenched with 100 mM glycine for 4 min, washed\nwith HEPES-buffered saline, and lysed in HEPES-RIPA buffer …”\nWe believe these steps effectively minimized the impact of residual PFA on downstream protein\ninteractions.\nRegarding the second point, we fully agree with the reviewer that PFA can stabilize proximal\nproteins, and therefore the interactions identified through this approach may include both direct and\nindirect associations. To further evaluate the MIC11–PLP1 interaction, we performed reciprocal co-\nimmunoprecipitation using parasites expressing PLP1-3HA (Fig. 4H). These experiments confirmed\nthat MIC11 was consistently co-immunoprecipitated with PLP1. While these findings strengthen a\nspecific association between the two proteins, our current data do not distinguish whether the\ninteraction is direct or mediated by other factors. Accordingly, we have revised our manuscript to\nstate that MIC11 and PLP1 are likely to interact either directly or indirectly. We have also included\nthis limitation in the Discussion as a direction for future investigation (L458–459).\n• The scanning mutagenesis approach generated extensive data, analyzing multiple motifs of MIC11.\nWhile the authors appropriately focused on the γ-propeptide’s role in localization, further analysis\nwas centered on mut_23, which showed the most pronounced mislocalization. However, other\nmutants such as mut_06 and mut_27 also showed altered localization, and mut_27 being essential for\negress. A clear rationale for not looking into these mutants is missing.\nAs the reviewer correctly noted, several other mutants, such as mut_06 and mut_27, also showed\naltered localization. However, the key distinction between these mutants and mut_23 is that other\nmutants still displayed microneme localization of MIC11. In contrast, mut_23 completely lost\nmicroneme localization, with MIC11 signal being entirely absent from micronemes. This unique and\npronounced phenotype was the primary reason why we selected mut_23 for further analysis. We\nagree that our rationale was not sufficiently clear in the original submission. To address this, we have\nrevised the text to explicitly state why we chose to further analyze mut_23 among the mutants\n(L241–245).\n• Concerning the scanning mutagenesis experiment analyzing egress of the mutants, are these clonal\npopulations? If not, how is the efficiency of the transfection and expression of the respective mutant\naccounted for in the egress analysis?\nTo clarify the experimental workflow, we have added a schematic figure (Fig. 3B) and revised the\nmethods sections to provide detailed explanations (L560–568).\nSpecifically, each MIC11 mutant was generated by transfecting the corresponding MIC11 expression\nconstruct into ∆MIC11 parasites, and parasites were maintained until stable polyclonal populations\nwere established. The expression vector consisted of pSAG1-MIC11 and pTub-Venus-P2A-\nHXGPRT, which served as a selectable marker. We think this construct integrates randomly into the\ngenome. Although we initially attempted a knock-in strategy into the UPRT locus but failed for\nunknown reasons, thus we adopted random integration for this study. We also chose to use\npolyclonal populations, as generating clonal lines for all ~40 mutants was impractical.\nTo assess transfection efficiency, we monitored Venus expression by flow cytometry for each\npopulation (please see Rebuttal Figure 3). In all mutants, 70–90% of parasites were Venus-positive,\nindicating mutated MIC11 expression in the majority of parasites. While these were not clonal lines,\nwe consider polyclonal populations sufficient for evaluating gain-of-function phenotypes in our\nmutagenesis screen, as the predominant Venus-positive fraction ensures that the observed egress\nphenotypes reflect the properties of the introduced mutants.\nRebuttal Figure 3. Flow cytometry analyses for transfection efficacy.\nParental ∆MIC11 and mutant parasites were analyzed by flow cytometry for Venus-positive\npopulation, assuring the majority of parasites expressed Venus. Table shows Venus-positive\npercentage of each mutant.\n• The scanning mutagenesis represents an impressive effort. However, the functional significance of\nthese findings remains unclear. The result that Mut 23 affects MIC11 localization is interesting, but\nthe authors do not explain why this matters or follow through with validation of the interaction (or\nlack of) with PLP1.\nFrom the observation of mut_23, we further refined our analysis by generating additional mutants\nwith two-residue alanine substitutions, which finally identified the IRPH motif as essential for\nMIC11 targeting to micronemes (Fig. 3F). Interestingly, the MIC11 paralogue MIC22 also harbors\nan IAPH motif within the corresponding γ-propeptide region, suggesting a conserved function,\nalthough this has not yet been experimentally validated.\nSoluble MICs are known to rely on escorter proteins for proper trafficking to micronemes. We\ntherefore hypothesize that MIC11 and MIC22 may interact with an unidentified escorter via the IxPH\nmotif to mediate their trafficking. However, this remains to be experimentally tested and represents\nan important future direction.\nImportantly, mut_23 was the only mutant in which MIC11 completely failed to localize to\nmicronemes, whereas other mutants (e.g., mut_06, mut_27) still showed microneme localization\ndespite impaired egress. As shown in Fig. 5, MIC11 localization to micronemes is not dependent on\nPLP1. Therefore, we consider that the phenotype of mut_23 is not due to a disrupted MIC11–PLP1\ninteraction, but rather to a trafficking defect likely caused by disruption of an interaction with\nunidentified escorters. We have clarified these points in the revised Discussion (L435–442).\n• The authors verified the interaction between PLP1 and MIC11 in Fig 4A. To validate their potential\ndirect interactions, recombinant PLP1 and MIC11 were purified, and IP was performed. To\nstrengthen the evidence for a specific interaction, the authors should include an anti-FLAG IP as a\nreciprocal control. Additionally, incorporating a negative control, such as rGFP-FLAG, would help\nrule out nonspecific binding. An equivalent assay with the mutant protein (mut_23 or other) could be\nincluded to further validate the interaction. This experiment would address if any of the motifs are\nimportant for this interaction giving some insights to the study.