Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis

Hugo Bisio; Aarti Krishnan; Jean-Baptiste Marq; Dominique Soldati-Favre

doi:10.1371/journal.ppat.1010438

. 2022 Mar 24;18(3):e1010438. doi: 10.1371/journal.ppat.1010438

Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis

Hugo Bisio ^1,^¤, Aarti Krishnan ¹, Jean-Baptiste Marq ¹, Dominique Soldati-Favre ^1,^*

Editor: Michael J Blackman²

PMCID: PMC8982854 PMID: 35325010

Abstract

Regulated microneme secretion governs motility, host cell invasion and egress in the obligate intracellular apicomplexans. Intracellular calcium oscillations and phospholipid dynamics critically regulate microneme exocytosis. Despite its importance for the lytic cycle of these parasites, molecular mechanistic details about exocytosis are still missing. Some members of the P4-ATPases act as flippases, changing the phospholipid distribution by translocation from the outer to the inner leaflet of the membrane. Here, the localization and function of the repertoire of P4-ATPases was investigated across the lytic cycle of Toxoplasma gondii. Of relevance, ATP2B and the non-catalytic subunit cell division control protein 50.4 (CDC50.4) form a stable heterocomplex at the parasite plasma membrane, essential for microneme exocytosis. This complex is responsible for flipping phosphatidylserine, which presumably acts as a lipid mediator for organelle fusion with the plasma membrane. Overall, this study points toward the importance of phosphatidylserine asymmetric distribution at the plasma membrane for microneme exocytosis.

Author summary

Biological membranes display diverse functions, including membrane fusion, which are conferred by a defined composition and organization of proteins and lipids. Apicomplexan parasites possess specialized secretory organelles (micronemes), implicated in motility, invasion and egress from host cells. Microneme exocytosis is already known to depend on phosphatidic acid for its fusion with the plasma membrane. Here we identify a type P4-ATPase and its CDC50 chaperone (ATP2B-CDC50.4) that act as a flippase and contribute to the enrichment of phosphatidylserine (PS) in the inner leaflet of the parasite plasma membrane. The disruption of PS asymmetric distribution at the plasma membrane impacts microneme exocytosis. Overall, our results shed light on the importance of membrane homeostasis and lipid composition in controlling microneme secretion.

Introduction

The phylum of Apicomplexa encompasses a diverse group of obligate intracellular protozoan parasites responsible for severe diseases in animals and humans. Plasmodium spp., the etiological agent of malaria, account for half a million deaths per year (World Malaria Report 2017, WHO). Cryptosporidium is one of the most important agents causing severe diarrhea in children [1]. Relevant for the farming industry, Eimeria and Theileria are responsible for a considerable economic burden [2,3]. Toxoplasma gondii is the most ubiquitous member of the phylum, capable of infecting humans and animals. The lytic cycle of apicomplexan parasites is tightly controlled to ensure parasite survival and dissemination [4,5]. Underpinning several steps of the lytic cycle is the release of apical secretory organelles called micronemes that are conserved in all motile and invasive stages of apicomplexans [4]. The micronemes secrete adhesins, perforins and proteases that allow gliding, invasion and egress of the parasites [6,7]. In a simplified model, microneme exocytosis is regulated and initiated by the production of cyclic guanosine monophosphate (cGMP) via a signaling platform composed of an atypical guanylate cyclase (GC) fused to a P type-IV ATPase (P4-ATPase) and associated to CDC50.1 as well as to a unique GC organizer, UGO [8–11]. The level of cGMP is tightly controlled by phosphodiesterases, which differential phosphorylation state upon depletion of the protein cAMP-dependent protein kinase (PKA-C1) indicates that are presumably regulated by cyclic adenosine monophosphate (cAMP) levels [12,13]. In turn, cGMP leads to the activation of the cGMP-dependent protein kinase (PKG) [14] which triggers a signaling cascade that involves the production of inositol-tri-phosphate (IP3) and diacylglycerol (DAG) by phosphoinositide phospholipase C (PI-PLC) [15]. IP3 is believed to mobilize calcium from an unknown intracellular store of the parasite [16] and activate calcium-dependent protein kinases (CDPKs) [17,18]. Both CDPK1 and CDPK3 contribute to microneme exocytosis while CDPK1 additionally extrudes the conoid and activates the actomyosin system [17–19], allowing parasite gliding motility, invasion and egress [20]. On the other hand, DAG is converted into phosphatidic acid (PA) through a reversible reaction catalyzed by the DAG kinase 1 (DGK1) and PA phosphatases (PAPs) [21]. Importantly, several feedback loops are expected to feed into different steps of this signaling pathway.

The endomembrane system of the cell displays diverse functions conferred by a defined composition and organization of proteins and lipids. In particular, the exocytosis of secretory organelles depends on specific phospholipids that select the target membranes and trigger fusion. In apicomplexan parasites, PA acts as an essential lipid mediator for microneme exocytosis [21,22]. PA is produced in the inner leaflet of the plasma membrane and allows the docking of micronemes with the assistance of the acylated pleckstrin-homology domain-containing protein (APH) on the microneme organelle surface [21,23]. Importantly, the asymmetric distribution of phospholipids (PLs) across the plasma membrane is known to generate a physical surface tension that is used to induce membrane curvature, favoring vesicle budding and fusion [24]. These gradients are set and maintained by different groups of proteins including P4-ATPases, which function as flippases and form heterodimeric complexes with cell division control protein 50 (CDC50), that act as cofactors and chaperones (Fig 1A) [25]. P4-ATPases possess ten predicted transmembrane spanning domains with cytosolic domains mediating nucleotide binding (N), phosphorylation (P) and dephosphorylation (A) (Fig 1A) [26]. The P4-ATPases couple the hydrolysis of ATP with inward PLs translocation via a Post-Albers mechanism [25], transitioning between two intermediate states: E1 and E2, with different affinities to substrates. During this process, the transmembrane region remainsstructurally rigid based on its interaction with the CDC50 partner [27]. Among the repertoire of P4-ATPases in apicomplexan parasites, some are essential for parasite survival [28–30] yet little is known about their biological roles and enzymatic functions. Beside the lipid composition, membrane fusion is a universal process that also involves a machinery composed of SNAREs (for “soluble N-Ethylmaleimide-sensitive factor (NSF)-attachment protein receptor”) [31]. During fusion, vesicular and target SNAREs assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together [31]. Additionally, this machinery is controlled by C2-containing proteins, like synaptotagmin/ferlin and DOC2, in a calcium-dependent manner [32,33]. The SNARE proteins appear to play pleiotropic functions in T. gondii with none identified to date to be uniquely associated to microneme secretion [34]. Contrastingly, both DOC2.1 and Ferlin 1 (FER1) are solely dedicated to microneme secretion in Plasmodium spp. and T. gondii [35–38]. While DOC2.1 function strictly participates in microneme exocytosis [35], FER1 is involved in microneme proteins trafficking in addition to exocytosis [37], although some of these phenotypes might result from indirect dominant negative effects.

Fig 1 — (A) Schematic representation of the domain architecture of the P4-ATPase-CDC50 heterocomplex. (B) Conservation of P4-ATPases and CDC50 cofactors across the Apicomplexa phylum. Blue circle: Absent in *Theileria*. *Eimeria* possesses two genes belonging to the CDC50.1/CDC50.2 subgroups but direct homology could not be deducted by blast analysis. See S1 Fig. Fitness scores associated to gene disruption in T. *gondii* are obtained from [28]. CM: Cyst-forming. PM: plasma membrane. G: Golgi apparatus. A: Apical. S.p: Secretory pathway. N.a: Not assessed. Accession numbers of all putative orthologs genes are included in S1 Table. (C) Indirect immunofluorescence assay (IFA) of intracellular ATP2A-mAID-HA and ATP2B-mAID-HA parasites. GAP45: parasite periphery. (D) IFA of intracellular ATP7B-SM-HA parasites. Actin: Parasite cytosol. (E) IFA of intracellular CDC50.2, CDC50.3 and CDC50.4-mAID-HA parasites. GAP45: parasite pellicle. Cam-Like protein: Golgi apparatus. The scale bars for the immunofluorescence images are 7μM, unless otherwise indicated.

In this study, we address the importance of several P type-IV ATPases and CDC50 chaperones in T. gondii. We show that ATP2B forms a heterocomplex with CDC50.4 and acts as an essential flippase to maintain phosphatidylserine (PS) enrichment in the inner leaflet of the parasite plasma membrane. ATP2B and CDC50.4 crucially contribute to microneme exocytosis indicating that PS is a key lipid participating in microneme fusion with the plasma membrane.

