Calreticulin (CALR) Promotes Ionophore-Induced Microneme Secretion in Toxoplasma gondii

doi:10.21203/rs.3.rs-3717986/v1

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Calreticulin (CALR) Promotes Ionophore-Induced Microneme Secretion in Toxoplasma gondii

https://doi.org/10.21203/rs.3.rs-3717986/v1

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published 21 Feb, 2024

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The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER), that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly down-regulated (p<0.05). The cellular biological functions of the down-regulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.

Toxoplasma gondii

Calreticulin

Ionophore-induced secretion

Microneme proteins.

Toxoplasma gondiiis one of the apicomplexans and is an obligate intracellular parasite as well as a cause of zoonosis. One third of the people on the planet are infected with T. gondii (Hill et al., 2005). The primary cause of clinical symptoms is the accelerated intracellular growth of tachyzoites. The lytic cycle includes invasion, proliferation, and egress. The microneme, which concludes perforin, protease, and adhesin that are crucial to the parasite's lytic cycle, plays a significant role in these processes, especially in invasion and egress (Soldati et al., 2001; Dubois and Soldati-Favre, 2019).

It is well known that high intracellular Ca²⁺ ([Ca²⁺]_i) levels coincide with enhanced microneme secretion during invasion and egress (Carruthers and Sibley, 1999). Microneme secretion was suppressed by the Ca²⁺ chelator, BAPTA-AM, but enhanced when Ca²⁺ was raised in the cytoplasm using ionophores (e.g., A23187 and ionomycin), which induced an instantaneous increase in Ca²⁺ and a rapid egress of the parasites (Song et al., 2005). The cGMP -initiated signaling cascade leads to the activation of PKG, which acts on PI-PLC and hydrolyzes PIP₂to generate the second messengers IP₃ and DAG (Fang et al., 2006; Brown et al., 2017; Jia et al., 2017). An Unknown IP₃-gated channel in the ER interacts with IP₃ to release Ca²⁺ into the cytoplasm and activate CDPK leading to microneme secretion and egress (Lourido et al., 2010). In summary, microneme secretion is a complex process involving multiple proteins and signaling pathways, and the main factor affecting microneme secretion is the change in intracellular Ca²⁺concentration. However, little is known about the molecular mechanisms of this regulation, particularly the exocytosis or egress.

T. gondii can take up Ca²⁺ from the host and release intracellular Ca²⁺, particularly from the endoplasmic reticulum (ER). Ca²⁺ is pumped into the ER by the sarcoplasmic-endoplasmic reticulum calcium-ATPase (SERCA) and released from the ER via the inositol triphosphate (IP₃) and ryanodine-like (Ryn) signaling pathways (Chini et al., 2005). Calcium-binding proteins (CaBPs) predominantly bind to and store Ca²⁺ in the ER. Calreticulin (CALR) is a kind of CaBP that is commonly found in the eukaryotic ER and is able to bind Ca²⁺ in the ER (Baksh and Michalak, 1991; Michalak et al., 2009b). In addition to regulating Ca²⁺ homeostasis, CALR is involved in a variety of cellular functions, including involvement in glycoprotein folding, Ca²⁺ signaling, and cell adhesion (Schurch et al., 2020).

Calreticulin consists of three conserved structural domains, N, P and C. The N-domain is a globular domain that containing primarily β-strands, contains predicted glycan-binding sites, and the amino acid sequence is highly conserved across all species. Due to the presence of proline residues, it has an arm-like structure known as the P-domain, which not only binds Ca²⁺but also has a chaperone function similar to that of lectins (Kozlov et al., 2010). The amino acid sequences IxDPxxxKP(E/D)DWD and GxWxxxxIxNP are each repeated three times in the P-domain, respectively. This region binds Ca²⁺with high affinity (Michalak et al., 1999). The C-terminal region of CALR contains many negatively charged amino acid residues with low affinity for Ca²⁺ but high capacity and is responsible for Ca²⁺ storage in the endoplasmic reticulum. The N and P domains mainly function as molecular chaperones, while the P and C domains perform the function of Ca²⁺ storage.