\n• The rationale for studying the interaction using recombinant proteins is unclear. Why the authors\ndid not use in-parasite approaches, like dual tagging of the genes followed with reciprocal IPs from\nparasite lysates or using antibodies against PLP1 for reciprocal IPs. It is not the same to study the\ninteraction between two recombinant proteins in vitro as it is to study their interaction inside the cell.\nThe recombinant proteins may not replicate the true cellular environment. Post-translational\nmodifications or folding might be different. The experiment may miss effects of cofactors, crowding,\nmembrane context, or additional interactors. A case in point would be the proposed cleavage of\nMIC11 that does not happen with the recombinantly generated protein.\nWe respond to the two comments above together, as they are closely related. After careful\nconsideration, we concluded that the in vitro co-immunoprecipitation using recombinant proteins\nmay not faithfully reflect the native interactions. This is due to several concerns: (i) the uncertainty\nregarding whether the proteins were correctly folded or post-translationally modified, and (ii) the\npossibility that additional co-factors or lipid membrane contexts are required for the interaction.\nIndeed, in Toxoplasma, several microneme protein complexes such as MIC1/MIC4/MIC6 or\nCLAMP/CLIP/SPATR consist of three components, suggesting that multi-protein assemblies are\ncommon in microneme proteins. Moreover, as the reviewer pointed out, the recombinant MIC11\nlacks evidence of physiological cleavage. Considering these concerns, and after consultation with the\neditor, we have decided to remove the recombinant protein-based data from the revised manuscript.\nInstead, to evaluate the MIC11–PLP1 interaction in a more physiological context, we generated\nPLP1-3HA expressing parasites and performed reciprocal co-immunoprecipitation followed by mass\nspectrometry using parasite lysates (Fig. 4H). These experiments revealed that MIC11 is consistently\nco-immunoprecipitated with PLP1-3HA, suggesting a specific association between the two proteins.\nHowever, this approach does not allow us to distinguish whether the interaction is direct or mediated\nvia other unidentified factors. Accordingly, we have revised the manuscript to state that MIC11 and\nPLP1 likely interact either directly or indirectly.\nWe believe this parasite-based approach provides more biologically relevant support for our\nconclusion regarding the MIC11–PLP1 association and have updated the manuscript accordingly.\n• Figure 4G does not clearly support a direct interaction between PLP1 and MIC11. Instead, the\nresults indicate that both proteins are important for egress and can not compensate for each other.\nAs the reviewer correctly noted, the data presented in Fig. 4G do not support a direct interaction\nbetween MIC11 and PLP1. Rather, the purpose of this complementation experiment was to assess\nthe functional relationship between the two proteins in the context of parasite egress. Our results\ndemonstrate that neither MIC11 nor PLP1 can compensate for the loss of the other, indicating that\nboth proteins are essential for efficient egress. We have revised the manuscript to more accurately\nreflect the intent and interpretation of this experiment (L292–295).\n• Figure 5: Based on IFA and microneme secretion assays, the authors conclude that ∆MIC11 does\nnot affect PLP1 localization or secretion. To further test their hypothesis that MIC11 facilitates\nPLP1's activity at PVM, it would be interesting to examine PLP1 localization in a DKO::PLP1-Ty\nmutant treated with cytochalasin D, with and without ionomycin. If MIC11 functions to promote\nPLP1’s activity at the PVM, one might expect PLP1 to accumulate in the PV, instead of the PVM\nupon ionomycin treatment in the presence of cytochalasin D.\nTo explore this possibility, we treated WT parasites with cytochalasin D for 5 min, followed by\nionomycin stimulation for additional 5 min. Fixed cells were permeabilized with 0.002% digitonin\nand processed for immunofluorescence assays, employing an anti-PLP1 antibody (one that can be\nused for immunofluorescence assay as shown in Kafsack et al., Science, 2009). Under these\nconditions, we were unable to detect any PLP1 signal or accumulation at the PVM (please see\nattached Rebuttal Figure 4 below).\nWhile currently accepted models suggest that discharged PLP1 forms pores on the PVM during\negress, to our knowledge, direct visualization of PLP1 at the PVM has not been reported in T. gondii.\nA likely explanation is that the amount of PLP1 on the PVM is extremely low and may fall below the\ndetection limit of conventional immunofluorescence methods. We agree that determining whether\nPLP1 localizes to the PVM will require further investigation, potentially using ultrasensitive imaging\ntechniques or proximity labelling approaches, which we intend to pursue in future studies.\nRebuttal Figure 4. Assessing PLP1 localization in ionophore-stimulated condition.\nWT parasites were inoculated on HFF monolayers and grown for 24 hours at 37°C. HFF monolayers\nwere pre-treated with 2 µM Cytochalasin D in DMEM for 5 min followed by the addition of an equal\nvolume of 2 µM Cytochalasin D and 4µM ionomycin in DMEM. Cells were fixed in 3.7% PFA and\npermeabilized with 0.002% digitonin and processed for immunofluorescence assays, with anti-PLP1\n(Kafsack et al., Science 2009) and anti-SAG1 antibodies. No PLP1 signal observed in both\nconditions.\n• Figure 5B: PLP1-myc signal looks slghtly reduced in ∆MIC11, please include loading and\nsecretion controls for quantification to validate the claim that ∆MIC11 does not affect PLP1\nsecretion.\nIn this revision, we used an anti-PLP1 antibody suitable for western blotting (Kafsack et al., Science,\n2009) and re-evaluated PLP1 secretion in WT and ∆MIC11 parasites. We included GRA1 as a\nsecretion control to ensure proper quantification. The analysis revealed no significant difference in\nPLP1 secretion between WT and ∆MIC11 parasites (Fig. 5D). We have updated the figure and revised\nthe main text accordingly (L328–329).\n• Figure 5D and 5E: PLP1 is between 140-180 kDa, however, the band that corresponds to the\nnormal size is not present in the western blot, even in the wild type parasites so this needs to be\nproperly explained. There is a band at 250 that apparently shows up and the authors claim that it is\nPLP1. A similar fainter band appears in the knockouts. The legend indicates that PLP1 has a non-\nspecific band of the same size as the high MW PLP1 band size the authors observed. This\nexplanation does not adequately address the issue. First the authors need to try a different antibody.