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  1. Author Response:

    Reviewer #2 (Public Review):

    Yu et al provide a comprehensive set of experiments to determine that bradyzoites have much slower cytosolic Ca2+ parameters, which impact on gliding motility, a key process of Toxoplasma spread and persistence.

    The only main criticism that I have is the use of the MIC2-GLuc reporter to measure microneme secretion in bradyzoites. Do bradyzoites have any appreciable level of MIC2 and its associated protein M2AP?? This is important that may affect the outcome. If bradyzoites do not, then the MIC2-GLuc reporter might not have appropriate levels of M2AP to correctly traffic to the micronemes. I recommend that the authors quantitate, either by western blot or IFA, the levels of MIC2 and M2AP in bradyzoites versus tachyzoites and also show that M2AP co-localises with MIC2-GLuc to give confidence that MIC2-GLuc is trafficked correctly and thus the low readings of secretion are not just a result of the reporter mistrafficked. It would also be pleasing to see, that 1hr incubation leads to restoration of MIC2-GLuc secretion.

    We acknowledge that the expression and localization of MIC2-Gluc reporter is a potential concern. We performed western blotting (Figure 2C) and IFA (Figure 2 supplement 1A) to confirm that bradyzoites express MIC2-Gluc and M2AP albeit at lower levels compared with tachyzoites. Moreover, MIC2-GLuc and M2AP were properly co-localized to the apical end in bradyzoites, ruling out the possibility of mis-localization of the MIC2-GLuc reporter. Based on these results, we believe that MIC2-GLuc provides a reliable read-out for microneme secretion in in vitro differentiated bradyzoites. Additionally, the conclusion that MIC secretion is dampened in bradyzoites is also supported by the studies using the FNR-Cherry reporter in Figure 2E,F,G.

    Reviewer #3 (Public Review):

    This is a first study that looks in detail at Ca-controlled gliding motility and ATP supply in bradyzoites. A comparison of such different parasite stage by manipulating Ca and ATP metabolism is challenging. Intervention by chemical compounds needs to overcome a prominent cyst wall and the usage of genetic tools needs to consider the broad changes in protein expression between tachyzoites and bradyzoites as well as a heterology between individual bradyzoites. The authors used excysted bradyzoites to exclude the cyst wall as a diffusion barrier as a major factor in the efficacy of different Ca agonists. To address differences in expression levels between tachyzoites and bradyzoite stages the authors developed a ratiometric Ca sensor based upon an autocleaved GCaMP6f-BFP dimer protein.

    Overall the conclusions are well supported but there are methodological questions that need to be addressed.

    Bradyzoites show a heterogenous expression of Bag1 / Sag1 markers as well as heterologous proteins. This is shown in Fig 1A and Fig 2b for example. However, in most time-dependent measurements of Ca-dependent fluorescence (Fig 2G, 3D the authors only average three cells. This appears to be insufficient to represent the bradyzoite population. How is the variance between the three measured cells?

    We have quantified more cells in all figures related to fluorescence measurements. For measurements of single parasites in Figure 5B, 5D, 5E, 6F, 8A, 8B and Figure 7 supplement 1A, we have now quantified 10 parasites for each condition and plotted the data as means ±S.D. to show the variance. For in vitro induced cysts or ex vivo cysts in Figure Fig 2G, 3D, 3E, 4C,4G, 6E, 7B and Figure 4 supplement 1A, we measured 5 cysts or vacuoles per condition. Because these samples contain many parasites within each vacuole or cyst, they represent a greater sample size. The data are also plotted a means ±S.D.

    In addition, the Mic2 promoter driven Gluc-myc protein is not expressed in all bradyzoites. This is perhaps not suprising as Mic2 seems to be downregulated in bradyzoites according to Pittman and Bucholz et al dataset in ToxoDB. If interpreted correctly the lower expression of Gluc in some bradyzoites would favour an underestimation of the RLUs in Fig 2D.

    We acknowledge that the expression and localization of MIC2-Gluc reporter is a potential concern. We performed western blotting (Figure 2C) and IFA (Figure 2 supplement 1A) to confirm that bradyzoites express MIC2-Gluc and M2AP albeit at lower levels compared with tachyzoites. Moreover, MIC2-GLuc and M2AP were properly co-localized to the apical end in bradyzoites, ruling out the possibility of mis-localization of the MIC2-GLuc reporter. Based on these results, we believe that MIC2-GLuc provides a reliable read-out for microneme secretion in in vitro differentiated bradyzoites. Additionally, the conclusion that MIC secretion is dampened in bradyzoites is also supported by the studies using the FNR-Cherry reporter in Figure 2E,F,G.