In recent years, the function of calreticulin in protozoa has been characterized and examined in Leishmania donovani and Trypanosoma cruzi. CALR is primarily involved in Ca²⁺ signaling and protein secretion in L.donovani, and overexpression of the P domain prevents the secretion of acid phosphatase, leading to accumulation of acid phosphatase in the parasite and growth inhibition(Debrabant et al., 2002). In recent years, research on CALR in T.cruzi has focused more on glycoprotein synthesis and found that T.cruzi expresses UDP-glucose glycoprotein glucosyltransferase (UGGT), which recognizes and facilitates the folding of glycoproteins (Labriola et al., 2010). Studies have shown that CALR has multiple roles in protozoa, including protein folding, Ca²⁺ signaling pathways, and modulating secretion processes.

Here, we identified CALR and probed its function in T. gondii by reverse genetics approaches. Our study confirmed that CALR is dispensable for the parasite’s lytic cycle, but it has a significant impact on Ca²⁺-mediated microneme secretion and egress induced by the ionophore A23187. We also provided potential evidence that CALR may contribute to Ca²⁺-mediated microneme secretion by influencing Ca²⁺ release from the ER.

Culture and collection of Toxoplasma

At 37°C with 5% CO₂, the parasite was serially passaged in DMEM medium containing 2% fetal bovine serum (FBS) and the culture medium was changed 24 h after inoculation. When the parasites were ready to be egress, the cells were scraped off and the culture was blown 3-5 times with 5 ml and 1 ml disposable sterile syringes, and then cellular debris was removed by passing through a 5 μM sterile filter. T. gondii was finally collected after centrifugation at 2800 rpm for 10 min.

Construction of transgenic parasites

The TgCALR-HA strain was created by inserting a 3HA tag before the target gene's stop codon, allowing the target protein and HA to be fused and produced. Plasmids were co-transfected into RHΔku80, positive strains were screened with pyrimethamine and recombinant strains were identified using Western blot and IFA.

Using the principle of homologous recombination for gene knockout, CALR 5'UTR and 3'UTR sequences were obtained from ToxoDB, and pTCR-TgCALR plasmid was constructed. The gRNA sequence was obtained through ToxoDB to construct CRISPR/Cas9-CALR plasmid. Positive strains were selected with chloramphenicol, and the recombinant strains were detected by PCR after the plasmid was co-transfected into RHΔku80.

The UPRT gene was specifically disrupted by a CRISPR/CAS9-UPRT specific gRNA and replaced by the cDNA sequence of TgCALR to complement calr-deficient parasites. The calr knockout strain (Δcalr) was co-transfected with a UPRT locus-targeted homologous recombinant plasmid containing the p5′UPRT-Tubulin promoter-DHFR-CALR-HA-3′UPRT and the CRISPR/CAS9-UPRT plasmid. Positive strains were screened using pyrimethamine.

Immunofluorescence assay

HFF cells were infected with parasites, then fixed with 4% paraformaldehyde, permeabilized with 0.25% TritonX-100, and blocked with 3% BSA for 1 h at room temperature. Subsequently, samples were treated for 1 h with mouse anti-TgBIP (1:100), mouse anti-HA (1:50), rabbit anti-FLAG (1:100), mouse anti-MIC4 (1:100), rabbit anti-MIC2 (1:500) and rabbit anti-TgGAP45 (1:300). The sample were then labeled using FITC- or Cy3-conjugated secondary antibodies (1:100) and DAPI (1:100). Images were captured using Olympus DP controller software and an Olympus IX70 inverted microscope.

Western blot

Parasites were lysed in RIPA buffer followed by SDS-PAGE for Western blot. Mouse anti-HA (1:500), anti-Flag (1:200), mouse anti-MIC4 (1:100), rabbit anti-MIC2 (1:500), mouse anti-GRA14 (1:100) and mouse anti-Actin (1:1000) were used as primary antibodies. HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG was diluted 1:10000 in 5% skim milk and incubated for 1 h at 37°C. Finally, the reactive bands were visualized using enhanced chemiluminescence reagents.

Plaque assay

Different strains of the same state were collected, purified and counted, and then 100 or 150 tachyzoites were introduced into a 12-well plate containing HFF cells in 3 duplicates per group. The free parasites were rinsed off with PBS after 7 days of static culture and fixed in paraformaldehyde for 30 min at room temperature, and then dyed with crystalline violet solution for 30 min. The size and number of plaques were measured using Photoshop to assess the in vitro growth of several parasite strains.