Results

Identification and localization of the putative flippases and their CDC50 partners in Toxoplasma gondii

Sequence homology search in T. gondii genome identified six genes predicted to code for P4- ATPases and four genes for CDC50 cofactors (Fig 1B and S1 Table). We utilized the nomenclature already established for Plasmodium species in order to name the putative homologues of P4-ATPases [39]. We have chosen this nomenclature, instead of the one presented in a recent overlapping report [30], in order to provide an integrative view of different apicomplexan parasites P4-ATPases and facilitate comparisons. ATP2A and ATP2B appear to be paralogues that are present either as one or two copies across all members of the Apicomplexa phylum. Similarly, ATP7A and ATP7B are putative paralogues found as a pair in the cyst-forming coccidian subgroup of Apicomplexa but absent in Theileria and Cryptosporidia. Plausible orthologs of T. gondii ATP8 and GC are found across the phylum (Fig 1B and S1 Table) with GC being duplicated in the Plasmodium and Eimeria spp., as previously reported [5] (Fig 1B and S1 Table). The CDC50 protein family is composed of four members in T. gondii. CDC50.1, CDC50.2 and CDC50.3 are clustered phylogenetically and may have arisen through gene duplication (S1 Fig). The presence of three individual genes belonging to this group is only found in coccidians (Fig 1B and S1 Table). A single gene assigned to the CDC50.1/2/3 group is present in Cryptosporidia while CDC50.3 is absent in Theileria. In contrast, CDC50.4 is conserved across the entire phylum (Figs 1B, S1 and S1 Table).

To determine their localization and scrutinize their function, the genes corresponding to the P4-ATPases and CDC50s were C-terminally tagged with 3-HA epitope tags and concomitantly fused to the auxin-inducible degron (mAID) [14] at their endogenous locus via CRISPR-Cas9 genome editing. The resulting mutants were cloned and confirmed by genomic PCR (S2A Fig). Similar to GC and its partner CDC50.1, which have previously been found at the apical cap of the parasites [8], ATP2A, ATP2B, CDC50.2 and CDC50.4 were also found at the apical cap (Fig 1C and 1E). Contrastingly, CDC50.3 localized to the Golgi apparatus (Fig 1E) while ATP7B could only be detected throughout the secretory pathway after tagging with the spaghetti monster-HA (SM-HA) [40] (Figs 1D and S3B and S3C). ATP8 was refractory to genetic modification, which hampered further investigation.

Toxoplasma gondii CDC50.4 forms promiscuously stable heterocomplexes with ATP2A and ATP2B

Given its conservation across the Apicomplexa, localization and predicted essentiality, we focus our attention on CDC50.4 [28]. In order to identify the complex formed by this protein, we performed immunoprecipitation of CDC50.4 coupled with mass spectrometry analysis (S2D Fig). ATP2A and ATP2B were identified as interacting partners of this protein (Fig 2A). Other proteins identified in pull-down are likely contaminants that correspond to highly abundant proteins or are predicted to be implicated in non-related functions. Moreover, endogenous epitope-tagging of ATP2B-Ty in CDC50.4-mAID-HA confirmed co-localization of the two proteins at the apical tip of the parasite (Fig 2B) and pull-down experiments provided further evidence of their stable association (Fig 2C). Compellingly, depletion of CDC50.4 led to a significant decrease in ATP2B protein level (Figs 2D and S2E). Partial colocalization and downregulation of ATP2A upon depletion of CDC50.4 were also shown by immunofluorescence (Fig 2E). Importantly, double tagging of ATP2A and CDC50.4 rendered a partial miss localization of the complex (Fig 2E), indicating some steric impediment for correct trafficking upon presence of both C-terminal tags. We also observed no decrease in ATP2A-Ty levels upon depletion of CDC50.4 (S2F Fig). The absence of CDC50.4 did not impact on the localization of GC-Ty (Fig 2F and 2G) and its level of expression (Fig 2H) although it shares the same localization as ATP2B but forms a heterocomplex with CDC50.1 [9]. Taken together, these data strongly indicate that CDC50.4 is forming a complex with ATP2A and ATP2B.

Fig 2 — (A) Gene ID and number of unique peptides identified as putative interactors of CDC50.4 upon coimmunoprecipitation and mass spectrometry analysis (B) IFA of intracellular ATP2B-Ty/CDC50.4-mAID-HA parasites. (C) Western blot of immunoprecipitation with anti-Ty from ATP2B-Ty/CDC50.4-mAID-HA lysate showing that ATP2B-Ty is associated with CDC50.4-mAID-HA. (D) Western blot of lysates ATP2B-Ty/CDC50.4-mAID-HA parasites treated with or without IAA for 24 hours. Actin: loading control. (E) IFA of intracellular RH ATP2A-Ty/CDC50.4-mAID-HA parasites with or without IAA. (F) IFA of intracellular RH GC-Ty/CDC50.4-mAID-HA parasites with or without IAA. (G) Quantification of representative pictures in (F). The ratio between the intensity of fluorescence at the basal pole versus the apical pole of the parasite is shown. Approximately 100 vacuoles were quantified. (H) Western blot of lysates GC-Ty/CDC50.4-mAID-HA parasites treated with or without IAA for 24 hours. Catalase: loading control. The scale bars for the immunofluorescence images are 7μM, unless otherwise indicated.

ATP2B-CDC50.4 complex and ATP7B are critical for the survival of Toxoplasma gondii

The three P-type IV ATPases (ATP2A, ATP2B and ATP7A) and CDC50s (CDC50.2, CDC50.3 and CDC50.4), were C-terminally fused to the auxin-inducible degron (mAID) at the endogenous locus [14] and efficiently depleted upon addition of 3-indoleacetic acid (IAA) as confirmed by western blot (Fig 3A and 3B). The relative fitness of each knockdown mutant was assessed by its ability to form plaques of lysis on host cell monolayers (Fig 3C and 3D). Parasite lacking ATP2A and CDC50.2 had no apparent fitness defect, whereas loss ATP2B, ATP7B and CDC50.4 in presence of IAA led to significantly smaller plaques compared to parasites grown in absence of IAA (Fig 3C and 3D). Down-regulation of CDC50.3 resulted in a moderate decrease in fitness (Fig 3C and 3D).

Fig 3 — (A) Western blot analysis of ATP2A-mAID, ATP2B-mAID and ATP7B-mAID regulation with IAA treatment for 24 hours. Actin: loading control. P: parental strain (Tir1 strain) (B) Western blot of lysates from CDC50.2-4-mAID parasites treated with or without IAA for 24 hours. Actin: loading control. (C) Images of plaques formed by RH Tir1 parental strain, ATP2A, ATP2B, ATP7B, CDC50.2, CDC50.3 and CDC50.4-mAID-HA lines on HFF monolayers with or without IAA treatment. (D) Quantification of plaque size relative to the parental control (Tir1), mean +/- SD of 1 representative experiment. Each parasite line was analysed individually for statistical significance using an unpaired t test. P values: **** = <0.0001, * = <0.05.

P-type IV ATPases and its CDC50 chaperones play distinct roles in the parasite biology

To dissect the fitness conferring role of ATP7B depletion, each of the steps of the lytic cycle were examined individually. Parasites depleted in ATP7B are impaired in intracellular growth (S3A Fig) but egress and invade normally (S3B and S3C Fig). Moreover, the organization of intracellular parasites in rosettes was disrupted in the absence of ATP7B (S3D and S3E Fig). Deeper characterization would be needed to understand the importance of ATP7B in the parasite biology.

Parasites depleted in either ATP2B or CDC50.4 showed a severe impairment in invasion (Fig 4A) and in egress (Fig 4B), without alteration of intracellular growth (Fig 4C). Importantly, microneme secretion of extracellular parasites depleted in either ATP2B or CDC50.4 triggered by BIPPO (PDE inhibitor which induces the accumulation of cGMP in the cell) was considerably reduced (Fig 4D and 4E). The defect in microneme exocytosis explains the impaired egress and invasion phenotype of ATP2B, suggesting a crucial role of the heterocomplex for the completion of the parasite’s lytic cycle. In contrast, depletion of the paralogue protein ATP2A did not affect any steps of the parasite lytic cycle including invasion (Fig 4F), egress (Fig 4G), intracellular growth (Fig 4H) or microneme secretion (Fig 4I and 4J).

ATP2B-CDC50.4, but not ATP2A-CDC50.4, is a phosphatidylserine flippase at the plasma membrane

Anchoring of ATP2A-CDC50.4 and ATP2B-CDC50.4 complex to the parasite plasma membrane was demonstrated by protease protection assay on non-permeabilized parasites. Proteins exposed to the outer leaflet of the plasma membrane are susceptible to cleavage by proteases. Concomitantly, the disappearance of the full length ATP2A, ATP2B and CDC50.4 upon protease treatment demonstrates that these complexes localize to the plasma membrane of the parasite (Fig 5A–5C). The presence of the complexes at the parasite plasma membrane, offers the convenient opportunity to assess its flippase activity using a live cells assay [41] as previously reported in T. gondii [8]. We focused our investigation on the analysis of phosphatidylserine (PS) since we previously demonstrated that it is the phospholipid that extracellular parasites majorly incorporate into the plasma membrane [8]. A bulk time-dependent increase in non-quenchable fluorescent analogues of PS was crucially dependent on the presence of ATP2B at the plasma membrane in extracellular (Fig 5D) or intracellular mimicking conditions (S3F Fig), whereas no changes were found upon depletion of ATP2A (Fig 5E). The depletion of CDC50.4 mimicked the effects of the depletion of ATP2B with respect to the bulk PS flipping activity (Fig 5F), in contrast to CDC50.1, which did not affect bulk PS activity upon depletion (Fig 5G).