\nSecond, they need to cut the band at 250 kDa and run mass spec to validate that it is indeed PLP1.\nAdditionally, the blot needs loading controls and quantifications.\nTo address concerns regarding the specificity of the PLP1 band, we evaluated four different anti-\nPLP1 antibodies, including one generated in this study, the antibody from Kafsack et al. (Science,\n2009), and N- and C-terminal antibodies from Roiko et al. (JBC, 2013) (Rebuttal Figure 5A). Using\nlysates from wild-type and ΔPLP1 parasites, all antibodies consistently detected a ~140 kDa band in\nwild type but not in ΔPLP1, confirming specificity (Rebuttal Figure 5B). Next, we tested using\nlysates of RH::PLP1-3HA parasites and host cell stimulated with cytochalasin D/ionomycin. Only\nthe antibody generated in this study detected a ~250 kDa band, while the other antibodies and anti-\nHA detected the expected ~140 kDa PLP1 band (Rebuttal Figure 5C). Further, mass spectrometry\nanalysis of the ~250 kDa band failed to identify PLP1, indicating that this band was non-specific\n(Rebuttal Table 1). We therefore retracted our initial interpretation that this band represented PLP1.\nAlthough these data have been removed, we think that this does not affect our conclusion that\nMIC11 is essential for parasite egress. We sincerely acknowledge the reviewer for pointing the\nconcerns to assure scientific rigor of our manuscript through peer-review.\nRebuttal Figure 5. Validation of anti-PLP1 antibodies specificity.\n(A) Schematic representation of PLP1 with a C-terminal 3HA tag. The epitopes recognized by the\nanti-PLP1 and anti-HA antibodies are indicated.\n(B) Validation of anti-PLP1 antibodies by immunoblot using WT and ∆PLP1 parasite lysates. All\nantibodies detected a specific band at ~140 kDa, corresponding to PLP1. GRA17 was used as a\nloading control.\n(C) Validation of anti-PLP1 antibodies by immunoblot using lysates of WT and ∆PLP1 parasite-\ninfected fibroblasts with or without ionophore stimulation. The anti-PLP1(1143–1157) antibody\ndetected a band at ~180–245 kDa, but failed to detect the ~140 kDa band under these conditions. In\ncontrast, anti-HA and other anti-PLP1 antibodies detected the ~140 kDa band but not the ~180–245\nkDa band. ROP18 was used as a loading control.\nRebuttal Table 1. List of proteins detected by mass spectrometry from the ~250 kDa band.\n• Complementation with MIC22 restores egress and virulence in the ∆MIC11 background,\nsuggesting that MIC22 may perform a similar function during the feline-restricted stage. While the\nauthors propose that PLP1–MIC11–mediated egress in tachyzoites is functionally replaced by PLP2–\nMIC22–mediated egress in the feline stage, this model would be strengthened by additional\nexperiments, such as assessing egress in a ∆PLP1∆MIC11::MIC22 mutant to directly test the\nfunctional relationship between these paralogs.\nTo explore potential redundancy between the PLP1–MIC11 and PLP2–MIC22 pathways, we\nattempted to express both MIC22 and PLP2 in the ∆MIC11∆PLP1 (DKO) background. While\nMIC22 was successfully expressed (Rebuttal Figure 6A), repeated attempts to ectopically express\nPLP2 from the UPRT locus were unsuccessful for unknown reasons (data not shown).\nAs an alternative approach, we swapped the endogenous PLP2 promoter with the SAG1 promoter in\nthe ∆PLP1 background to drive PLP2 expression in tachyzoites (Rebuttal Figure 6B). Although RT-\nqPCR confirmed elevated PLP2 mRNA expression (Rebuttal Figure 6C), this strain failed to egress\n(Rebuttal Figure 6D). This phenomenon may reflect failure in PLP2 protein expression or\nincompatibility between PLP2 and MIC11, or the requirement for feline-specific cofactors that are\nabsent in human cells or tachyzoites. As expected, DKO::MIC22-Spot-HA parasites also failed to\negress (Rebuttal Figure 6D).\nGiven the preliminary nature of these results and the time constraints of this revision, we have not\nincluded these data in the manuscript. Nevertheless, we appreciate the reviewer’s suggestion and\nagree that this represents an important direction for our future studies, particularly in the context of\nthe feline-restricted sexual stage.\nRebuttal Figure 6. Investigating MIC22 and PLP2 function in tachyzoites.\n(A) Immunofluorescence images of ∆MIC11∆PLP1(DKO)::MIC22-Spot-HA showing co-\nlocalization of MIC22-Spot-HA (magenta) with MIC2 (cyan) in micronemes.\n(B) Schematic of promoter swapping of PLP2. To generate PLP2-expressing strains, the HXGPRT-\npSAG1 cassette was PCR amplified with 40 bp homology arms to the 5' UTR region of the PLP2\nlocus. The resulting repair templates were co-transfected into ∆PLP1 parasites with gRNA vectors\ntargeting the 5' UTR region of PLP2. Transfected parasites were selected with 25 µg/ml\nmycophenolic acid and 50 µg/ml xanthine.\n(C) Validation of PLP2 mRNA expression by RT-qPCR. Data are presented as mean ± SEM (n = 3).\nStatistical comparisons were performed using Welch’s t-test on ΔCt values.\n(D) Quantification of ionophore-induced egress assay of WT, ∆PLP1, ∆PLP1::pSAG1-PLP2, DKO,\nand DKO::MIC22-Spot-HA parasites. Data are displayed as mean ± SEM (n = 3). Significance was\nanalyzed by ordinary one-way ANOVA with Dunnett test vs ∆PLP1 or DKO, respectively. ****p <\n0.0001, ns, not significant.\n• Fig S1D and Fig S6B: To validate the KOs, the authors performed qRT-PCR. However, it was not\nindicated how many biological replicates were tested.\nIn the original manuscript, RT-qPCR validation of the knockout lines was performed using\nbiological duplicates. In the revised manuscript, we have repeated these experiments and now\npresent data from biological triplicates (Figs. S1E and S5B). This information has been included in\nthe figure legends to clarify the number of replicates used.\n• While the statistical analysis methods are described in the materials and methods section, figures\nand legends do not clearly show the application of these analyses. Statistical analysis should be\nincluded in the figure and legends. Legends in general are incomplete. It is not possible to understand\nthe experiments by reading the legends.\nWe have now ensured that all figures include appropriate statistical analyses, which are clearly\nindicated within the figures and legends. In addition, we have revised all figure legends to provide\ndetailed descriptions of the experiments, including information on the statistical tests used, sample\nsizes, and error bars where applicable.