Invasion assay

After collecting the parasites, 1×10⁷ tachyzoites were placed in each well. After culturing for 25 min, the medium was removed. The IFA experiment was performed after fixation with paraformaldehyde. External parasites (noninvaded) were stained with anti-rabbit SAG1 before permeabilization, and internal parasites (invaded) were detected by anti-mouse IMC1. Secondary antibodies were FITC goat anti-rabbit and Cy3 goat anti-mouse. Parasites in the red and green channels were counted using a fluorescence microscope. Parasites in the red channel are those that adhere to the surface of HFF cells, while the green channels are all parasites. The number of parasites invading the host cell is given by the count in the green channel X minus the count in the red channel Y, while the count in the host cell nucleus is given by Z. (X-Y)/Z is the invasion rate.

Egress assay

1×10⁶ pure tachyzoites were infected and cultured for 24-30 h on a 12-well plate containing HFF cells. When the majority of tachyzoites began to egress, 5 μM A23187 was added and fixed with paraformaldehyde. The proportion of ruptured parasites or integrity parasitophorous vacuoles (PV) in each strain was measured using the IFA assay.

Microneme secretion assay

Intracellular parasites were collected, washed twice with PBS, resuspended in extracellular buffer containing 5 μM A23187, incubated for 10 min, and centrifuged at a temperature of 4℃ and 1500 rpm for 15 min. The supernatant and precipitate were collected and examined for secretion of MIC proteins by Western blot.

Fluo-4AM calcium monitoring

Ca²⁺ release was detected using Fluo-4AM. Fresh Parasites were lysed, labeled with 250 nM Fluo-4AM (Beyotime, China) at 37℃ for 20 min, centrifuged for 5 min, washed twice, and resuspended in Ringer's solution. The parasites were then dispersed in 96-well plates that had been pre-treated with lysine and centrifuged at 100 g for 1 min to allow parasites adsorption. After finding the field of view, the parasites were treated with 5 μM A23187 and the samples were photographed within 5 min using a fluorescence microscope from Olympus Co., Japan. Utilizing the ImageJ measurement module, picture pixel intensities were measured and visualized in Graph Pad.

Statistical analysis

GraphPad Prism was used for plotting and statistical analysis. Means are shown by graphs, while standard errors of the means are represented by error bars. A t-test was performed on the data to determine the differences between the groups. Where p>0.05 (n.s.) indicates that there is no significant difference, p<0.05 (*) suggests a significant difference, and p<0.01 (**) denotes a highly significant difference.

T. gondii genome encodes a calreticulin

The protein sequence information of calreticulin (CALR) was obtained from the ToxoDB, and the related protein sequences were obtained by comparing them with the database using the BLAST online tool. Finally, phylogenetic analyses of the newly discovered CALR with selected animal and plant CALRs as well as protozoan CALRs were performed using the MEGA7 program. The findings revealed that CALR is conserved only in a few apicomplexan species, such as Neospora, Besnoitia, and Cystoisospora (Fig. 1A). Additionally, we discovered that TgCALR (TGGT1_ 310320) and several apicomplexan parasites, including Neospora caninum (NCLIV_054410), Besnoitia besnoiti (BESB_071810), Cystoisospora suis (CSUI_004467), Eimeria tenella (ETH_00022665)and Eimeria necatrix (ENH_00019190) contain one conserved calreticulin domain, hereafter referred to as CALR (Fig. 1B).

The 3D structure of CALR was modelled using the SWISS-MODEL server with the amoeboid protozoan CALR as the structural template. Judging from the structural modelling, CALR is conserved in that it contains a globular lectin domain with predicted glycan and high-affinity Ca²⁺ binding sites (Fig. 1C, D), however, CALR possesses only one similar repeat of the GXWXPPXIXNPXYX motif (Supplementary Figure 1).

CALR is not required for the growth of T. gondii

With the use of CRISPR/Cas9 gene editing, the mutants were created to figure out the biological role of CALR (Fig. 2A). The PCR results showed that the 5' homology arm and 3' homology arm of the obtained strain were recombined correctly, and both pairs of primers could not amplify the calr gene, proving that the calr gene deletion strain was constructed successfully (Fig. 2B).