Fig 5 — (A-C) Full length ATP2A, ATP2B and CDC50.4 are digested by proteinase K (PK) in non-permeabilized parasites, respectively. C-terminally HA-tagged ATP2B and CDC50.4 were used for the assay. ROM4; plasma membrane protein. GAPM3; alveolar protein. Catalase; cytosolic marker. (D-G) Flow cytometry measurement of residual fluorescence upon addition of DPX to NBD-PS incubated extracellular ATP2B, ATP2A, CDC50.4 and CDC50.1-mAID strain of T. *gondii*, respectively. Data represents mean +/- SD of three independent experiments. (H) LactC2 stains plasma membrane and internal vesicular organelles even upon downregulation of ATP2B. GAP45: parasite periphery. (I) LactC2 localized to the cytoplasm of the parasite upon mutation of PS binding sites. GAP45: parasite periphery. (J) Flow cytometry measurement of Annexin V staining of extracellular parental and ATP2B-mAID strain T. *gondii*. Data represents mean +/- SD of three independent experiments. Each parasite line was analysed individually for statistical significance using an unpaired t test. P values: **** = <0.0001, * = <0.05. The scale bars for the immunofluorescence images are 7μM, unless otherwise indicated.

The impact of ATP2B on microneme secretion implicates the importance of a pool of PS at the inner leaflet of the plasma membrane. Such a pool can be detected using the genetically encoded molecular probe lactadherin C2 domain (Lact-C2) fused to GFP known to bind to PS [42]. GFP-Lact-C2 specifically labelled the parasite periphery (Fig 5H) as well as some endomembrane compartments where PS synthesis possibly takes place. Mutation in the specific binding site of Lac-C2 for PS inhibited the plasma membrane localization of the protein (Fig 5I) [42]. Importantly, no changes were observed in Lac-C2 localization upon depletion of ATP2B (Fig 5H). These results are not surprising since PS is the most abundant anionic phospholipid in eukaryotic membranes (accounting up to 10% of the total cellular lipids) and it is highly concentrated at the inner leaflet of the plasma membrane [43]. Concordantly, due to the high affinity of LacC2 to PS [44], low concentration of PS would be sufficient for its binding and re-localization.

We then reasoned that fluctuation in PS levels would be easier to measure in the outer leaflet of the plasma membrane, where concentration in wild type parasites is low. Compellingly, parasite depleted in ATP2B failed to restore PS asymmetric distribution in natural conditions, leading to an accumulation of PS in the outer leaflet of the plasma membrane that can be detected in extracellular parasites via binding to Annexin V (Figs 5J and S3G and S3H).

Taken together, these results demonstrate that ATP2B-CDC50.4 complex, but not ATP2A complex, controls flipping of PS at the plasma membrane of T. gondii.

Discussion

Phospholipid asymmetry plays a key role in several indispensable cellular functions including membrane potential [45], receptor based signaling [46] and secretion of vesicles [47]. P4-ATPases are central flippases that help maintain lipids asymmetry [25]. Most P type-IV ATPases usually require CDC50 partners acting as chaperones for correct localization and activity [48], and complexes formed between these proteins have been shown to be either highly promiscuous or specific [48]. Here we demonstrate that T. gondii encodes for 6 type IV ATPases and 4 CDC50 cofactors with different functions and fitness associated to their deletion. Importantly, some of the data presented here are supported by an overlapping study [30].

P4-ATPases and CDC50 complex formation in Apicomplexa

Here, we demonstrate that in T. gondii, CDC50.4 forms heterocomplexes with ATP2B and ATP2A but not with GC despite sharing a similar localization [8], demonstrating some level of specificity in complex formation independent of protein localization. ATP2B acts as a PS flippase at the plasma membrane which plays a crucial role in microneme exocytosis. The promiscuity of ATP2B substrates was not assessed in this study and would require further analysis. Importantly, a recent report has indicated that P. chabaudi recombinant ATP2 is capable of flipping PS and PE [49], which would suggest that T. gondii ATP2B or ATP2A would also flip PE. Importantly, the authors of this study identified CDC50A and CDC50B as interactors of ATP2, while CDC50C (homologue of CDC50.4) could not be produced recombinantly [49]. This promiscuity of binding remains to be confirmed in vivo since we show here that T. gondii ATP2B is incapable of using other CDC50 chaperones to compensate for the lack of CDC50.4.

Assignment of other pairs will await further investigation but ATP7 and CDC50.3 might form another heterocomplex which is absent in Theileria and Cryptosporidia. ATP7 has previously been shown to be essential and localize to the parasite–host interface in Plasmodium parasites [29]. Despite not being the main focus of this study, we show here that ATP7B is important for T. gondii intracellular growth while ATP7A has been reported to be dispensable based on its fitness score deduced from the genome wide analysis [28]. In addition, a recent report indicates that mutations in the ATP7B are associated with increase resistance of T. gondii to extracellular environment during in vitro evolution studies [50]. The mechanistic details for this emergent resistance are obscure.

ATP8 belongs to the class 2 of P4-ATPases, which includes ATP9A and ATP9B in mammals and Neo1p in yeast [48]. These proteins do not appear to use CDC50 as β-subunit which might indicate that apicomplexan ATP8 can function independently of any CDC50 subunit. In yeast and mammals, these proteins translocate PS and affect the Golgi/endosomal system [51] and recycling of endosomes [52]. A similar function of ATP8 in PS flipping at the Golgi/endosomal compartment in apicomplexans remains to be assessed.

With the group of alveolates, the apicomplexan parasites as well as some ciliates [53] have directly fused a P4-ATPase with GC catalytic domains to form a large GC protein [5]. In T. gondii, CDC50.1 was shown to be essential for GC localization and the sensing and integration of external signals, notably phosphatidic acid [8]. Plasmodium species possess two genes that harbor a fusion with P4-ATPases GCα and GCβ. Importantly, in P. yoelii, CDC50A has been demonstrated to interact with GCβ and to be essential for ookinete gliding motility [54]. The Plasmodium CDC50A groups with the members of CDC50.3 (S1 Fig). On the other hand, the β-subunit associated to GCα remains to be identified. Plasmodium CDC50A and CDC50B belong to the same phylogenetic subgroup that T. gondii CDC50.1 and CDC50.2, and would be good candidates to bind GCα. Conflictingly, neither of these proteins are essential for Plasmodium yoelii erythrocytic stages (PY17X_0619700 and PY17X_0916600) [55] and, despite discrepancies with a genome wide screening on P. falciparum [56], would suggest a possible functional redundancy between CDC50A and CDC50B.

Maintenance of PS asymmetry by ATP2B-CDC50.4 at the plasma membrane is crucial for efficient microneme secretion

Plasma membrane asymmetry is an essential need in cell biology [24–26] and flipping of PS is likely to be maintained across the entire lytic cycle of the parasite for survival. Coherently, ATP2B flippase activity is maintained even in intracellular mimicking conditions (S3F Fig). Here, we demonstrate that the first repercussion of the dysregulation of PS asymmetry at the plasma membrane are during egress, invasion and egress. Compellingly, PS in the inner leaflet of the plasma membrane is known to play a critical role in neurotransmitter release [47]and insulin secretion [57] in mammalian cells. Moreover, Candida albicans strains impaired in PS biosynthesis display decreased ability to secrete proteases and phospholipases [58]. As in most eukaryotic cells, PS is synthesized at the cytosolic leaflet of the ER in T. gondii [59] and asymmetry is predictably maintained by flippases at the Golgi and plasma membrane [41,60]. Golgi localized PS flippases are key players in exocytic vesicle sorting [60]. Once PS reaches the parasite plasma membrane, the ATP2B-CDC50.4 heterocomplex presumably ensures an enrichment of PS at the inner leaflet at the apical tip of the parasites. Any excess of PS at the plasma membrane is rapidly converted into PE as recently demonstrated [30]. The disruption of this homeostasis might lead to overall changes in plasma membrane tension, curvature and could also affect the activity of important signaling components (i.e. GC [8–11], PKG [14], PI-PLC or DGK1 [21]), explaining the phenotype associated to the depletion of ATP2B-CDC50.4 complex. In addition to the previously reported role of PA [21], PS is a second anionic PL implicated in the docking and/or fusion of the micronemes with the plasma membrane. PA is recognized by APH, an acylated protein at surface of the micronemes [21]. Hypothetically, a plausible candidate binding to PS could be DOC2.1 [35] or Ferlin 1 (FER1) [37], and since this phospholipid is enriched at the inner leaflet of the plasma membrane (Fig 5H), might contribute to the mechanism of recognition and fusion of micronemes with the plasma membrane for exocytosis, similarly to well-studied mechanisms proposed in model organisms [32,61,62].

In addition to its role in exocytosis, ‘healthy’ exposure of PS has been previously associated to pathogenesis and immune regulation by T. gondii [63], as well as other eukaryotic parasites [64,65]. The regulation of PS exposure at the plasma membrane of T. gondii and the role of ATP2B-CDC50.4 in this process remain to be investigated. Remarkably, T. gondii is known to secrete a soluble PS decarboxylase which might contribute to a decrease of PS concentration at the outer leaflet of the plasma membrane [66].