\nMinor Comments:\n• Figure 2F: MIC2 signal from ∆MIC11 in ESA seems to be stronger when compared to WT. Is this\nan actual phenotype or is this due to uneven loading? Please include a secretion control with GRA1\nand calculate the ratio.\nIn the revised manuscript, we repeated the microneme secretion assay and included GRA1 as a\nsecretion control. We quantified the MIC2/GRA1 ratio to assess potential differences in secretion\nlevels. The analysis revealed no significant difference in MIC2 secretion between WT and ∆MIC11\nparasites (Fig. 2F), indicating that the apparent signal variation in the previous figure was likely due\nto uneven loading or blotting rather than a true phenotype.\n• To validate their potential direct interactions, recombinant PLP1 and MIC11 were purified, and IP\nwas performed. However, the observation that rPLP1-FLAG can be pulled down in an anti-HA IP\neven without rMIC11-HA (Fig 4H, fainter band) raises some questions about the specificity of the\nassay.\nAs noted above, we recognize the limitations of the assays using recombinant proteins. Accordingly,\nwe have removed these data from the revised manuscript following consultation with the editor.\nTo address this issue in a more physiological context, we performed reciprocal co-IP experiments\nusing endogenously tagged PLP1-3HA parasites as described above (Fig. 4H). We believe this in-\nparasite approach better reflects the native interaction environment and provides more biologically\nrelevant evidence for the association between MIC11 and PLP1.\n• Figure S3A: plaque area should be presented as mm2 not mm3.\nWe have corrected the unit of plaque area from mm³ to mm² in the revised figure (Fig. S3A).\n• Figure 6F: What is the band between 25-30 kDa that is present in +2ME sample?\nThe ~25–30 kDa band that appears exclusively under reducing conditions (+2ME) is a consistent\nfeature in our western blots. To assess whether this band is specific to MIC11, we probed wild-type\nand ΔMIC11 samples using an anti-MIC11 antibody. Under non-reducing conditions, the anti-\nMIC11 antibody detected a ~16 kDa band consistent with the mature species, whereas under\nreducing conditions two bands of ~10–15 kDa and ~20–25 kDa were observed (Fig. S2D). We\nspeculate that the ~25–30 kDa band seen only under reducing conditions may represent a dissociated\nform of MIC11. Although the precise identity of this band remains unclear, its absence in the\nΔMIC11 sample suggests that it is MIC11-specific. We have not further characterized this band at\nthis stage.\nConclusion:\nThis study presents a well-designed in vivo genetic screen that builds on prior work to identify and\nfunctionally characterize MIC11 as a contributor to Toxoplasma gondii egress. Although MIC11 was\nidentified in earlier datasets, this study investigates its potential interaction with PLP1. The study\npresents an impressive number of experiments. However, some critical controls are missing, which\nare important for interpreting the data. Most important, the conclusion that PLP1 and MIC11 interact\nis not convincingly supported by the presented evidence. Additionally, while the authors suggest\nMIC11 as a promising therapeutic target, this claim is not yet supported by compound inhibition\ndata. The study raises interesting questions about the broader biological and clinical relevance,\nespecially concerning the feline stage and host specificity.\nIn response to the reviewer’s comments, we have incorporated appropriate experimental controls to\nstrengthen the validity of our findings. To further support the potential interaction between MIC11\nand PLP1, we performed reciprocal co-IP using parasite lysates. These experiments consistently\nrevealed co-IP of MIC11 with PLP1, supporting a specific association. However, as the reviewer\nrightly noted, our current data do not allow us to determine whether this interaction is direct or\nmediated by unidentified co-factors. Accordingly, we have revised the manuscript to reflect that\nMIC11 and PLP1 may interact either directly or indirectly.\nRegarding our earlier claim that MIC11 might serve as a therapeutic target, we agree that this\nassertion was premature and not yet experimentally substantiated. We have therefore removed this\nclaim from the revised manuscript. We hope that these revisions address the reviewer’s concerns.\nReviewer #3 (Remarks to the Author):\nThis study reported by Tachibana et al. reveals an unexpected role for MIC11 in PLP1-mediated\negress in Toxoplasma gondii. Using a targeted CRISPR/Cas9 screen, MIC11 was highly enriched for\nin vivo essentiality. Mutants lacking MIC11 grow poorly in vitro and are avirulent in vivo.\nMechanistically, MIC11 was determined to regulate host cell egress by cooperating with PLP1 for\nPVM disruption. This model was supported by interactome analysis and genetic approaches.\nImpressively, the authors screened the full MIC11 sequence by mutational analysis to pinpoint\ndomains required for host cell egress. Additionally, a paralog of MIC11 expressed in merozoites was\nshown to rescue loss of MIC11 in tachyzoites, suggesting similar mechanisms are used in cat stages.\nSince MIC11 orthologs are not present in Plasmodium, MIC11 may represent an adaptation to\nsupport PLP1-dependent egress in coccidians. Additional studies are needed to determine how\nMIC11 promotes PLP1 activity post secretion, but that does not dampen the key advances described\nin this report.\nWe appreciate the reviewer’s overall evaluation of our manuscript and the recognition of the novelty\nand significance of our findings. Below, we provide a point-by-point response to each of the\nreviewer’s comments.\nSuggestions for revision:\nIt is interesting that MIC11 had a neutral in vitro fitness score in Fig 1C, yet MIC11 deletion reduced\nparasite growth in vitro Fig. 2A. Please comment on this discrepancy in the results or discussion.\nAs shown in Fig. 2 and Fig. S3, deletion of MIC11 leads to smaller plaques, yet ∆MIC11 parasites\nshow no significant defects in invasion or replication. This suggests that the reduced plaque size\nprimarily results from impaired egress. This phenotype is consistent with previous reports showing\nthat ∆PLP1 parasites, which invade and replicate normally but fail to egress efficiently, also form\nsmaller plaques (Roiko et al., 2013, PMID: 23376275).