The plaque assay was used to assess the growth of T. gondii throughout the lytic cycle, and we used this assay to determine whether CALR is required for parasite survival. There were no significant differences in the area and number of plaques in HFF cells between wild-type, Δcalr, and iΔcalr, implying that CALR is not required for the parasite lytic cycle (Fig. 2C, D). Further, examination of the invasion efficiency, intracellular proliferation capacity, and natural egress ability of Δcalr parasites showed no difference compared with the wild-type (Fig. 2E, F, G). Similarly, deletion or complementary of calrhad no significant effect on the growth of T. gondii in vitro (Fig. 2H).

CALR impedes induced-egress by reducing microneme secretion

The calcium ionophore A23187 induces Ca²⁺ flux, which is required for parasite egress (Black et al., 2000; Caldas et al., 2010). The finding that CALR has Ca²⁺-binding sites let us to investigate whether CALR is involved in ionophore-induced egress. For this purpose, we used A23187 to assess the induced-egress ability of the parasites. When the Δcalrstrain was stimulated with A23187 for 3 min, the majority of parasites were unable to quickly egress from the host cell (Fig.3A). However, the stimulus lasted longer than 10 min and all parasites were egressed (data not shown). This finding indicates that parasites lacking calr are less sensitive to A23187 treatment than wild-type and complementary parasites.

The ability of T. gondii to egress is mainly dependent on microneme secretion, and its secreted perforin, protease and adhesion protein are essential for egress (Roiko et al., 2014; Dogga et al., 2017). According to the findings, calr deletion influences parasite induced-egress. To better understand the involvement of CALR in parasite egress, we investigated the secretion of MIC2 and MIC4. As shown by Western blot, calr deletion resulted in a significant reduction in MIC2 and MIC4 secretion (Fig. 3B). Furthermore, we question as to whether the secretion of microneme was related to the mis-trafficking of the microneme proteins. However, our findings demonstrated that calr deletion had no effect on the location of MIC2 and MIC4 (Fig. 3C).

Based on these results, we conclude that CALR plays a role in the Ca²⁺-dependent signal transduction pathway in microneme secretion and affects the induced-egress of T. gondii.

CALR localizes in the ER

CALR influences ionophore-induced secretion and it has a conserved Ca²⁺ binding site, which suggests that CALR may regulate Ca²⁺ homeostasis, and it will be fascinating to study the localization of CALR in T. gondii.

Therefore, we generated an epitope-tagged CALR strain with C-terminal 3HA to investigate CALR localization (Fig. 4A). Western blot analysis using the anti-HA antibody showed bands of approximately 67 kDa, which corresponded to the predicted size of the CALR (Fig. 4B). Using IFA, the CALR was discovered around the nucleus, and subsequently CALR was found to co-localized with BIP polyclonal antibodies (Fig. 4C and Supplementary Fig 2). To further investigate the localization of CALR, the study used endoplasmic reticulum retention signal receptor (ERD2) as an endoplasmic reticulum marker, created a TgCALR1-3HA-TgERD2-FLAG endogenous strain (Supplementary Fig 3), and confirmed that CALR and ERD2 are co-localized in the endoplasmic reticulum of T. gondii (Fig. 4D).

CALR regulates calcium signaling pathway

Calreticulin plays an important role in the regulation of Ca²⁺ homeostasis in other species, and we discovered that CALR is localized in the ER, which is the main Ca²⁺ repository of T. gondii. As a result, we examined the effect of CALR on Ca²⁺ release. The cytosolic Ca²⁺ levels of the parasites were measured after being loaded with the Ca²⁺-sensitive fluorescent dye fluo-4AM. Temporal variations in intracellular Ca²⁺were tracked using time-lapse video microscopy in the absence of Fluo-4AM labeling and upon addition of A23187 to the parasites. When the parent strains were exposed to 5 μM A23187, a high amplitude was induced, but the amplitude was modestly diminished in the calr-deficient strains, indicating that CALR influences the release of Ca²⁺ from T. gondii (Fig. 5A, Supplementary video).