A possible implication of ATP2B in phosphatidylthreonine (PT) homeostasis has not been investigated due to the lack of commercially available tools to study this phospholipid. PT was previously described as highly enriched phospholipid in Apicomplexa [59] and was shown to be associated to calcium homeostasis in T. gondii [67]. It is possible that the phenotype of ATP2B is, at least partially, associated to an unexplored capacity of ATP2B to translocate PT. On the other hand, PT synthesis was previously shown to impact specifically natural egress [59], while induced egress reminds unaltered [67]. In addition, since the lack of PT could not be complemented nor aggravated by excess or reduction of PS [59], it is not likely that PS and PT have redundant functions for secretion. Taken together, this data strongly indicates that lack of PS translocation is the main responsible of the phenotype associated to the depletion of ATP2B-CDC50.4 complex showed here.

Overall, this study identified the complex ATP2B-CDC50.4, which is a PS flippase that crucially contributes to motility, invasion and egress. A model by which PS concentration at the inner leaflet of the plasma membrane contribute to microneme docking and exocytosis could imply the participation of lipid binding proteins. However, this hypothesis awaits further investigations.

Materials and methods

Bacteria, parasite and host cell culture

E. coli XL-10 Gold chemo-competent bacteria were used for all recombinant DNA experiments. Parental T. gondii strain Ku80 KO (genotype RHΔhxgprtΔku80) and parental parasites expressing the Tir1 protein were used in this study [14]. T. gondii tachyzoites parental and derivative strains were grown in confluent human foreskin fibroblasts (HFFs) maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 5% fetal calf serum (FCS), 2 mM glutamine and 25 mg/ml gentamicin. Depletion of mAID fusion proteins was achieved with 500 μM of IAA [14].

Preparation of T. gondii genomic DNA

Genomic DNA (gDNA) was prepared from tachyzoites of RH or RH ΔKu80 (here referred as Δku80) strains using the Wizard SV genomic DNA purification (Promega) according to manufacturer’s instructions.

DNA vector constructs and transfection

All primers used in this study are listed in S2 Table. Auxin-inducible degradation of ATP2A, ATP2B, ATP7B, CDC50.2, CDC50.3 and CDC50.4 were generated using a PCR fragment encoding the mAID–HA and the HXGPRT cassette produced using the KOD DNA polymerase (Novagen, Merck) with the vector pTUB1:YFP-mAID-3HA as template and the primers indicated in S2 Table. A specific sgRNA was generated to introduce a double-stranded break at the 3′ of each gene (primers used to generate the guide are indicated in S2 Table).

Parasite transfection and selection of clonal stable lines T. gondii tachyzoites were transfected by electroporation as previously described [68]. Selection of transgenic parasites were performed either with mycophenolic acid and xanthine for HXGPRT selection [69], pyrimethamine for DHFR selection [70] or chloramphenicol for CAT selection [71]. Stable line for all expressing strains were cloned by limited dilution and checked for genomic integration by PCR and analysed by IFA and/or WB.

Antibodies

The monoclonal antibodies against the Ty tag BB2 (1:10 dilution by WB, 1:20 by IFA) [72], actin (1:20 WB) [72], SAG1 (1:20 IFA) (generous gift from J-F. Dubremetz), GRA1 T5-2B4 (1:50 WB, 1:100 IFA), GRA3 (1:50 WB, 1:100 IFA) (generous gift from J-F. Dubremetz), MIC2 (1:20 WB, 1:50 IFA) (generous gift from J-F. Dubremetz), MIC3 T4-2F3 (1:20 WB, 1:50 IFA), anti-Catalase (1:2000 WB) [73], anti-IMC1 (1:2000 WB, 1:1000 IFA), anti-ARO (1:3000 IFA). For western blot analysis, secondary peroxidase-conjugated goat anti-rabbit-IgG, anti-mouse-IgG antibodies and secondary Alexa-Fluor-680-conjugated goat anti-rabbit IgG antibodies (Thermofisher) were used. For immunofluorescence analysis, the secondary antibodies Alexa-Fluor-488-conjugated goat anti-rabbit IgG antibodies, Alexa-Fluor-488-conjugated goat anti-mouse-IgG antibodies and Alexa-Fluor-594-conjugated goat anti-mouse-IgG antibodies (Thermofisher) were used.

Immunofluorescence assay (IFA)

HFFs seeded on coverslips in 24-well plates were inoculated with freshly egressed parasites. After 24 h, cells were fixed with 4% paraformaldehyde (PFA) and 0.005% glutaraldehyde (GA) in PBS for 10 min and processed as previously described [8]. Confocal images were acquired with a Zeiss confocal laser scanning microscope (LSM700 or LSM800) using a Plan-Apochromat 63x objective with NA 1.4 at the Bioimaging core facility of the Faculty of Medicine, University of Geneva. Final image analysis and processing was done with Fiji [74].

Western blotting

Freshly egressed parasites were pelleted after complete host cell lysis. SDS-PAGE, wet transfer to nitrocellulose and proteins visualized using ECL system (Amersham Corp) were performed as described previously [8].

Plaque assay

A confluent monolayer of HFFs was infected with around 50 freshly egressed parasites for 7 to 8 days before cells were fixed with PFA/GA. Plaques were visualized by staining with Crystal Violet (0.1%) as previously described [8]. Quantification was performed using the Fiji [75].

Intracellular growth assay

Parasites were allowed to grow on HFFs for 24 h prior to fixation with PFA/GA. IFA was performed as described previously [8].

Invasion assay

Freshly egressed parasites were inoculated on coverslips seeded with HFFs monolayers and centrifuged at 1100 x g for 1 min. Invasion was allowed for 20 min at 37°C +/- ATc prior to fixation with PFA/GA. Extracellular parasites were stained first using monoclonal anti-SAG1 Ab in non-permeabilized conditions. After 3 washes with PBS, cells were fixed with 1% formaldehyde/PBS for 7 min and washed once with PBS. This was followed by permeabilization with 0.2% Triton/PBS and staining of all parasites with polyclonal anti-GAP45 Ab. Appropriate secondary Abs were used as previously described. 100 parasites were counted for each experiment, the ratio between red (all) and green (invaded) parasites is presented. Results are presented as mean ± standard deviation (SD) of three independent biological replicate experiments.

Induced egress assay

Freshly egressed tachyzoites were added to a new monolayer of HFFs, washed after 30 min and grown for 30 h. The infected HFFs were washed once in serum-free DMEM and then incubated with 50 μM BIPPO in serum-free DMEM for 7 min at 37°C. Cells were fixed with PFA/GA and processed for IFA using anti-GAP45 Ab. 100 vacuoles were counted per strain and scored as egressed or non-egressed. Results are presented as mean ± standard deviation (SD) of three independent biological replicate experiments. Control experiment with DMSO showed no egress. For live video microscopy of induced egress, parasites were grown on glass bottom plates seeded with HFFs monolayers for 30 h at 37°C and egress was induced as described above.

Microneme secretion assay

Microneme secretion assay was performed on freshly egressed parasites, pre-treated 24 or 48 h +/- ATc. Parasites were pelleted at 1000 rpm for 5 min and resuspended in extracellular (EC) buffer (142 mM NaCl, 5.8 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5.6 mM glucose, 25 mM HEPES, pH to 7.2 with NaOH). After centrifugation, the pellets were resuspended in 100 μL of extracellular (EC) buffer containing +/- 2% ethanol and incubated for 30 min at 37°C. Then, parasites were pelleted at 1000 x g for 10 min at 4°C, the supernatant was transferred to a new Eppendorf tube (the pellet from this step serves as the pellet fraction) and centrifuged again at 2000 x g for 10 min at 4°C. The final supernatant, containing the excreted/secreted antigens (ESA), and pellet fraction were resuspended in SDS loading buffer and boiled prior to immunoblotting.

Flippase assay

NBD-phospholipid incorporation (NBD-PS) was assessed by flow cytometry as described before [8,41]. In brief, 5 × 10⁶ extracellular parasites were washed in Hank’s balanced salt solution (pH 7.4) containing 1 g l−1 glucose. Subsequently, 1 μM NBD-PS was incubated at room temperature. At the designated time point, 20 mM DPX (p-xylene-bis-pyridinium bromide) was added to quench fluorescence of lipids localized in the outer leaflet. Then, 10,000 cells were analysed with a Gallios (4-laser) cytometer. The mean fluorescence intensities of the cells were calculated.

