Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

  1. Simon Butterworth
  2. Francesca Torelli
  3. Eloise J. Lockyer
  4. Jeanette Wagener
  5. Ok-Ryul Song
  6. Malgorzata Broncel
  7. Matt R. G. Russell
  8. Joanna C. Young
  9. Moritz Treeck

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Abstract

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii , has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.

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    Reply to the reviewers

    We thank all reviewers for their input and suggestions.

    In the discussion section, the reviewers agreed on four major points which we have addressed as follows:

    *In vivo *validation of ROP1. We have now carried out mouse infections using the PRUΔKU80, PRUΔROP1, and complemented lines, showing that PRUΔROP1 is completely avirulent. This matches our *in vivo *CRISPR screen and in vitro IFNγ restriction assays results, and confirms that ROP1 is an important T. gondii virulence factor. We highlight the discrepancy with Soldati et al 1995, and suggest that this important role of ROP1 was previously overlooked in T. gondii RH due to the “hypervirulence” of this strain in laboratory mice. Clarify discrepancies in Irgb6 recruitment compared to published data: We revisited our image analysis pipeline for this data and corrected an error in the host cell segmentation step which was causing erroneously high calling of Irgb6 recruitment. The recruitment we now measure is now less variable and is consistent with published data, confirming that ROP1 does not affect Irgb6 recruitment or rhoptry bulb protein secretion. Conduct specific assays to measure host cell death or remove claims about this. We have carried out kinetic propidium iodide uptake assays as suggested by Reviewer #3. These have clarified that there is minimal parasite-induced host cell death at low MOI which cannot explain the host cell loss observed in the high-content imaging restriction assays. At an MOI of 0.3, ROP1 has no detectable effect on host cell death, while at a much higher MOI of 3, ROP1 knockout moderately increases cell death of PRU-infected BMDMs. Since cell death in macrophages is reported to result from exposure of parasite-derived PAMPS to host cytosolic sensors, we suggest that this increased host cell death at high MOI is a secondary effect of vacuole disrustion. Alternatively, these findings raise the interesting possibility of a differential phenotype for ROP1 at low versus high parasite burden. Queries regarding the restriction assays in C1QBPflox/flox/-/- MEFs. We included these data as we found statistically significant differences between the C1QBPflox/flox and C1QBP-/- MEFs that were dependent on the presence of ROP1 in the parasites. However, given the concerns raised by all three reviewers regarding the apparent lack of T. gondii restriction in these cells, we have withdrawn all conclusions relating to the putative role of C1QBP in parasite restriction. The data are included now in a supplementary figure only as a reference for other researchers working on the role of C1QBP in innate immunity who may use these previously published cell lines. We have made additional attempts to explore the link between ROP1 and C1QBP that have been unsuccessful, which are now mentioned in the discussion section. Although the co-immunoprecipitation of C1QBP with ROP1 is interesting, given the putative role of C1QBP in regulating autophagy and innate immune responses, further exploration of this potential interaction will require new tools and substantially more work that is beyond the scope of this manuscript. Further responses to comments raised by individual reviewers and details of further revisions are described below.

    Reviewer #1

    The authors identified ROP1 as a significant hit from their in vivo screen. However, they have not done any validation experiments using rop1 KO parasites in mice. Previous studies have shown no virulence defect in mice for rop1 KO in the type I background (PMID: 8719248). The result could be different in the type II strain used here, but this needs to be tested and shown.

    Soldati et al 1995 (PMID: 8719248) demonstrated that there was no virulence defect associated with ROP1 knockout in the RH strain. However, the RH strain is extremely virulent in most laboratory mice strains, which can mask phenotypes observed in a type II strain. We have now carried out in vivo infections of C57BL/6J mice using the type II Prugniaud strain, and have shown a severe virulence defect for PRUΔROP1 parasites which is rescued by complementation. This matches our in vivo CRISPR screen and in vitro IFNγ restriction assays results, and confirms that ROP1 is a virulence factor in vivo.

    The data in Fig. 2 and S3 do support that reduced parasitemia was due to decrease in number of vacuoles rather than their size or host cell death. However, it is important to control for invasion and/or egress differences of rop1 KO parasites in IFN-g activated cells.

    Soldati et al 1995 (PMID: 8719248) demonstrated that ROP1 has no role in invasion in HFFs, therefore it would be very surprising if ROP1 were to have a specific IFNγ-dependent role in invasion in macrophages. There is no involvement of rhoptry bulb-localised proteins in the predominant model of invasion, only rhoptry neck and microneme proteins.