Next, a comparative transcriptomic analysis between the Δcalr and the parent strains was performed to explore which pathways are affected by CALR. The results showed that 226 genes were significantly down-regulated in Δcalr parasite (Fig. 5B). GO enrichment analysis showed that the cellular biological function of the down-regulated genes mainly involved in calmodulin-dependent protein kinase activity (Fig. 5C). The study conducted bioinformatics analysis on gene-matched DEGs of the functional domains of the Ca²⁺ pathway in GO and found that the differential genes mainly consisted of protein kinases and Ca²⁺ binding proteins (Fig. 5D, E). These results may indicate that CALR affects Ca²⁺ flux, which regulates kinases and binding proteins downstream of Ca²⁺ and thus regulates microneme secretion.

In mammalian cells, the Ca²⁺-buffering molecular chaperone (known as calreticulin) participates in endoplasmic reticulum Ca²⁺ storage via its P and C domains (Michalak et al., 2009a; Giraldo et al., 2010). The P domain binds Ca²⁺ with high affinity and low capacity, whereas the C domain has a low-affinity, high-capacity Ca²⁺-binding site (Ellgaard et al., 2001). Despite the large Ca²⁺ storage capacity of the C domain, the regulation of Ca²⁺ release by calreticulin persists in the absence of the C domain, suggesting that the P and C domains function synergistically in Ca²⁺ signaling (Jia et al., 2010). Besides, CALR increases the folding rate of glycoprotein intermediates and promotes their folding by recruiting two folding enzymes, ERp57 and CypB (Oliver et al., 1999; Bedard et al., 2005; Chouquet et al., 2011).

However, the presence of CALR has only been reported for a few parasites, mainly T. cruzi and L. donovani. The purpose of this study was to characterize the CALR of T. gondii and to explore the function of CALR in parasite. The study predicted the CALR structure and its conserved domains and confirmed that it may bind glycoproteins and Ca²⁺, suggesting that TgCALR may have a dual role in the synthesis of glycoproteins and regulation of Ca²⁺homeostasis. Using gene editing techniques, we created Δcalr strain and discovered that CALR had no effect on the parasite's ability to proliferate in vitro. On the other hand, CALR influenced ionophore-induced microneme secretion, which could explain its effect on induced-egress.

Ca²⁺-mediated microneme secretion is a key event required for successful egress of T. gondii from the host cell. The study determined that deletion of calr impairs microneme secretion triggered by a high Ca²⁺ concentration through via stimulation with A23187. The Ca²⁺-mediated microneme secretion pathway begins with the parasitesensing exogenous egress signals or generating internal egress signals through a series of signal pathways that ultimately lead to the exit of tachyzoites from the host cell (Millholland et al., 2013; Paul et al., 2015; Brown et al., 2016). In brief, the second messenger cyclic guanosine monophosphate (cGMP) activates cGMP-dependent protein kinase G (PKG), which is a protein kinase, to produce inositol triphosphate (IP₃) and diacylglycerol (DAG) (Bisio et al., 2019; Brown and Sibley, 2018). In eukaryotes, IP₃ binds to the IP₃ receptor (inositol triphosphate, IP₃R), which acts as a Ca²⁺ channel, and releases Ca²⁺ from the ER. In T. gondii, IP₃ stimulates the release of Ca²⁺from an unknown calcium channel, and intracellular Ca²⁺ activates Ca²⁺-dependent protein kinase, which in turn mediates microneme secretion (Mccoy et al., 2012; Treeck et al., 2014). Nevertheless, no significant growth defects were found in calr-deficient parasites. We propose a model here (Fig. 6), CALR binds Ca²⁺ in the ER and its function depends on the different Ca²⁺requirements of the parasite. In the absence of pharmacological stimulation, we hypothesized that there was no significant difference in egress ability between Δcalrand parent parasites due to the fact that Ca²⁺ in the cytoplasm at this time is available to complete the parasite's lytic cycle. However, when stimulated with A23187, a large amount of Ca²⁺ was required to be released, whereas the deletion strain released a reduced amount of Ca²⁺, which could not fully satisfy the need for egress and microneme secretion, resulting in an impaired ability to induce microneme secretion and egress. In addition, comparative transcriptomic results also showed that CALR predominantly affects some Ca²⁺ kinases and Ca²⁺-binding proteins downstream of Ca²⁺. Thus, our data suggest that CALR may affect induced-microneme secretion and egress by influencing Ca²⁺ signaling pathways.