Immunoprecipitation assay

Extracellular tachyzoites were harvested, washed in PBS and lysed in co-immunoprecipitation buffer (0.2% v/v Triton X-100, 50 mM Tris-HCl, pH 8, 150 mM NaCl) in the presence of a protease inhibitor cocktail (Roche). Cells were sonicated on ice and centrifuged at 14,000 r.p.m. for 30 min at 4°C. Supernatants were then subjected to immunoprecipitation using anti-HA antibodies as previously described [8]. 2 μl of DTT (50 mM in liquid chromatography–mass spectrometry-grade water) were added and the reduction was carried out at 37°C for 1 h. Alkylation was performed by adding 2 μl of iodoacetamide (400 mM in distilled water) for 1 h at room temperature in the dark. Protein digestion was performed overnight at 37°C with 15 μl of freshly prepared trypsin (Promega; 0.2 μg μl−1 in ammonium bicarbonate). After beads were removed, the sample was desalted with a C18 microspin column (Harvard Apparatus), dried under speed vacuum, and redissolved in H2O (94.9%), CH3CN (5%) and FA (0.1%) before liquid chromatography–electrospray ionization-tandem mass spectrometry analysis (LC–ESI-MS/MS). LC–ESI-MS/MS was performed on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an Easy nLC 1000 system (Thermo Fisher Scientific). Peptides were trapped on an Acclaim PepMap 100, C18, 3 μm, 75 μm × 20 mm nano-trap column (Thermo Fisher Scientific) and separated on a 75 μm × 500 mm, C18, 2 μm Easy-Spray column (Thermo Fisher Scientific).

Annexin V staining

Annexin V (ThermoFisher, 88-8005-72) labelling was performed as indicated by supplier. Briefly, 1x10⁶ freshly egressed parasites were resuspended in binding buffer provided by supplier. 5 uL of Annexin V was added for labelling and incubated during 10–15 minutes. Upon washing once, 10,000 cells were analyzed with a Gallios (4-laser) cytometer. The mean fluorescence intensities of the cells were calculated.

In silico analysis of proteins and modelling

Sequences of Apicomplexan P-type ATPases and CDC50s were procured from EuPathDB and aligned using MUSCLE sequence alignment software [76,77]. The resulting sequence alignment was manually curated utilizing BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) to edit out uninformative alignment positions. Phylogeny tree was generating utilizing PhyML [78] on the curated MUSCLE alignment, using LG model of amino acids substitution with NNI topology search. Phylogeny.fr [78] platform was utilized for much of the above analysis. All accession numbers are provided in S1 Table.

C2 domain modelling of TgDOC2.1 was performed using the automated server i-TASSER [79] and visualized using PyMOL (www.pymol.org). Modelling was performed using the residues 569 to 653, using the sequences 4ihbA, 3jzyA, 4icw, 5ixcA, 4rj9A, 3pfqA and 5ixcA (Swissmodel templates) with normalized Z-scores of 0.87 to 2.51. Overall model possesses an estimated C-score of -0.41, TM score of 0.66 ± 0.13 and RMSD of 4.4 ± 2.9Å.

Statistics and reproducibility

All data are presented as the mean ± s.d. of 3 independent biological replicates (n = 3), unless otherwise stated in the figure. The mean of each independent biological replicate was generated by counting 100 vacuoles/parasites. All data analyses were carried out using GraphPad Prism. The null hypothesis (α = 0.05) was tested using unpaired two-tailed Student’s t-tests and significant P values are shown.

Supporting information

S1 Fig

(A) Apicomplexan CDC50s cluster into 2 phylogenetic groups. An unrooted maximum likelihood tree of apicomplexan ASPs was generated using PhyML v3.0, using WAG model of amino acids substitution with NNI topology search, based on an amino acid alignment by MUSCLE. The genes are represented by the EuPathDB accession numbers. Node support values are indicated.

(TIF)

Click here for additional data file.^{(3.8MB, tif)}

S2 Fig

(A) PCR demonstrates correct integration of ATP2A, ATP2B and ATP7B and CDC50.2–4 mAID. Primers used are listed in S2 Table. (B) PCR demonstrates correct integration of ATP7B-SM-HA. Primers used are listed in S2 Table. (C) Immunoblot of lysates from RH parental and ATP7B-SM-HA parasites. HA antibodies were used to detect tagged ATP7B. MIC2: loading control. (D) Western blot showing enrichment of CDC50.4-mAID-HA upon immunoprecipitation. Anti-HA antibodies were used to detect CDC50.4-mAID-HA in the different fractions. S: soluble fraction (input), P: pellet fraction. (E) IFA of intracellular RH ATP2B-Ty/CDC50.4-mAID-HA parasites with or without IAA. The scale bars for the immunofluorescence images are 7μM, unless otherwise indicated. (F) Western blot of lysates ATP2A-Ty/CDC50.4-mAID-HA parasites treated with or without IAA for 24 hours. Catalase: loading control.

(TIF)

Click here for additional data file.^{(51.6MB, tif)}

S3 Fig

(A) Parasites lacking ATP7B display a delay in intracellular replication. Error bars represent ±SD from three independent experiments. (B) Egress assay of Tir1 parental strain and ATP7B-mAID-HA parasites grown for 30hs treated with or without IAA. Egress was induced with BIPPO or DMSO for 7 minutes. Percentage of egressed vacuoles is shown as means+/- SD of 3 independent replicates. (C) Invasion assay of Tir1 parental strain and ATP7B-mAID-HA parasites treated with or without IAA for 24 hours. Data represents mean +/- SD. (D) Representative images of vacuole organization in parasites depleted (or not) of ATP7B. Quantification is shown in (E). (F) Flow cytometry measurement of residual fluorescence upon addition of DPX to NBD-PS incubated extracellular ATP2B-mAID in intracellular buffer. Parasites were mechanically released from intracellular condition to avoid activation of egress signalling. (G-H) Histograms corresponding to one experiment of Annexin V binding to parasites are shown in (G) and representative images in (H). The scale bars for the immunofluorescence images are 7μM, unless otherwise indicated.

(TIF)

Click here for additional data file.^{(4.7MB, tif)}

S1 Table. Gene accession numbers of the homologs of the studied genes within the Apicomplexa phylum.

(XLSX)

Click here for additional data file.^{(13.3KB, xlsx)}

S2 Table. Oligonucleotide sequences used in this study.

(XLSX)

Click here for additional data file.^{(10.5KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the Swiss National Foundation to D.S.-F. (FN3100A0-116722) and by the Scientific & Technological Cooperation Programme Switzerland-Rio de Janeiro (IZRJZ3_164183). H.B. is the recipient of a Swiss Government Excellence Scholarship with Uruguay. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1010438.r001

Decision Letter 0

Vern B Carruthers, Michael J Blackman

23 May 2021

Dear Dominique,

Thank you very much for submitting your manuscript "Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

As you will see, all three reviewers are excited by the study and agree that it makes an important contribution to understanding of the role of P4-ATPase flippases in phospholipid asymmetry, microneme discharge and host cell invasion by Toxoplasma. However, the reviewers also share a number of substantial concerns with the manuscript in its current form. In particular, they agree that not all the conclusions are currently supported by the experimental data. Areas in which additional experimental evidence is requested include the suggestion that PS binds directly and specifically to DOC2.1. Examining the effects of ATP2B knockdown on DOC2.1 localisation would also inform the major conclusions, as would analysis of the effects of CDC50.4 knockdown on subcellular localisation of ATP2A and ATP2B. An examination of whether PS flipping occurs in intracellular parasites would also address the proposed link with egress. Whether these P4-ATPases can flip phospholipids other than PS has not been examined, although we do not feel this is an essential requirement. More detailed analysis of the calcium dependency of DOC2.1-PS binding is warranted. The inclusion of statistical analysis of the quantitative data (including full details in the Methods section of the statistical methods used), as well as information on numbers of experimental replicates, is essential. Finally, Figure 6 in particular requires attention and corrections, whilst a number of textual changes and corrections are requested throughout the manuscript that we agree in most cases would help to clarify the narrative.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael J Blackman

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript 'Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis' presents some nice cell biological work utilising reverse genetics to detail the role of flippases in the asexual cycle of Toxoplasma. The authors identify P4-ATPase flippase proteins and their partner CDC50 chaperones and identify a potential, novel and essential role for an ATP2B-CDC50.4 complex in microneme secretion required for host cell invasion. Overall, the work present is well designed and the conclusions are well supported by the data presented.

Reviewer #2: The work by Bisio and colleagues provides important insights into P4-ATPases in Toxoplasma gondii - a class of enzyme that is involved in flipping phospholipids across membrane bilayers. Given previous findings that have shown that phosphatidic acid is important for mediating egress, this piece of work looks into the involvement of several P4-ATPases and their CDC50 chaperones, focusing mainly on egress and invasion. Epitope tagging and immunoprecipitation revealed that two ATPases, ATP2A and ATP2B, interact with the chaperone CDC50.4. Using the conditional auxin degradation system, Bisio et al reveal that CDC50.4 is important for ATP2B stability, but does not affect another P4-Type ATPases, GC. Using this system the authors go on to reveal that conditional knockdown of CDC50.4 and ATP2B leads to egress and invasion defects as well as a defect in microneme secretion. On the other hand, ATP7B knockdown leads to a disruption of intracellular growth and vacuole organisation, while disruption of ATP2A shows no defect at all.