    The maturation of bradyzoite takes several weeks. This cannot be accomplished with currently available system in vitro and the authors use 1 week matured bradyzoites. To facilitate comparability to data from other manuscripts it would be helpful if the authors could quantify the differentiation stage of the in vitro bradyzoites. This could be done by measuring the fractions of Bag1-positive and Sag1-negative bradyzoites.

    We thank the reviewer for this useful comment. We have quantified the percentage of BAG1-positive SAG1-negative bradyzoites within each cyst induced for 3, 5 or 7 days by IFA and spinning disc confocal microscopy (Figure 3 supplement 1A). This analysis demonstrated that the percentage of BAG1-positive and SAG1-negative bradyzoites reached ~70% at day 7 after induction (Figure 3 supplement 1B). For this reason, we used a 7 day induction treatment for the majority of experiments. Also, where imaging was used in the analysis, we focused on regions of in vitro differentiated cysts that expressed high levels of BAG1-mCherry.

    The mcherry and GCaMP6f signal in fig 3B seem mutually exclusive. This may be due to difference in calcium signalling between Bag1 pos or neg parasites or due to expression differences of GCaMP6f.

    To test the possibility of expression differences in GCaMP6f, we quantified the fluorescence of BAG1-mCherry and GCaMP6f in different bradyzoites within the cyst shown in Figure 3B. At time 0 prior to stimulation, we observed heterogenous expression of BAG1- mCherry while the signal for GCaMP6f expression was relatively constant (Figure 3B supplement 1C and 1D). In contrast, when in vitro differentiated bradyzoites were stimulated with A23187, they showed reduced levels of GCaMP expression in cells that were strongly positive for BAG1-mCherry (Figure 3B). Collectively, these findings are consistent with the difference in GCaMP fluorescence being due to dampened calcium responses in bradyzoites rather than expression differences. This conclusion is supported by studies on GCaMP responses in cells where we normalized for expression level using a dual-expression BFP reporter in Figure 6. Therefore, we do not think that heterogeneity in the expression of GCaMP is responsible for the observed dampened response in bradyzoites.

    The authors use syringe, trypsin-released and FACS sorted bradyzoites in multiple Ca assays. How can it be excluded that this procedure affects (depletes) Ca stores?

    In all the figures except Figure 2C-2D, we did not use FACS to sort bradyzoites. Instead, we scraped cells cultured at pH 8.2, used syringe passage through 25g needle followed by centrifugation. Cyst pellets were resuspended and digested with trypsin to liberate bradyzoites. For tachyzoites, all procedures were similar except that we did not use trypsin digestion. As a control, we have now treated tachyzoites similarly with trypsin and monitored the calcium stores using ionomycin. We found that trypsin digestion did not affect the calcium stores or response as shown in Figure 7 figure supplement 1A.

    In my opinion several experiments in this manuscript would benefit from clarification of this point. For example: In Fig 7A Fu et al measure Ca for 5min during trypsin digestion, however, for gliding assays cysts are digested for 10min. The Ca monitoring should cover the complete 10min off trypsin digest.

    We understand the concern but there were practical reasons for the slightly different times used. In panel A where we are monitoring calcium during trypsin digestion, the majority of cysts are dispersed after 5 min resulting the parasites being out of focus. As such, it is not practical to monitor beyond this time point. In the panel C, we were interested in observing parasites after the cysts where fully digested and hence we used a slightly longer time period to allow complete digestion and for the parasites to settle to the bottom of the dish before further recording. In this instance, similar to the result in A, most parasites remained dormant and did not show elevated calcium levels. In the figure, we are selectively showing a rare example where calcium signaling was observed in order to compare the patterns to what is normally observed with tachyzoites. These combined panels are not meant to be a comparison of kinetics, as this aspect is tested more directly in later experiments. We have modified the text to make the rationale for this experiment clear.

    In Fig 2B Fu et al digest infected monolayers with trypsin to release mcherry from cysts matrices. How can the authors exclude that trypsin is not digesting mCherry protein in this assay?

    I think the reviewer means 2F as in 2B we are using BAG1 mCherry to visualize bradyzoites – but they are not being liberated in this image. In 2F we use a different construct, FnR-mCherry that directs the reporter to be constitutively secreted to either the PV (surrounding tachyzoites) or the cyst matrix (surrounding bradyzoites). When the cysts are disrupted with trypsin, the mCherry is likely to disperse and may also be digested. However, this would not happen if it remains inside the parasite. This control is provided to show that the protein is secreted into the matrix. We have revised the text to clarify the use of this control.