Furthermore, the transcriptomic results revealed that CALR affects the fatty acid oxidation pathway. The study examined the differentially expressed genes involved in this pathway and discovered that they are primarily homologous to acyl-coenzyme A dehydrogenase, located in mitochondria. Based on this analysis, we hypothesized that CALR may have an impact on fatty acid metabolism in mitochondria. Mitochondria, as essential organelles in eukaryotes, are crucial to the cellular metabolic network, which is responsible for the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, nucleotide synthesis, and amino acid metabolism. Acyl CoA is an activated fatty acid that is required for oxidative catabolism occurring in mitochondria. The protein quality control of mitochondria is one of the many factors that influence the oxidation of fatty acids in mitochondria (Kusum et al., 2019). To preserve mitochondrial homeostasis and prevent cellular damage, eukaryotes have evolved a number of quality control mechanisms. Mitochondrial Ca²⁺ regulates mitochondrial dynamics, mitophagy, and mitochondrial biogenesis, all of which contribute to maintaining mitochondrial quality control (MQC) in cellular homeostasis (Marchi et al., 2018; Srimanta et al., 2021). The mitochondria-associated membrane (MAM) provides a platform of proteins and channels involved in Ca²⁺ transport between the ER and mitochondria. Chaperone proteins such as calnexin, calreticulin and sigma 1 receptor are present in the MAM and play significant roles in Ca²⁺ buffering (Lan et al., 2019). Several studies have found that CALR is not only important for retaining Ca²⁺ within the ER, but also controls ER Ca²⁺ flux into mitochondria (Arnaudeau and S., 2002; Gutiérrez et al., 2020). Thus, calreticulin may affect fatty acid metabolism by modifying mitochondrial Ca²⁺ concentration, which in turn regulates MQC, and thus acyl CoA dehydrogenase activity.

The study sought to examine whether TgCALR influences glycoprotein expression, as it is expected to have a globular lectin structural domain and glycoprotein binding site, but this experiment has not been completed due to the lack of appropriate glycoproteins and antibodies. CALR is a lectin-like chaperone involved in the production of a wide range of molecules in other species, including ion channels, surface receptors, integrins, and transporters (Peterson et al., 1995). The research looked at the expression of MIC2 and MIC4 and discovered that CALR had no influence on either (results not shown), but this does not necessarily mean that CALR is not involved in protein folding. Further research is required to determine whether CALR is involved in protein synthesis.

In conclusion, we have discovered the CALR of T. gondii, which is present in the endoplasmic reticulum. This study shows that CALR plays a role in converting a substantial rise in cytoplasmic Ca²⁺ concentration into a burst of microneme secretion. The gene knockout lines also confirmed there was no significant growth defect in the parasite following calr deletion. However, the ability of induced-egress was reduced, which was related to the suppression of ionophore-induced secretion of MIC2 and MIC4. Other studies have showed that CALR influences Ca²⁺ release, thereby controlling possible downstream genes, which in turn affects microneme secretion. The above findings imply that CALR controls the homeostasis of Ca²⁺ and thus affects ionophore-induced secretion and egress of T. gondii.

Ethics approval

Animal experiments were conducted in strict compliances with the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. All experimental protocols were approved by the Institutional Animal Care and Use Committee of China Agricultural University (under the certificate of Beijing Laboratory Animal staff ID: 18049).

Consent for publication

All authors gave their consent before submitting this work.

Competing interests

The authors declare no conflict of interest.

Author contributions

Qun Liu and Zhili Shan conceived and designed this study. Zhili Shan, Xingju Song and Xu Yang analysed the data and generated the figures. Zhili Shan, Xingju Song, Xianmei Wang, Yayun Wu and Yangfei Xue performed the experiments. Zhili Shan, Qun Liu and Jing Liu wrote the manuscript.

Funding

This work was supported by the the National Natural Science Foundation of China (31172309) and National Key Research and Development Program of China (2017YFD0500400).

Availability of data and materials

All datasets generated for this study are included in the manuscript/supplementary files.

Acknowledgments

We appreciate Prof. Shaojun Long's advice and assistance (China Agricultural University). We are also grateful to Prof. Bang Shen (Huazhong Agricultural University) for providing CRISPR/Cas9 plasmid.