Further characterisation of ATP2B and CDC50.4 revealed that ATP2B is involved in flipping phosphatidyl serine from the outer to the inner leaflet of the plasma membrane and that disruption of ATP2B leads to an accumulation of PS on the surface of tachyzoites. Next the authors take a side step and investigate the role of Doc2.1, a C2 containing protein involved in mediating microneme secretion, in sensing PS. The authors show that this protein does indeed bind to PS in a calcium independent manner. Overall these findings are important and show that regulating PS asymmetry is important in regulating processes such as egress and invasion and will be of great value to the Toxoplasma field. Despite these important findings, the manuscript in its current state requires significant improvement and amendments for publication. Most importantly: more experiments are required for ATP2A and CDC50.4 given that these are the major focus of this study, while the Doc2.1 data feels a bit like an add-on.

Reviewer #3: This work studies the role of a Toxoplasma gondii complex formed between the predicted flippase ATP2B with the cell division control protein (CDC) 50.4 for the flipping and asymmetric distribution of phosphatidylserine, which may act as lipid mediator for organelle fusion and microneme exocytosis. Authors first identified and localized putative flippases and CDC50s in T. gondii. They did this by C-terminal tagging of the respective proteins. A number of them localized to the apical end of the parasite. They decided to focus on CDC50.4 because of its predicted essentiality and conservation across the apicomplexans. They first demonstrated the interaction of CDC50.4 with ATP4A and ATP4B by immunoprecipitation coupled with mass spectrometry analysis. They tagged ATP2B in the CDC50.4-AID-HA background for co-localization studies. To study the function of these proteins they tagged them with the auxin-inducible degron (mAID) for downregulation. Downregulation of CDC50.4 led to degradation of ATP2B as shown by western blot analysis. They did not check for ATP4A. They next determined that the complex between ATP2B-CDC50.4 and also ATP7B were critical for T. gondii growth. They examined every step of the lytic cycle: invasion, egress and replication. Depletion of ATP2B or CDC50.4 caused impairment in invasion and egress but did not alter intracellular replication. Microneme secretion of extracellular parasites was reduced upon downregulation of either ATP2B or CDC50.4. No motility assays were shown. They showed that both proteins localize to the parasite membrane using a protease protection assay in non-permeabilized parasites. They measured flippase activity in situ with PS analogues, which was dependent on the presence of ATP2B at the PM. They also used a genetically encoded lactadherin C2 domain fused to GFP, known to bind PS. No controls for specific labeling of PS were shown. In conclusion authors claim to have identified a PS flippase that contributes to motility, invasion and egress. The authors claim that DOC2.1, a previously described key egress and invasion factor, senses changes in cytosolic calcium in intracellular parasites and may be the plausible sensor of PS at the inner leaflet of the plasma membrane.

The work has some weaknesses concerning statistical analysis of the data and only partial information on the number of biological replicates for each experiment. Some statements are not supported by the data presented like the Ca2+ sensing of DOC2.1 or the motility defects of the mutants. Some improvements are suggested, and more explanations and discussion of results obtained are needed.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The work characterising the essentiality of identified CDC50 and P4-ATPase proteins has been carried out well. This has been recently published in an overlapping study (Ref 30) and therefore there should be further discussion of this published work included in the manuscript. However, the present study adds a detailed view of the role of these proteins at specific stages of the asexual cycle. The authors clearly demonstrate that ATP2B and CDC50.4 forms an essential complex and in some very nice experimental work, show that this complex translocates phosphatidylserine (PS). They then go on to mechanistically characterise the role of the ATP2B-CDC50.4 complex and PS translocation. The authors indicate that PS translocation by ATP2B-CDC50.4 is required for microneme secretion, and this is carried out by DOC2.1 binding of PS. Whilst this is a nice conclusion, the data presented to support the premise of DOC2.1-PS binding is weak and requires more substantial affirmation. Given that liposome-binding experiments were performed with parasite lysate, it cannot be discounted that DOC2.1 binding is not direct but mediated by another protein factor, the text should be amended to reflect this.

Additionally, since PS is an anionic lipid, it is possible that liposome binding of DOC2.1 from lysate is simply due to charge-charge interaction. The group’s previous work has demonstrated the production of the anionic lipid phosphatidic acid (PA) at the plasma membrane preceding egress, which is required for microneme secretion. Given this, the authors should repeat the sedimentation experiment of tagged DOC2.1 using liposomes containing PA to rule out that interaction is not PS specific but due to binding of anionic charge.

Furthermore, it would add more support to the hypothesis of DOC2.1-PS interaction if the study could visualise the localisation of tagged DOC2.1 in egressed parasites with and without knock-down of ATP2B. Presumably, the localisation of DOC2.1 would become increased in plasma membrane proximity which would likely be lacking upon ATP2B knock-down.

The authors outline in Figure 5A and 5B that invasion and egress are impaired in ATP2B and CDC50.4 knock-down parasites. However, looking at the data it seems that invasion is similarly impaired to the extent of the lack of egress. Can the authors rule out that invasion occurs normally, and the reduction seen is merely a result of the reduction in ATP2B and CDC50.4 knock-down in parasite egress?

The authors suggest that there is no significant effect on microneme secretion by ATP2A knock-down, however, in Figure 5I there appears to be a ~50% reduction in MIC2 secretion in ATP2A knock-down parasites in the presence of BIPPO. Could the authors comment on this? Is this reproducible? If so, this needs to be acknowledged in the main text.

Figure 6H is hard to interpret. It is unclear what the significance value relates to. Additionally, it is difficult to clearly see the increase in annexin V binding in ATP2B knock-down tachyzoites. Perhaps displaying the data as a box plot would increase clarity.

Figure 6I shows the potential localisation of PS in WT parasites, however was the visualisation of internal PS attempted in ATP2B or CDC50.4 knock-down parasites? If so, was there any difference? If not, a comment should be included in the manuscript.

Lines 87-89 Although there are references cited for the statement regarding phosphodiesterases (PDEs) : ‘…presumably regulated by PKA-C1 and cAMP…’, it leaves the reader wondering why the authors presume this. The statement either should be omitted or modified to outline briefly any evidence that regulation of TgPDEs may be by cAMP/PKA.

Line 186, this results section would benefit from brief mention of some of the other proteins identified in the pull down/MS experiment shown in Figure 2.

Line 220, should explain that proteins exposed at the surface of the PM are susceptible to the protease (rather than this just indicating PM localisation).

Line 296-298, it should be clarified that this statement refers to P. yoelii erythrocytic stages (Ref 52) rather than ‘Plasmodium species’ in general.

The section describing the results shown in Figure 5 would benefit from briefly mentioning and explaining the use of the PDE inhibitor BIPPO. I note that its use is mentioned but not explained in the figure legend.

Figure 7, what is the likely explanation for the fact that mutation of the two aspartates in DOC2.1 causes reduced fitness but not invasion or microneme secretion? Could it e.g. mean that these residues (and perhaps calcium binding) have a role in parasite growth, but not at the stage of invasion/microneme secretion?

Line 277, were different concentrations of ‘calcium’ tested to decide on the use of 0.1 mM? If not, can it be concluded from this ~30% reduction in bound protein that binding is independent of calcium?

Reviewer #2: Major Issues:

• Line 173: in this section, the authors focus on ATP2B but not ATP2A and it is unclear why the experiments were done solely on ATP2B, despite evidence of ATP2A also binding to CDC50.4. It would be interesting to see if ATP2A mislocalises when CDC50.4 is knocked down or whether there are other chaperones that can compensate for loss of CDC50.4.

• Line 183: the authors demonstrate that knockdown of CDC50.4 leads to a knockdown of ATP2B levels but they do not show an IFA of localisation of ATP2B-Ty following treatment with IAA. Does the remaining protein localise to the apical end of the parasite or is the localisation affected?

• Line 189: the authors offer no explanation of the lines that were generated. Please add a sentence explaining to the readers that the proteins of interest were fused to a mAID tag

• Line 208 & Figure 5D: the authors state in Line 208 that they assessed microneme secretion by stimulating with ethanol, however the figure legend of Figure 5D states that the parasites were stimulated with BIPPO. Can the authors correct either the legend or the main text since it is unclear which stimulant was used.

• Line 223: The authors decide to focus solely on PS experiments on ATP2A and ATP2B. Given their proven ability to do these assays with other phospholipids (PA and PC in the Bisio et al 2019 paper) and also given that they later state in the discussion that these ATPases may be involved in flipping PE, the authors should do these flipping experiments with PA, PC and PE as these would be highly informative and reduce the some speculation in the discussion section. Despite ATP7B not being involved in egress or invasion, it would also be useful for the greater Toxoplasmosis field and our understanding of Toxoplasma biology to know whether this ATPase is flipping any phospholipids, so it would also be important to include these experiments too since these lines are available.

• Line 227: the authors look at PS flipping in extracellular parasites. It would also be important to look at whether this flipping occurs in intracellular parasites i.e. by performing this experiment on parasites lysed in ENDO buffer. This is an important experiment to do to support the author’s model of PS accumulating on the inner leaflet prior to egress, since if this flipping is also occurring in intracellular parasites without any stimulation then in theory there shouldn’t be any PS flipping onto the inner leaflet.