\nIn the in vitro CRISPR screen, however, each passage involves mechanical disruption of host cells\nby syringe lysis, which releases parasites regardless of their egress capacity. Therefore, mutations\nthat specifically impair egress do not impose a selective disadvantage under these conditions and\nyield neutral in vitro fitness scores. Indeed, both PLP1 and MIC11 scored as neutral in vitro. By\ncontrast, in vivo CRISPR screens lack such artificial interventions, and parasites defective in egress\nare strongly depleted. These considerations likely explain the apparent discrepancy between the\nneutral in vitro fitness score of MIC11 and the plaque phenotype, and we have clarified this point in\nthe revised Discussion (L415–419).\nIf PLP1 was screened in Fig. 1C, please label it. It seems like it should have been a hit as stated on\nline 69. I assume it was not in the apical-pellicle library?\nAs the reviewer correctly inferred, PLP1 was not included in the apical-pellicle library used in this\nstudy. This is because PLP1 had already been included as a control gene in our previous in vivo\nCRISPR screen using the ROP/GRA gRNA library (Tachibana et al., Cell Reports, 2023), where it\nwas validated as a strong hit.\nLoss of MIC11 results in remarkably poor egress in response to calcium ionophore. Is this true for\ncGMP (zaprinast/BIPPO) induced egress as well?\nTo address this point, we performed a zaprinast-induced egress assay, which provides a more\nphysiologically relevant stimulus than calcium ionophore. Consistent with the phenotype observed\nupon ionophore stimulation, ΔMIC11 parasites exhibited a severe defect in egress following\nzaprinast treatment. This phenotype was fully rescued in the complemented strain. These results\nsuggest that MIC11 is required for efficient egress downstream of both calcium- and cGMP-\nmediated signaling pathways. We have now included these data in the revised manuscript (Fig. S3E)\nand updated the Results section accordingly (L182–184).\nThe main conclusion of Fig. 2 is that MIC11 is essential for parasite egress and PVM\npermeabilization. Please quantify the data in Fig. 2I to properly support this claim.\nIn response, we have quantified PVM permeabilization to strengthen our conclusion that MIC11 is\nrequired for this process. Specifically, we measured the percentage of vacuoles exhibiting PVM\nrupture within 5 minutes following calcium ionophore stimulation. The results show that, unlike\nwild-type parasites, ΔMIC11 parasites fail to induce PVM permeabilization upon stimulation. These\nquantification data have now been incorporated into the revised figure (Fig. S3H).\nAn Alphafold structure model of MIC11 with chains colored by domains depicted in Fig 3A would\nhelp visualize the mutated regions and inform how they might disrupt MIC11 function.\nIn response, we have generated an AlphaFold-predicted structural model of mature MIC11,\ncomprising the α- and β-chains (Fig. 3A). Since the signal peptide and γ-propeptide are removed\nduring maturation, they are excluded from the model. The predicted structure revealed that both the\nα- and β-chains adopt helical conformations.\nIt’s not clear when MIC11 and PLP1 interact. Do they reside in the same vesicles or do they only\ninteract following secretion? Do the mass spec data suggest what residues MIC11 and PLP1 may\ninteract with?\nAt present, our data do not allow us to definitively determine the timing or subcellular context of the\nMIC11–PLP1 interaction. To address this question, we performed immunofluorescence assays (IFA)\nusing the PLP1-3HA strain, which revealed co-localization of MIC11 and PLP1 (Fig. 5A). While\nthis suggests they may reside in the same vesicles, we understand that standard IFA lacks the\nresolution to conclusively define sub-organellar localization. Higher-resolution methods such as\nultrastructure expansion microscopy (U-ExM) will be necessary to more accurately determine\nwhether MIC11 and PLP1 are stored in the same vesicles.\nRegarding the identification of potential interaction residues, our mass spectrometry data do not\nallow us to determine which specific residues in MIC11 and PLP1 may mediate the interaction. As\nReviewer #2 also noted, it remains unclear whether MIC11 and PLP1 interact directly or via\nadditional co-factors. Notably, several known microneme complexes in Toxoplasma—such as\nMIC1/MIC4/MIC6 and CLAMP/CLIP/SPATR—consist of three components, suggesting that multi-\ncomponent complexes are common. Therefore, in the revised manuscript we have revised our\nconclusion to state that MIC11 and PLP1 are likely to interact either directly or indirectly.\nWe agree that further experiments are needed to address these mechanistic questions. Elucidating the\nprecise mode of interaction and the spatiotemporal dynamics of the MIC11–PLP1 association will be\na focus of future studies, as now stated in the Discussion (L459–461).\nThe data presented in this study point to an interesting model whereby MIC11 acts as a regulator of\npost-secretion PLP1 activity for egress. Please include a model figure for the different mechanisms\nfor how this might occur (e.g. membrane insertion, oligomerization, pore formation, processing\netc.).\nIn response, we have created a schematic model illustrating the cooperation between MIC11 and\nPLP1 during parasite egress (Fig. 7). This model highlights potential mechanisms by which MIC11\nmay facilitate PLP1 function—such as promoting membrane insertion, oligomerization, or pore\nformation on the parasitophorous vacuole membrane (PVM). We hope that this figure will help\nreaders better understand our working hypothesis.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate the reviewer #4 for co-reviewing our manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate the reviewer #5 for co-reviewing our manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have done extensive revisions and there are no concerns left.\nThe only thing to make sure is that in the final version, all antibodies used are cited.\nCongratulations to a nice piece of work.\nWe have ensured that all antibodies used are properly cited. We would like to thank the reviewer for their\npositive assessment of our revised manuscript and for recommending its publication.\nReviewer #2 (Remarks to the Author):\nThis resubmission presents an improved version of the previous manuscript. However, two important issues\nstill need to be addressed to strengthen the impact of the study:\nWe thank the reviewer for acknowledging the improvements in our revised manuscript. We have carefully\naddressed the two remaining important issues to further strengthen the impact of our study, as detailed\nbelow.