Arnaudeau, S., 2002. Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic Reticulum and Mitochondria. Journal of Biological Chemistry 277, 46696-46705.
Baksh, S., Michalak, M., 1991. Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains. Journal of Biological Chemistry 266, 21458-21465.
Bedard, K., Szabo, E., Michalak, M., Opas, M., 2005. Cellular Functions of Endoplasmic Reticulum Chaperones Calreticulin, Calnexin, and ERp57. International Review of Cytology-a Survey of Cell Biology 245, 91-121.
Bisio, Hugo, Lunghi, Matteo, Brochet, Mathieu, Soldati-Favre, Dominique, Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform.
Black, M.W., Arrizabalaga, G., Boothroyd, J.C., 2000. Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress. Molecular & Cellular Biology 20, 9399-9408.
Brown, K.M., Long, S., Sibley, L.D., Weiss, L.M., 2017. Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii. mBio 8, e00375-00317-00317.
Brown, K.M., Lourido, S., Sibley, L.D., 2016. Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. Journal of Biological Chemistry 291, 9554-9565.
Brown, K.M., Sibley, L.D., 2018. Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase. Cell Host & Microbe 24.
Caldas, L.A., Souza, W.D., Attias, M., 2010. Microscopic analysis of calcium ionophore activated egress of Toxoplasma gondii from the host cell. Veterinary Parasitology 167, 8-18.
Carruthers, V.B., Sibley, L.D., 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Holt, Rinehart and Winston.
Chini, E.N., Nagamune, K., Wetzel, D.M., Sibley, L.D., 2005. Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii. Biochemical Journal 389, 269-277.
Chouquet, A., Païdassi, H., Ling, W.L., Frachet, P., Houen, G., Arlaud, G.J., Gaboriaud, C., 2011. X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism. PLoS ONE 6.
Debrabant, A., Lee, N., Pogue, G.P., Dwyer, D.M., Nakhasi, H.L., 2002. Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages. International Journal for Parasitology 32, 1423-1434.
Dogga, S.K., Mukherjee, B., Jacot, D., Kockmann, T., Soldati-Favre, D., 2017. A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. Elife 6.
Dubois, D.J., Soldati-Favre, D., 2019. Biogenesis and secretion of micronemes in Toxoplasma gondii (vol 21, e13018, 2019). Cell Microbiol, 21.
Ellgaard, L., Riek, R., Herrmann, T., 2001. NMR structure of the calreticulin P-domain. Proceedings of the National Academy of Sciences of the United States of America 98, 3133-3138.
Fang, J., Marchesini, N., Moreno, S.N.J., 2006. A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol. Biochemical Journal 394, 417-425.
Giraldo, A.M.V., Medus, M.L., Lebrero, M.G., Pagano, R.S., Labriola, C.A., Landolfo, L., Delfino, J.M., Parodi, A.J., Caramelo, J.J., 2010. The Structure of Calreticulin C-terminal Domain Is Modulated by Physiological Variations of Calcium Concentration. Journal of Biological Chemistry 285, 4544-4553.
Gutiérrez, T., Qi, H., Yap, M.C., Tahbaz, N., Milburn, L.A., Lucchinetti, E., Lou, P.-H., Zaugg, M., LaPointe, P.G., Mercier, P., Overduin, M., Bischof, H., Burgstaller, S., Malli, R., Ballanyi, K., Shuai, J., Simmen, T., 2020. The ER chaperone calnexin controls mitochondrial positioning and respiration. Science Signaling 13.
Hill, D.E., Chirukandoth, S., Dubey, J.P., 2005. Biology and epidemiology of Toxoplasma gondii in man and animals. Animal Health Research Reviews 6, 41-61.
Jia, X.Y., He, L.H., Jing, R.L., Li, R.Z., 2010. Calreticulin: conserved protein and diverse functions in plants. Physiol Plant 136, 127-138.
Jia, Y., Marq, J.B., Bisio, H., Jacot, D., Mueller, C., Yu, L., Choudhary, J., Brochet, M., Soldatifavre, D., 2017. Crosstalk between PKA and PKG controls pH‐dependent host cell egress of Toxoplasma gondii. Embo Journal 36, e201796794.