• Line 234: the authors provide little commentary on the results from the Lact-C2 experiment - is there a difference in binding between the ATP2BmAID mutants +/-IAA? It also appears they have mixed up the Figures since Figure 6H is the annexin experiment mentioned further down and figure 6I is the Lact-C2 experiment. Furthermore in Figure 6I it is unclear which parasites are shown in the image and there should be images comparing ATP2B parasite - and + IAA. Not to mention that this subfigure (6I) has no figure legend.

• Line 235: the authors state that ATP2B knockdown parasites are unable PS asymmetric distribution - are there images to support this? The graph shown (Figure 6H shows that there is maybe more binding of Annexin V) but it would aid the reader if images of these parasites are shown. Is this increased accumulation of Annexin at the apical tip where ATP2B is localised to? Please provide these images as this is critical information that the reader should be able to see.

• Line 273: have the sedimentation experiments been done with the mutant versions of Doc2.1? If not the authors should perform these experiments since there appears to be a difference in the binding of PS + and - Ca2+ (despite it not being the pattern that the authors expected).

• Line 298: the authors reference the Jian et al paper from 2020 as the source for CDC50A and CDC50B not being essential for Plasmodium - however these experiments are nowhere to be found in this source. The authors should reference the correct source here. As far as I am aware there is only evidence of CDC50A not being essential in P. yoelii (Gao et al 2018) but I couldn’t find one for CDC50B

• Line 298: after checking PlasmoDB and looking at the mutagenesis screen by John Adams lab, it appears that CDC50B is essential and CDC50C is important for asexual growth. The authors state here that CDC50B is not essential and do not mention CDC50C in the discussion

• Line 353: the authors mention the recent FER1 pre-print, but this text seems out of place and needs more explanation or to be rewritten to link it to the rest of the discussion

• The discussion needs substantial improvements. At times it is difficult to follow the order and reasoning behind the information included. The authors should re-write this and also include summaries of their experiments. For example there is no mention of ATP7B or any of the Doc2.1 experiments which constitute a small yet significant part of the paper.

• Line 595: the authors should change "(D-F)" to (D-G) since figure G is also a part of this series of experiments.

• Line 597: the authors state in the legend that figure 6G represents the staining oft he plasma membrane and internal organs of the parasite with LactC2 but the actual figure doesn’t correspond to this. Can the authors please add the appropriate image and fix the legend.

• Line 599: in Figure 6 there is a subfigure 6I, however there is no reference to this subfigure in the figure legend. Please add a description of this image explaining what the parasite line is.

• Figure 5: no statistical analysis for any of the graphs after figure 4 are provided. The authors should provide this and if there is a statistically significant difference in the levels of PS binding of Doc2.1 + and - Ca2+ (which it looks like there is), then this should be commented on in the manuscript.

• Throughout the manuscript no statistical tests were performed. I would expect the appropriate stats tests to be performed for all graphs without exception. Without this data, the reader is missing crucial bits of information to make their own judgements on the data.

• Methods section: there is no methods section for the AnnexinV binding experiments - please add this.

Reviewer #3: Major issues

1. Figure 2E shows a lot of background with the GC-TY. Any explanation for that?

2. The IFA image shown for the downregulation of CDC50.4 appears to show some effect on the expression/distribution of GC and it concentrates in the residual body?. This result needs quantification and a better description in the legend.

3. There are no attempts to localize ATP2B in the CDC50.4 knock downs.

4. Figure 3C: the plaques for CDC50s are strange. Either there are no plaques in the image shown or they are not fully lysed. It will be good to explain what was measured and quantified for part D. Indicate in the legend the number of biological replicates.

5. Figure 4 presents the phenotype of ATP7B and is not clear how it fits in the story. Authors do not give an explanation of the disrupted rosette phenotype observed.

6. Figure 5, please indicate in the legend the number of biological replicates for all panels (some are missing) and show statistical significance analysis for the ones that are relevant.

7. Figure 6: The ATP2B-CDC50.4 complex localizes to the apical end or the plasma membrane? This should be consistent in the description of the results. The proteolysis experiment shows that the HA epitope is degraded but not necessarily the whole proteins.

8. Figure 6D-F: indicate the number of biological replicates and any statistical significance obtained in the legend. Labeling of panels need to be corrected in the figure which does not agree with the legend.

9. Figure 6H: The flow cytometry charts could be shown as supplemental. The differences in the violin plots results are not clearly evident from the distribution. Need to detail in the legend, the number of independent replicates and explain more how the statistical analysis was done. The p value shown is for which comparison?

10. Figure 6H and I are swapped in the text.

11. Figure 6I: Some evidence of specific binding is needed. Is there a cell line depleted of PS that could serve as control for the binding of the lactadherin-GFP? Reference 59 generated mutants for PS synthesis although the PS content of the mutants was not clear, and it was a double mutant. Specificity could also be shown by transfecting parasites with the mutated version of the Lac-gfp gene as shown in reference 42.

12. Figs 7 D and E need quantification of plaque sizes and statistical analysis

13. Figure 7F needs statistical analysis and the legend needs to indicate the number of biological replicates for G.

14. Figure 7H needs quantification and statistical analysis. Indicate the number of replicates. Could this part be re-organized with the legends at the top? Also, separate the bottom panel since it is very confusing as presented.

15. Figure 8: Legend needs to indicate the number of biological replicates. The data in B needs statistical analysis.

16. Figure 8: Authors show that DOC2.1 binding to PS liposomes is independent of calcium. The concentration of free calcium is difficult to predict from the information provided. EDTA mainly chelates Mg2+ and EGTA would be more appropriate. The concentration of Ca2+ of the lysate could be high considering that Ca2+ bound to proteins would be released during lysis of the cells. 0.1 and 1 mM EGTA should be tested to be sure that calcium is not relevant. In addition, there is no information of the Ca2+ binding affinity of DOC2.1

17. Abstract says that DOC2.1 senses changes in cytosolic Ca and that is a sensor of PS at the inner leaflet of the PM. The data only shows that mutating putative calcium binding domains results in growth defects due to egress defect. There is no evidence for Calcium binding of DOC2.1 so the mutation could impact other aspects of the protein. May be increases in cytosolic Ca affects the localization of DOC2.1 making it more defined? This would be interesting to explore.

18. The methods section does not have an explanation for the statistical analysis of the data.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 33, insert ‘the’ parasite.

Line 64, insert ‘the’ plasma membrane.

Line 67, Perhaps modify ‘inner enriched gradient’ which doesn’t sound quite right. It reads well in line 133.

Line 68, delete ‘the’.

Line 73, 79 should be ‘phylum Apicomplexa’. Delete ‘of’.

Line 82, ‘stages’.

Line 89, define PKA as the cAMP-dependent protein kinase.

Line 90, delete ‘G’ so that PKG is defined correctly.

Line 96, Perhaps replace ‘system’ with ‘motor’

Line 117, ‘based on’.

Line 128, Perhaps replace ‘participating’ with ‘involved’ or ‘strictly participates’.

Line 136-139 This section containing two presumptions would benefit from a re-write and isn’t a strong end to the Introduction.

Line 137, ‘presumable’ should be replaced by e.g. ‘putative’.

Line 157, replace ‘aroused’ with ‘arisen’

Line 175, ‘focused’.

Line 177, e.g. delete ‘an’.

Line 199, e.g. delete ‘the’.

Line 200, replace ‘every’ with ‘each of’.

Line 201, delete ‘in repaired’.

Line 205, ‘parasites’.

Line 217, ‘of the complexes’.

Line 222, ‘cell’.

Line 224-225, please re-write the last half of this sentence.

Line 225, e.g. ‘A bulk time’.

Line 235, ‘parasites’.

Line 236, ‘the asymmetric PS distribution’.

Line 238, ‘taken together these results indicate that…’.

Line 242, ‘micronemes’.

Line 247, delete ‘the’.

Line 251, Delete ‘A’.

Line 293, specify that in P. yoelii it is GCβ

Line 294, ‘groups with’ would benefit from slight expansion.

Line 363, ‘remained’?

Line 629, state here what calcium salt was used.

Reviewer #2: Minor Notes:

• Line 157: please change “aroused" to “have arisen”

• Line 166: please consider changing sentence to “Similar to GC and its partner CDC50.1, which have previously been found at the apical tip of the parasites (8), ATP2A, ATP2B, CDC50.2 and CDC50.4 are also found at the apical tip”

• Line 191: why do some of the western blots have a parental sample while some don’t?

• Line 217: the authors provide no rationale behind the protease protection experiments. A short sentence here explaining the reasoning behind this or the line of thought would greatly aid the readers in following the story.

• Line 353: unclear what is meant by “tonic”

• The authors have size bars for all IFAs but they fail to state what size this bar corresponds to in any of the figure legends. Please include this

• The authors should consider merging Figures 7 & 8

• Figure 6: the scale bars of 6E should be amended to be the same as the scale bars the other figures

• Figure 6: the statistical analysis of Figure 6H is missing despite there being a p value

Reviewer #3: Minor issues:

1. Tables S1 and S2 are mislabeled.

2. The links in Table S2 (should be S1) all lead to a “page not found”

3. Figure 2C needs markers

3. Although is not relevant to the story, the authors mention in the introduction that the signaling cascade starts with cGMP, which activates PKG, PI-PLC, IP3, calcium and CDPKs. However, it should also be taken into consideration that PIPLC needs calcium for activity, so the pathway may not be that linear and calcium could also be upstream to PIPLC.