    Fig 7 E,F: the authors measure shorter gliding distances of bradyzoite as compared to tachyzoites. Trails of both parasites however, are detected by visualizing using different antigens that may have different shedding behavior on the FBS-coated glass surface. The Bag1 trail also depends on Bag1 expression, which is shown in numerous images to not be equal among individual bradyzoites. This point is very challenging to address but should at least be discussed.

    BAG1 is used here to discern the bradyzoites, not to detect the trail. Trails are stained with either SAG1 or SRS9 – corresponding to the most abundant surface GPI anchored antigen in each stage. Since these proteins are part of the same C-C fold family and are similarly anchored, we feel they are comparable. We have added the following statement to the results: “These two surface markers are both members of the cysteine rich SRS family that are tethered to the surface membrane by a GPI anchor, thus they represent comparable reporters for each stage.”

    Fig 7E: Bradyzoites are considered to satisfy their ATP needs mostly via glycolysis and the data shown do support this capability. I find the ability of OligomycinA to block glucose-dependent gliding surprising as this suggests a necessary mitochondrial transport chain for ATP-production from glucose. This result should be mentioned clearly in the text and its implications discussed.

    The Discussion has been revised as suggested.

    Figure 8: The authors claim a recovery of bradyzoite ATP and Ca levels after 1hr incubation with carbon sources and Ca, that together enable efficient gliding. However, the elevation of bradyzoite ATP occurs after the parasites spend 2 hours in glucose-free and Ca-free conditions, whereas gliding assays are done after a short 10min trypsin digest. I am not entirely convinced that low ATP levels post-egress are responsible for the low gliding activity. Ideally gliding assays should be done after a similar purification procedure to correlate the two experiments.

    We have repeated the gliding assays using bradyzoites purified in the same manner as for the ATP measurements and found the same result that a combination of exogenous calcium and glucose enhance recovery of gliding motility (Figure 8D, 8F). In addition, we used the same time point to purify bradyzoites for MIC2-Gluc secretion and found exogenous calcium and glucose also led to an increase in MIC2-GLuc secretion, indicative of the recovery of microneme secretion (Figure 8C).

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  2. Evaluation Summary:

    The cyst-forming stages of Toxoplasma gondii that perpetuate chonic infections in more than a quarter of the world's human population exist in a metabolically quiescent state. This study provides evidence that metabolic quiescence in bradyzoite cysts is associated with a profound dampening of calcium signalling, including uptake and release from internal stores, which is reversed following bradyzoite egress and exposure to exogenous calcium and carbon sources.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This study investigates calcium signalling in Toxoplasma gondii bradyzoites, which form persistent cysts in brain and muscle tissues in more than a quarter of the world's human population. Unlike the fast growing (and intensively studied) tachyzoites, relatively little is known about the signalling pathways that regulate the intracellular growth and host cell egress of these slow growing, metabolically quiescent stages. The authors use a variety of different approaches to stimulate calcium signalling and monitor calcium transients in bradyzoites stages and show that every aspect of calcium signalling is repressed in these stages. They show that repressed calcium signalling is associated with reduced motility and microneme secretion (important for infection of new host cells) and that calcium uptake and replenishment of stores occurs rapidly after egress and exposure to exogenous calcium and either glucose or glutamine carbon sources. Based on these findings, it is proposed that repression of calcium signalling might be intimately linked to metabolic quiescence (or visa versa). This is a very nice and carefully performed study which utilizes multiple agonists/inhibitors, reporter lines and calcium sensors together with advanced imaging techniques to undertake the comparative analysis of calcium signalling in tachyzoites and bradyzoite stages. A major strength of the study is the use of bradyzoites from different sources e.g. in vitro differentiated in fibroblasts to more 'physiologically' relevant bradyzoites formed in myoblasts or isolated from murine brain lesions. The conclusions are generally justified. One point that is not addressed is the source of energy used by bradyzoites to egress naturally (before they are exposed to high concentrations of extracellular glucose/glutamine). The possibility that bradyzoites mobilize internal carbohydrate stores is strongly suggesed by the finding that bradyzoite motility is increased in vitro in the presence of exogenous calcium but no glucose/glutamine. In this context, it would be important to discuss the possible role of calcium signalling in amylopectin mobilization and activation of glycolysis/OxPhos from internal stores.

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  4. Reviewer #2 (Public Review):

    Yu et al provide a comprehensive set of experiments to determine that bradyzoites have much slower cytosolic Ca2+ parameters, which impact on gliding motility, a key process of Toxoplasma spread and persistence.