Kozlov, G., Pocanschi, C.L., Rosenauer, A., Bastos-Aristizabal, S., Gehring, K., 2010. Structural Basis of Carbohydrate Recognition by Calreticulin. Journal of Biological Chemistry 285, 38612-38620.
Kusum, Y., Anurag, Y., Priyanka, V., P, P.V., N, D.U., 2019. Protein Misfolding Diseases and Therapeutic Approaches. Current protein & peptide science 20.
Labriola, C.A., Conte, I.L., Máximo, L.M., Parodi, A.J., Caramelo, J.J., Martins, B.E., 2010. Endoplasmic Reticulum Calcium Regulates the Retrotranslocation of Trypanosoma Cruzi Calreticulin to the Cytosol. PLoS ONE 5, e13141.
Lan, B., He, Y., Sun, H., Zheng, X., Gao, Y., Li, N., 2019. The roles of mitochondria-associated membranes in mitochondrial quality control under endoplasmic reticulum stress. Life sciences 231, 116587.
Lourido, S., Shuman, J., Zhang, C., Shokat, K.M., Hui, R., Sibley, L.D., 2010. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359.
Marchi, S., Patergnani, S., Missiroli, S., Morciano, G., Pinton, P., 2018. Mitochondrial and endoplasmic reticulum calcium homeostasis and cell death. Cell Calcium 69.
Mccoy, J.M., Whitehead, L., Van Dooren, G.G., Tonkin, C.J., Soldati-Favre, D., 2012. TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells. Plos Pathogens 8, e1003066.
Michalak, M., Corbett, E.F., Mesaeli, N., Nakamura, K., Opas, M., 1999. Calreticulin: one protein, one gene, many functions. Biochemical Journal 344 Pt 2, 281.
Michalak, M., Groenendyk, J., Szabo, E., Gold, L.I., Opas, M., 2009a. Calreticulin, a Multi-Process Calcium-Buffering Chaperone of the Endoplasmic Reticulum. Biochemical Journal 417, 651-666.
Michalak, M., Groenendyk, J., Szabo, E., Gold, Leslie I., Opas, M., 2009b. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochemical Journal.
Millholland, M.G., Mishra, S., Dupont, C.D., Love, M.S., Greenbaum, D.C., 2013. A Host GPCR Signaling Network Required for the Cytolysis of Infected Cells Facilitates Release of Apicomplexan Parasites. Cell Host & Microbe 13, 15-28.
Oliver, J.D., Roderick, H.L., Llewellyn, D.H., High, S., 1999. ERp57 Functions as a Subunit of Specific Complexes Formed with the ER Lectins Calreticulin and Calnexin. Mol Biol Cell 10, 2573.
Paul, A.S., Saha, S., Engelberg, K., Jiang, R.H.Y., Coleman, B.I., Kosber, A.L., Chen, C.T., Ganter, M., Espy, N., Gilberger, T.W., 2015. Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans. Cell Host & Microbe 18, 49-60.
Peterson, J.R., Ora, A., Van, P.N., Helenius, A., 1995. Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol Biol Cell 6, 1173-1184.
Roiko, M.S., Svezhova, N., Carruthers, V.B., Knoll, L.J., 2014. Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity. Plos Pathogens 10, e1004488.
Schurch, P., Malinovska, L., Wildschut, T., Lakkaraju, A., Lysenko, V., Aguzzi, A., Picotti, P., Theocharides, A.P.A., 2020. Homozygous Calreticulin mutations affect chaperone function and perturb the glycoproteome. Oncology research and treatment., 43.
Soldati, D., Dubremetz, J.F., Lebrun, M., 2001. Microneme proteins:: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. International Journal for Parasitology 31, 1293-1302.
Patra, K.K. Mahapatra, P.P. Praharaj, D.P. Panigrahi, C.S. Bhol, S.R. Mishra., 2021. Intricate role of mitochondrial calcium signalling in mitochondrial quality control for regulation of cancer cell fate. Mitochondrion 57, 230-240.
Song, H.O., Ahn, M.H., Ryu, J.S., Min, D.Y., Lee, Y.H., 2005. Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii. The Korean Journal of Parasitology 42, 185-193.
Treeck, M., Sanders, J.L., Gaji, R.Y., Lafavers, K.A., Boothroyd, J.C., 2014. The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis. PLoS Pathogens 10, e1004197.

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Calreticulin (CALR) Promotes Ionophore-Induced Microneme Secretion in Toxoplasma gondii

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