**********

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Reviewer #2: No

Reviewer #3: No

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PLoS Pathog. 2022 Mar 24;18(3):e1010438. doi: 10.1371/journal.ppat.1010438.r002

Author response to Decision Letter 0

21 Nov 2021

Attachment

Submitted filename: Flippases_Rebuttal_17.11.21.docx

Click here for additional data file.^{(59.4KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1010438.r003

Decision Letter 1

Vern B Carruthers, Michael J Blackman

22 Dec 2021

Dear Dominique,

Thank you very much for submitting your manuscript "Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

As you will see, the revised version of this manuscript has been examined by 3 reviewers. The reviewers came to widely differing views, including a recommendation to reject the submission. After extensive consideration, the editorial view is that you should be given an opportunity to respond to these comments in a further round of revision. Whilst minor points of revision are raised by Reviewer #3, Reviewer #1 points out that in the absence of any data showing that DOC2.1 binds PS, there is now no obvious link between the DOC2.1 data in the revised manuscript and the data on the ATP2B/CDC50.4 complex. We agree that this substantially reduces the impact and breadth of the manuscript since it means that ther is now no experimentally validated functional link between DOC2.1 function and the function of the ATP2B/CDC50.4 complex. This conclusion is further strengthened by the fact that mutagenesis of DOC2.1 residues presumed to be involved in calcium binding affected egress but not invasion or microneme discharge. We would be grateful if you could address this major comment, as well as those of Reviewer #3. In addition, it is noted that the localisation by immunofluorescence of CDC50.4-mAID-HA in panels E and F of Figure 2 appears different from that of HA-tagged CDC50.4 (Fig 2B). Can you please address this issue too?

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael J Blackman

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have dealt with the reviewer comments comprehensively. It is good that they decided to pull back on the direct interaction of DOC2.1 and PS in light of further work being needed to substantiate this.

Reviewer #2: The revised manuscript by Bisio and colleagues incorporates several of the requested experiments and changes. However, some major comments were not addressed by the authors due to technical difficulties, even for experiments previously performed and published by the lab, such as assessing the involvement of the ATPases identified in flipping phospholipids other than PS. They also have not been able to reproduce consistently the DOC2.1 liposome binding experiments. As a result, the authors have removed a significant portion of the results section relating to DOC2.1. This is a very honest response, which I highly value, however, this leave the manuscript in a weaker position and slightly disjointed.

As the manuscript currently stands, the authors have convincingly shown that ATP2A and ATP2B bind to CDC50.4, and that ATP2B and CDC50.4 play an important role in egress, invasion and microneme secretion. They also show that knockdown of these two proteins leads to a block in PS flipping. While it is possible that ATP2B is directly involved in PS flipping, this has not been shown experimentally. The authors also show that ATP7B is important for lytic growth, but do not characterise this line further. Finally, the authors show that knockdown of DOC2.1, a protein thought to be involved in microneme secretion, leads to a block in lytic growth due to the inability of the parasites to egress, invade or secrete micronemes. By mutating the calcium binding sites of DOC2.1, they also show that these sites seem to be important for egress but not invasion or microneme secretion. This is a surprising result, but not further pursued. Since the authors removed the DOC2.1 liposome binding experiments, which showed binding of DOC2.1 to PS, there is no longer a link between the role of DOC2.1 and ATP2B/CDC50.4. This weakens the manuscript significantly. Another concern is that in Figure 2, the localisation of CDC50.4-HA in panel B appear to occupy a very different localisation compared to CDC50.4-mAID-HA in panel E. This seems to indicate that mAID-tagging of CDC50.4 mislocalises the protein.

Reviewer #3: This work studies the role of a complex formed between the flippase ATP2B with the cell division control protein (CDC) 50.4 for the flipping and uneven distribution of phosphatidylserine, which may act as lipid mediator for organelle fusion and microneme exocytosis. Authors demonstrated the interaction of CDC50.4 with ATP4B and focused the work on the function of these proteins. Downregulation of CDC50.4 led to degradation of ATP2B and the complex was critical for T. gondii growth. Invasion and egress was impaired in the mutants while intracellular replication was not. Microneme secretion of extracellular parasites was reduced upon downregulation of either ATP2B or CDC50.4. The authors claim that DOC2.1, a previously described key egress and invasion factor, senses changes in cytosolic calcium in intracellular parasites and may be the plausible sensor of PS at the inner leaflet of the plasma membrane.

This work is interesting, and it is an important contribution to our knowledge of the role of P4-ATPases flippases in T. gondii. Authors responded to most of the previous critique of this reviewer. Two minor concerns are indicated.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: No major issues

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Two minor concerns that could easily be clarified in the legends to the figures.

The legend for Figure 2C needs an explanation for how the westerns were done. It is mentioned the IPs with anti-Ty but the antibodies used for the western are not mentioned.

The PCRs presented in FigS4B are a little confusing. Could the authors include the size of the expected bands? There is a shadow/smear in part B. Does it mean anything? Please clarify what is expected and the result.

This figure is important to understand how the mutation of DOC2.1 was made.

**********

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Reviewer #1: No

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Reviewer #3: No

Figure Files:

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Reproducibility:

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLoS Pathog. 2022 Mar 24;18(3):e1010438. doi: 10.1371/journal.ppat.1010438.r004

Author response to Decision Letter 1

9 Mar 2022

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PLoS Pathog. doi: 10.1371/journal.ppat.1010438.r005

Decision Letter 2

Vern B Carruthers, Michael J Blackman

11 Mar 2022

Dear Dominique,

We are pleased to inform you that your manuscript 'Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael J Blackman

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Dear Dominique

During the manuscript proofing stage, there are a small number of grammatical errors in the Abstract and Author summary that should be attended to, as follows:

Second sentence of Abstract: please alter 'micronemes' to 'microneme'

Penultimate sentence of Abstract: please delete 'the' from '...for the organelle fusion...'

3rd sentence of Author summary: replace 'depends' with 'depend'

4th sentence of Author summary: alter to '...act as a flippase...' (insert 'a')

Final sentence of Author summary: alter to '...the importance of membrane homeostasis...' (insert 'of')

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1010438.r006

Acceptance letter

Vern B Carruthers, Michael J Blackman

21 Mar 2022

Dear Dr Soldati-Favre,

We are delighted to inform you that your manuscript, "Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(TIF)

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S2 Fig

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S3 Fig

(TIF)

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S1 Table. Gene accession numbers of the homologs of the studied genes within the Apicomplexa phylum.

(XLSX)

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S2 Table. Oligonucleotide sequences used in this study.

(XLSX)

Click here for additional data file.^{(10.5KB, xlsx)}

Attachment

Submitted filename: Flippases_Rebuttal_17.11.21.docx

Click here for additional data file.^{(59.4KB, docx)}

Attachment

Submitted filename: rebuttal_2022.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis

Hugo Bisio

Aarti Krishnan

Jean-Baptiste Marq

Dominique Soldati-Favre

Roles

Abstract

Author summary

Introduction

Fig 1. Members of the Apicomplexa phylum encode a plethora of P4-ATPases and CDC50 cofactor at different parasite locations.

Results

Identification and localization of the putative flippases and their CDC50 partners in Toxoplasma gondii

Toxoplasma gondii CDC50.4 forms promiscuously stable heterocomplexes with ATP2A and ATP2B

Fig 2. ATP2B and CDC50.4 form an heterocomplex.

ATP2B-CDC50.4 complex and ATP7B are critical for the survival of Toxoplasma gondii

Fig 3. Fitness conferring and dispensable P-type IV ATPases and CDC50 subunits are encoded in the T. gondii genome.

P-type IV ATPases and its CDC50 chaperones play distinct roles in the parasite biology

Fig 4. ATP2B-CDC50.4 heterocomplex facilitates microneme secretion.

ATP2B-CDC50.4, but not ATP2A-CDC50.4, is a phosphatidylserine flippase at the plasma membrane

Fig 5. ATP2A and ATP2B are phospholipid specific flippases at the parasite plasma membrane.

Discussion

P4-ATPases and CDC50 complex formation in Apicomplexa

Maintenance of PS asymmetry by ATP2B-CDC50.4 at the plasma membrane is crucial for efficient microneme secretion

Materials and methods

Bacteria, parasite and host cell culture

Preparation of T. gondii genomic DNA

DNA vector constructs and transfection

Antibodies

Immunofluorescence assay (IFA)

Western blotting

Plaque assay

Intracellular growth assay

Invasion assay

Induced egress assay

Microneme secretion assay

Flippase assay

Immunoprecipitation assay

Annexin V staining

In silico analysis of proteins and modelling

Statistics and reproducibility

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Vern B Carruthers

Michael J Blackman

Roles

Author response to Decision Letter 0

Decision Letter 1

Vern B Carruthers

Michael J Blackman

Roles

Author response to Decision Letter 1

Decision Letter 2

Vern B Carruthers

Michael J Blackman

Roles

Acceptance letter

Vern B Carruthers

Michael J Blackman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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