    “Natural” egress after ~48 h would not occur here as the cells are fixed after 24 h. IFNγ-induced “early” egress has been documented in HFFs, A549s, and macrophages in vivo, and is apparent through increased host cell death/lysis (Niedelman et al 2013 PMID: 24042117, Rinkenberger et al 2021 PMID: 34871166, Tomita et al 2009 PMID: 19846885). We have now carried out prodium iodide uptake assays to more accurately quantify parasite-induced host cell death, and find no differences between strains at an MOI of 0.3, the targeted MOI we use in our restriction assays. At a higher MOI of 3, we find a moderate increase in host cell death in PRUΔROP1-infected BMDMs, which we suggest results from increased exposure of parasite-derived PAMPs to cytosolic sensors (Fisch et al 2019 PMID: 31268602, Zhao et al 2009 PMID: 19197351). Alternatively, this increased host cell death could result from increased rates of early egress, or from direct inhibition of programmed cell death pathways.

    It is important and informative to depict absolute parasite/size when making multiple comparisons. For example, the data in Fig. 4E shows C1QBP-/- MEFs can clear both RH and PRU better than the WT. However the authors do not comment on what is the meaning of > 100% parasite numbers in IFN-g treated MEFs with respect to untreated in WT. Since the data are normalized, it is difficult to appreciate what the actual differences are. Additionally, C1QBP-/- MEFs show close to equal survival in control and IFN-g treated condition (approximately 100%). Is it correct to infer that C1QBP has no effect on parasite survival? This should be considered in light of the comment below on colocalisation of C1QBP.

    It is standard practise to show IFNγ-dependent restriction as a percentage of unstimulated cells as this reflects the hypothesis being tested and the statistical tests carried out, as for example in Wang et al 2020 PMID: 33067458, Matta et al 2019 PMID: 31413201, Gay et al 2016 PMID: 27503074, Bando et al 2018 PMID: 30283439, Fleckenstein et al 2012 PMID: 22802726. Absolute numbers are included in the supplementary data for further reference.

    The >100% survival observed in the C1QBPflox/flox and C1QBP-/- MEFs is puzzling. We conclude that these cell lines have largely lost the ability to restrict T. gondii parasites as a result of the immortalisation process and/or passage history, and what little restriction we observe is at the limit of detection in our assay. MEFs are otherwise known to restrict both RH and PRU parasites (Niedelman et al 2012 PMID: 22761577). We included these data as we found statistically significant differences between the C1QBPflox/flox and C1QBP-/- MEFs which were dependent on the presence of ROP1 in the parasites. However, after the concerns raised by all three reviewers we agree that it is better to not to draw conclusions from these assays given the lack of parasite restriction. We will include these data only in the supplementary figures as a reference for other researchers working on the role of C1QBP in innate immunity who may use these previously published cell lines.

    The authors observed good restriction of both RH and PRU in IFN-g activated THP1s without cell death (Fig S1D). It is important to incorporate this information into the main result and discuss their implications in contrast to a previous report from 2019 (Fisch et al, PMID: 31268602).

    As mentioned above, we have carried out propidium iodide uptake assays to address questions regarding host cell death more precisely, which are now included as a main figure in the revised manuscript. While Fisch et al measured host cell death during infection of stimulated THP-1s at an MOI of 3, our restriction assays were carried out at a targeted MOI of 0.3. The Howard lab has shown that host cell death is directly proportional to MOI in BMDMs (Zhao et al 2009 PMID: 19197351), and we also find that at an MOI of 0.3 there is little detectable host cell death in BMDMs. While we are not aware of a similar study in THP-1s, it is likely also the case that at low MOIs there is little detectable cell death.

    The authors should consider conducting C1QBP functional assays to explore potential roles in parasite survival/growth within host cells. For example, it would be informative to measure the extent of autophagy or transcriptomic profile of the KO to deduce or suggest possible mechanisms of restriction.

    All reviewers have raised concerns regarding the restriction assays in the C1QBPflox/flox/C1QBP-/- MEFs, particularly that they do not appear to restrict parasite growth as would be expected. As a result, we have decided to withdraw conclusions from these assays regarding the role of C1QBP. For the same reason, we feel that further functional assays using these cells would be of limited value. As an alternative, we attempted siRNA-mediated knockdown of C1QBP in primary BMDMs using a pool of three commercial siRNAs, but were able to achieve only

    The authors state in their text that "C1QBP localised primarily to the mitochondria (Figure S7A) and therefore did not see any co-localisation with ROP1". The authors should discuss in more detail as this finding seems to contradict the interaction studies. Is there any independent evidence to corroborate the interaction studies and show they are not simply an in vitro artifact?