\nFigure S2E needs quantification:\nThe data do not fully support the conclusion that MIC11 is an ASP3 substrate. While the reduction of the\nmature MIC11 species in the ASP3-iKD strain is suggestive, no quantification supports that claim. The\nauthors present results with MIC6 as a positive control and a negative control, such as a secretory protein\nknown not to be processed by ASP3, which would also be important to include to confirm the specificity of\nthe effect. As presented, the results do not conclusively demonstrate that MIC11 is a direct substrate of\nASP3.\nWe performed three independent biological replicates of the ASP3 knockdown experiment and quantified\nthe western blot signals. Following the reviewer's suggestion, we included MIC4 as a negative control, as it\nis a microneme protein known not to be processed by ASP3 (Dogga et al., eLife, 2017). As shown in the\nrevised Figures S2E and S2F, the levels of the mature forms of both MIC11 and the positive control MIC6\nwere significantly reduced upon ATc treatment. In contrast, the levels of MIC4 remained unchanged,\nconfirming the specificity of the effect. We agree that these results do not definitely prove that MIC11 is a\ndirect substrate of ASP3. Therefore, we have modified the text to state that \"MIC11 is processed in an\nASP3-dependent manner\" (Lines 162-172).\nThe conclusion that MIC11 interacts with PLP1 to facilitate egress is central to the mechanistic model but is\nnot supported by the data presented. The evidence relies primarily on reciprocal IP-mass spec, which\nindicates co-purification within a complex but does not demonstrate a specific physical interaction between\nthe two proteins. Reciprocal co-IPs would be necessary to confirm this claim. In addition, the double\nknockout and complementation analyses demonstrate that MIC11 and PLP1 function in the same pathway\nbut do not prove molecular interaction. As presented, the data support a functional association but not a\nprotein-protein interaction.\nWe performed reciprocal co-IP experiments under the same conditions used for the IP-MS analysis\n(Rebuttal Figure). We successfully detected the co-IP of PLP1 with MIC11-HA. Conversely, MIC11 was\nalso detected in PLP1-3HA immunoprecipitates. Although the signal was weaker, this is likely due to\nlimited antibody accessibility caused by epitope masking within the cross-linked complex.\nWe agree with the reviewer that, given the use of cross-linking agents, these data demonstrate co-\npurification within a complex rather than a definitive direct physical interaction, which would require\nstructural analysis such as cryo-EM. Accordingly, we have tempered our conclusion to state that MIC11 and\nPLP1 co-purify within a complex rather than claiming a direct protein-protein interaction (Lines 296-299).\nRegarding the second point, we agree that the double knockout and complementation analyses indicate a\nfunctional link where both proteins are essential, but do not prove a molecular interaction. To avoid\nmisleading the readers, we have reorganized the text and moved these data to the end of the paragraph\n(Lines 301–306; Figs. 4F–H). This structure presents the genetic data as support for a functional connection\nwithout implying direct interaction.\nRebuttal Figure. Co-immunoprecipitation analysis by western blot.\nIndicated parasite lysates were prepared under the same conditions used for the IP-MS analysis described in\nthe manuscript and subjected to immunoprecipitation targeting the HA tag. GRA17 was used as a loading\ncontrol for inputs and as a negative control for immunoprecipitation specificity.\nMinor comments:\nThe introduction lists several key regulators of egress (GC, PKG, CDPK1/3, DGK1) but does not mention\ncalcium signaling, which is central to the activation of these pathways and is later presented as a trigger for\nmicroneme secretion and egress. Is there a specific reason why calcium signaling is not introduced here?\nWe thank the reviewer for pointing this out. We have rewritten the Introduction to include a proper\ndescription of calcium signaling as a central trigger for microneme secretion and egress (Lines 42–49).\nThe statement that MIC11 secretion occurs in a “Ca²⁺-dependent manner” may not be technically accurate.\nThe experiments used ethanol to stimulate secretion and BAPTA-AM to inhibit it, which indicates that\ncalcium mobilization is involved but does not directly demonstrate calcium involvement. Considering that\nBAPTA-AM has multiple broad and sometimes nonspecific effects, it would be more accurate to say that\nsecretion is “sensitive to calcium chelation” or “stimulated by calcium-mobilizing treatments” rather than\nstrictly “Ca²⁺-dependent.”\nWe agree with the reviewer regarding the technical accuracy. We have modified the text to state that \"its\nsecretion was stimulated by calcium-mobilizing treatments\" (Line 157).\nReviewer #3 (Remarks to the Author):\nThe revised manuscript demonstrates careful consideration of reviewer comments and often goes above and\nbeyond to thoroughly address them through robust new experimentation, re-analyses of the original data,\naddition of model figures, and revisions of the text and figures for clarity and accuracy. All original\nconcerns have been adequately addressed and no new concerns have been raised.\nWe are grateful for the reviewer's generous comments regarding our revision efforts and the additional\nexperiments. We thank the reviewer for recommending the publication of our manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for co-reviewing our revised manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for co-reviewing our revised manuscript.\nReviewer #2 (Remarks to the Author):\nThank you for providing the reciprocal co-IP data, which shows potential interaction between MIC11 and\nPLP1. The IP with MIC11 appears to show a band that corresponds to PLP1. However, the reverse is not\nvery convincing, and it almost appears to be a spill-over from the input band. The second blot is confusing\nbecause there are three lanes in the top panel, only two in the middle, and again three in the bottom. This\nmismatch in lane number between the top and lower panels is concerning. It is my understanding that in a\nco-IP experiment, all panels should derive from the same membrane or from membranes with identical lane\nstructure. As presented, it is not possible to determine whether the signals correspond to the same samples,\nwhich complicates the interpretation of the result. I also noticed that the elution band in the first blot is\nbroader than the corresponding bands in the other lanes. Was this because of uneven loading? I also wonder\nwhy the authors named this figure a rebuttal figure and why it is not included in the manuscript. In the\nmanuscript, they refer to Fig. S5D and S5E, which show a cartoon in D and an IFA in E. I am not sure if I\nam looking at the figures correctly.