    The only main criticism that I have is the use of the MIC2-GLuc reporter to measure microneme secretion in bradyzoites. Do bradyzoites have any appreciable level of MIC2 and its associated protein M2AP?? This is important that may affect the outcome. If bradyzoites do not, then the MIC2-GLuc reporter might not have appropriate levels of M2AP to correctly traffic to the micronemes. I recommend that the authors quantitate, either by western blot or IFA, the levels of MIC2 and M2AP in bradyzoites versus tachyzoites and also show that M2AP co-localises with MIC2-GLuc to give confidence that MIC2-GLuc is trafficked correctly and thus the low readings of secretion are not just a result of the reporter mistrafficked. It would also be pleasing to see, that 1hr incubation leads to restoration of MIC2-GLuc secretion.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    This is a first study that looks in detail at Ca-controlled gliding motility and ATP supply in bradyzoites. A comparison of such different parasite stage by manipulating Ca and ATP metabolism is challenging. Intervention by chemical compounds needs to overcome a prominent cyst wall and the usage of genetic tools needs to consider the broad changes in protein expression between tachyzoites and bradyzoites as well as a heterology between individual bradyzoites. The authors used excysted bradyzoites to exclude the cyst wall as a diffusion barrier as a major factor in the efficacy of different Ca agonists. To address differences in expression levels between tachyzoites and bradyzoite stages the authors developed a ratiometric Ca sensor based upon an autocleaved GCaMP6f-BFP dimer protein.

    Overall the conclusions are well supported but there are methodological questions that need to be addressed.

    Bradyzoites show a heterogenous expression of Bag1 / Sag1 markers as well as heterologous proteins. This is shown in Fig 1A and Fig 2b for example.

    However, in most time-dependent measurements of Ca-dependent fluorescence (Fig 2G, 3D the authors only average three cells. This appears to be insufficient to represent the bradyzoite population. How is the variance between the three measured cells?

    In addition, the Mic2 promoter driven Gluc-myc protein is not expressed in all bradyzoites. This is perhaps not suprising as Mic2 seems to be downregulated in bradyzoites according to Pittman and Bucholz et al dataset in ToxoDB. If interpreted correctly the lower expression of Gluc in some bradyzoites would favour an underestimation of the RLUs in Fig 2D.

    The maturation of bradyzoite takes several weeks. This cannot be accomplished with currently available system in vitro and the authors use 1 week matured bradyzoites. To facilitate comparability to data from other manuscripts it would be helpful if the authors could quantify the differentiation stage of the in vitro bradyzoites. This could be done by measuring the fractions of Bag1-positive and Sag1-negative bradyzoites.

    The mcherry and GCaMP6f signal in fig 3B seem mutually exclusive. This may be due to difference in calcium signalling between Bag1 pos or neg parasites or due to expression differences of GCaMP6f.

    The authors use syringe, trypsin-released and FACS sorted bradyzoites in multiple Ca assays. How can it be excluded that this procedure affects (depletes) Ca stores? In my opinion several experiments in this manuscript would benefit from clarification of this point. For example: In Fig 7A Fu et al measure Ca for 5min during trypsin digestion, however, for gliding assays cysts are digested for 10min. The Ca monitoring should cover the complete 10min off trypsin digest.

    In Fig 2B Fu et al digest infected monolayers with trypsin to release mcherry from cysts matrices. How can the authors exclude that trypsin is not digesting mCherry protein in this assay?

    Fig 7 E,F: the authors measure shorter gliding distances of bradyzoite as compared to tachyzoites. Trails of both parasites however, are detected by visualizing using different antigens that may have different shedding behavior on the FBS-coated glass surface. The Bag1 trail also depends on Bag1 expression, which is shown in numerous images to not be equal among individual bradyzoites. This point is very challenging to address but should at least be discussed.

    Fig 7E: Bradyzoites are considered to satisfy their ATP needs mostly via glycolysis and the data shown do support this capability. I find the ability of OligomycinA to block glucose-dependent gliding surprising as this suggests a necessary mitochondrial transport chain for ATP-production from glucose. This result should be mentioned clearly in the text and its implications discussed.

    Figure 8: The authors claim a recovery of bradyzoite ATP and Ca levels after 1hr incubation with carbon sources and Ca, that together enable efficient gliding. However, the elevation of bradyzoite ATP occurs after the parasites spend 2 hours in glucose-free and Ca-free conditions, whereas gliding assays are done after a short 10min trypsin digest. I am not entirely convinced that low ATP levels post-egress are responsible for the low gliding activity. Ideally gliding assays should be done after a similar purification procedure to correlate the two experiments.

    Was this evaluation helpful?