\nIn summary, the authors need to redo the second IP or present it more clearly and include it in the main\nfigures of the paper. Please include the number of biological replicates in the legend.\nTo address the concerns regarding lane consistency, potential spillover, and loading quality, we performed\nnew reciprocal co-immunoprecipitation experiments. In the revised manuscript, we have added new results,\nnow presented as Figure 4F in the main figures. We observed reciprocal co-immunoprecipitations of MIC11\nand PLP1, consistent with their association. We ensured that all panels in Figure 4F derive from membranes\nwith identical lane structures to allow for a direct comparison of signals across samples. As requested, we\nhave also specified in the figure legend that these results are representative of n=3 independent biological\nexperiments. Furthermore, we added a sentence for clarity regarding generation of endogenously tagged\nPLP1-HA and ROP9-HA strains for IP-MS (Fig. S5D and S5E) (Lines 298–299).\nMinor comment for the introduction: Please link calcium signaling and microneme secretion.\nFollowing the suggestion, we have rewritten the introduction to explicitly link calcium signaling and\nmicroneme secretion (Lines 42–51)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done extensive revisions and there are no concerns left.\nThe only thing to make sure is that in the final version, all antibodies used are cited.\nCongratulations to a nice piece of work.", "reviewer_2": "This resubmission presents an improved version of the previous manuscript. However, two important issues still need to be addressed to strengthen the impact of the study:\nFigure S2E needs quantification:\nThe data do not fully support the conclusion that MIC11 is an ASP3 substrate. While the reduction of the mature MIC11 species in the ASP3-iKD strain is suggestive, no quantification supports that claim. The authors present results with MIC6 as a positive control and a negative control, such as a secretory protein known not to be processed by ASP3, which would also be important to include to confirm the specificity of the effect. As presented, the results do not conclusively demonstrate that MIC11 is a direct substrate of ASP3.\nThe conclusion that MIC11 interacts with PLP1 to facilitate egress is central to the mechanistic model but is not supported by the data presented. The evidence relies primarily on reciprocal IP-mass spec, which indicates co-purification within a complex but does not demonstrate a specific physical interaction between the two proteins. Reciprocal co-IPs would be necessary to confirm this claim. In addition, the double knockout and complementation analyses demonstrate that MIC11 and PLP1 function in the same pathway but do not prove molecular interaction. As presented, the data support a functional association but not a protein-protein interaction.\nMinor comments:\nThe introduction lists several key regulators of egress (GC, PKG, CDPK1/3, DGK1) but does not mention calcium signaling, which is central to the activation of these pathways and is later presented as a trigger for microneme secretion and egress. Is there a specific reason why calcium signaling is not introduced here?\nThe statement that MIC11 secretion occurs in a “Ca²⁺-dependent manner” may not be technically accurate. The experiments used ethanol to stimulate secretion and BAPTA-AM to inhibit it, which indicates that calcium mobilization is involved but does not directly demonstrate calcium involvement. Considering that BAPTA-AM has multiple broad and sometimes nonspecific effects, it would be more accurate to say that secretion is “sensitive to calcium chelation” or “stimulated by calcium-mobilizing treatments” rather than strictly “Ca²⁺-dependent.”", "reviewer_3": "The revised manuscript demonstrates careful consideration of reviewer comments and often goes above and beyond to thoroughly address them through robust new experimentation, re-analyses of the original data, addition of model figures, and revisions of the text and figures for clarity and accuracy. All original concerns have been adequately addressed and no new concerns have been raised.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for acknowledging the improvements in our revised manuscript. We have carefully addressed the two remaining important issues to further strengthen the impact of our study, as detailed below.\nFigure S2E needs quantification:\nThe data do not fully support the conclusion that MIC11 is an ASP3 substrate. While the reduction of the mature MIC11 species in the ASP3-iKD strain is suggestive, no quantification supports that claim. The authors present results with MIC6 as a positive control and a negative control, such as a secretory protein known not to be processed by ASP3, which would also be important to include to confirm the specificity of the effect. As presented, the results do not conclusively demonstrate that MIC11 is a direct substrate of ASP3.\nWe performed three independent biological replicates of the ASP3 knockdown experiment and quantified the western blot signals. Following the reviewer's suggestion, we included MIC4 as a negative control, as it is a microneme protein known not to be processed by ASP3 (Dogga et al., eLife, 2017). As shown in the revised Figures S2E and S2F, the levels of the mature forms of both MIC11 and the positive control MIC6 were significantly reduced upon ATc treatment. In contrast, the levels of MIC4 remained unchanged, confirming the specificity of the effect. We agree that these results do not definitely prove that MIC11 is a direct substrate of ASP3. Therefore, we have modified the text to state that \"MIC11 is processed in an ASP3-dependent manner\" (Lines 162-172).\nThe conclusion that MIC11 interacts with PLP1 to facilitate egress is central to the mechanistic model but is not supported by the data presented. The evidence relies primarily on reciprocal IP-mass spec, which indicates co-purification within a complex but does not demonstrate a specific physical interaction between the two proteins. Reciprocal co-IPs would be necessary to confirm this claim. In addition, the double knockout and complementation analyses demonstrate that MIC11 and PLP1 function in the same pathway but do not prove molecular interaction. As presented, the data support a functional association but not a protein-protein interaction.\nWe performed reciprocal co-IP experiments under the same conditions used for the IP-MS analysis (Rebuttal Figure). We successfully detected the co-IP of PLP1 with MIC11-HA. Conversely, MIC11 was also detected in PLP1-3HA immunoprecipitates. Although the signal was weaker, this is likely due to limited antibody accessibility caused by epitope masking within the cross-linked complex.\nWe agree with the reviewer that, given the use of cross-linking agents, these data demonstrate co-purification within a complex rather than a definitive direct physical interaction, which would require structural analysis such as cryo-EM. Accordingly, we have tempered our conclusion to state that MIC11 and PLP1 co-purify within a complex rather than claiming a direct protein-protein interaction (Lines 296-299).\nRegarding the second point, we agree that the double knockout and complementation analyses indicate a functional link where both proteins are essential, but do not prove a molecular interaction. To avoid misleading the readers, we have reorganized the text and moved these data to the end of the paragraph (Lines 301–306; Figs. 4F–H). This structure presents the genetic data as support for a functional connection without implying direct interaction.\nRebuttal Figure. Co-immunoprecipitation analysis by western blot.\nIndicated parasite lysates were prepared under the same conditions used for the IP-MS analysis described in the manuscript and subjected to immunoprecipitation targeting the HA tag. GRA17 was used as a loading control for inputs and as a negative control for immunoprecipitation specificity.\nMinor comments:\nThe introduction lists several key regulators of egress (GC, PKG, CDPK1/3, DGK1) but does not mention calcium signaling, which is central to the activation of these pathways and is later presented as a trigger for microneme secretion and egress. Is there a specific reason why calcium signaling is not introduced here?\nWe thank the reviewer for pointing this out. We have rewritten the Introduction to include a proper description of calcium signaling as a central trigger for microneme secretion and egress (Lines 42–49).\nThe statement that MIC11 secretion occurs in a “Ca²⁺-dependent manner” may not be technically accurate. The experiments used ethanol to stimulate secretion and BAPTA-AM to inhibit it, which indicates that calcium mobilization is involved but does not directly demonstrate calcium involvement. Considering that BAPTA-AM has multiple broad and sometimes nonspecific effects, it would be more accurate to say that secretion is “sensitive to calcium chelation” or “stimulated by calcium-mobilizing treatments” rather than strictly “Ca²⁺-dependent.”\nWe agree with the reviewer regarding the technical accuracy. We have modified the text to state that \"its secretion was stimulated by calcium-mobilizing treatments\" (Line 157).\nReviewer #3 (Remarks to the Author):\nThe revised manuscript demonstrates careful consideration of reviewer comments and often goes above and beyond to thoroughly address them through robust new experimentation, re-analyses of the original data, addition of model figures, and revisions of the text and figures for clarity and accuracy. All original concerns have been adequately addressed and no new concerns have been raised.\nWe are grateful for the reviewer's generous comments regarding our revision efforts and the additional experiments. We thank the reviewer for recommending the publication of our manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for co-reviewing our revised manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for co-reviewing our revised manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "Thank you for providing the reciprocal co-IP data, which shows potential interaction between MIC11 and PLP1. The IP with MIC11 appears to show a band that corresponds to PLP1. However, the reverse is not very convincing, and it almost appears to be a spill-over from the input band.\nThe second blot is confusing because there are three lanes in the top panel, only two in the middle, and again three in the bottom. This mismatch in lane number between the top and lower panels is concerning. It is my understanding that in a co-IP experiment, all panels should derive from the same membrane or from membranes with identical lane structure. As presented, it is not possible to determine whether the signals correspond to the same samples, which complicates the interpretation of the result.\nI also noticed that the elution band in the first blot is broader than the corresponding bands in the other lanes. Was this because of uneven loading?\nI also wonder why the authors named this figure a rebuttal figure and why it is not included in the manuscript. In the manuscript, they refer to Fig. S5D and S5E, which show a cartoon in D and an IFA in E. I am not sure if I am looking at the figures correctly.\nIn summary, the authors need to redo the second IP or present it more clearly and include it in the main figures of the paper. Please include the number of biological replicates in the legend.\nMinor comment for the introduction: Please link calcium signaling and microneme secretion."}, "author_response": "To address the concerns regarding lane consistency, potential spillover, and loading quality, we performed new reciprocal co-immunoprecipitation experiments. In the revised manuscript, we have added new results, now presented as Figure 4F in the main figures. We observed reciprocal co-immunoprecipitations of MIC11 and PLP1, consistent with their association. We ensured that all panels in Figure 4F derive from membranes with identical lane structures to allow for a direct comparison of signals across samples. As requested, we have also specified in the figure legend that these results are representative of n=3 independent biological experiments. Furthermore, we added a sentence for clarity regarding generation of endogenously tagged PLP1-HA and ROP9-HA strains for IP-MS (Fig. S5D and S5E) (Lines 298–299).\nFollowing the suggestion, we have rewritten the introduction to explicitly link calcium signaling and microneme secretion (Lines 42–51)."}, {"round": 4, "reviewer_comments": {"reviewer_2": "The authors have appropriately addressed the concerns raised."}, "author_response": ""}]

Decision letter