    We have now added immunofluorescence images of C1QBP and ROP1 in infected MEFs and HFFs to the main figure and discuss this in further detail. We find that C1QBP primary localises to the mitochondria, therefore there is some overlap with ROP1 signal at the PVM in RH as type I strains recruit mitochondria to the vacuole via MAF1B (Pernas et al 2014 PMID: 24781109, Adomako-Ankomah et al 2016 PMID: 26920761). However, type II strains do not recruit host mitochondria therefore we see little overlap with ROP1 in PRU. Furthermore, the precise localisation of C1QBP is a matter of some debate, such that it is unclear whether ROP1 and C1QBP are topologically able to interact. One study reported that C1QBP is exclusively localised to the mitochondrial matrix and therefore would not be able to interact with ROP1 even when the mitochondria are recruited to the vacuole (Muta et al 1997 PMID: 9305894). Others have asserted that there is an additional cytosolic pool of C1QBP which can be recruited to the outer membrane of the mitochondria, thus allowing interaction with ROP1 immediately following rhoptry secretion into the cytosol or at the PVM (Xu et al 2009 PMID: 19164550). From these IFAs we are therefore unable to draw any firm conclusions.

    We attempted proximity biotinylation to validate this potential interaction in cellulo, but C-terminal fusion of TurboID to ROP1 caused mislocalisation of the protein and prevented secretion of ROP1 to the parasitophorous vacuole. Based on the current evidence, we are unable to exclude that the interaction is an in vitro artefact as a result of cell lysis during the immunoprecipitation, and we clearly state this in the discussion. However, given this pulldown data is highly reproducible and technically sound, we believe it is important to include this result in the manuscript.

    The authors state in their text that "Enhanced restriction of Δrop1 parasites is primarily mediated through increased vacuole destruction". Their data is more suggestive of growth restriction. The authors should provide more direct data for destruction of the vacuoles or change the wording to indicate it is due to growth restriction.

    In the absence of specific results for vacuole destruction, which is technically challenging to determine quantitatively, we concluded by a process of elimination that restriction of ΔROP1 parasites at low MOI mostly likely occurs primarily through vacuole destruction, as we did not see strong evidence for vacuole size reduction and (now added in revision) do not see differences in parasite-induced host cell death. Reviewer #1 agrees in an earlier comment that vacuole destruction is the most likely mechanism. We note some subtle indications of strain- and host species-dependent differences, namely that RHΔROP1 parasites in THP-1 macrophages appear to have reduced vacuole size compared to RHΔUPRT (but not the complemented strain), but in all other cases the most likely mechanism consistent with our data is destruction of vacuoles. We have revised our conclusions to reflect that this is an inference from the data rather than a definitive finding.

    The authors should provide high resolution IFA images and decrease their size to an equivalent size of other sub-figures. Empty white space between sub-figures can be minimized. Font size of the figure labels/axis/titles should be matched and increased slightly.

    Thank you for these suggestions. Each IFA image is only 2x2 cm so we feel that decreasing their size further would make them difficult to see.

    Reviewer #2

    The title indicates a positive role for ROP1 in subverting innate immune restriction, but the data indicate that a deficiency in ROP1 causes susceptibility to innate immune restriction. This might seem like a subtle discord, but in the absence of identifying the mechanism of ROP1 subversion of innate immune restriction it seems more appropriate to provide a title that better reflects the findings i.e., that ROP1 deficient parasites are more susceptible to innate immune restriction.

    We recognise this point and have changed the title to reflect both this and new results added in revision: “*Toxoplasma gondii *virulence factor ROP1 reduces parasite susceptibility to murine and human innate immune restriction”

    The reference strains and complement strains lack UPRT, but from the available information it appears the KO strains have UPRT. If this is the case, it is necessary to rule out that the presence of UPRT doesn't render parasites more susceptible to IFNg mediated killing by performing additional experiments comparing RH∆ku80 with RH∆ku80∆uprt and Pru∆ku80 with Pru∆ku80∆uprt.

    This is correct, the reference and complemented strains lack UPRT while the ROP1 knockout strains have a functional UPRT. For the high-content imaging assay it was necessary to use a reference strain that expressed a fluorophore, as the knockout and complemented strains do, to allow for accurate segmentation of the parasites. We considered it preferable to insert the mCherry fluorophore at the well-established, non-essential UPRT locus rather than integrate it randomly. Complementation at the UPRT locus is widely used in the literature with no impact on virulence phenotypes: Shen et al 2014 (PMID: 24825012) Fig 4F, Wang et al 2020 (PMID: 33067458) Fig 5F, Wang et al 2020 (PMID: 31908049) Fig 5, Fox et al 2019 (PMID: 31266861) Fig 5, Olias et al 2016 (PMID: 27414498) Fig 6. Moreover, the genome-wide CRISPR knockout screen of Wang et al 2020 (PMID: 33067458) has also demonstrated that knockout of UPRT does not affect growth of RH parasites in IFNγ-stimulated vs. naive BMDMs. UPRT has 100% amino acid identity between RH and PRU so no functional differences are expected.

    Although it is understandable why the authors chose MEFs to test the role of C1QBP because MEFs were used for the Co-IP, the MEFs do not appear to be responding to IFNg for parasite growth restriction (-/+ IFNg % survival is at or above 100% for WT MEFs). As such, the authors are potentially blind to the role of C1QBP in the context of IFNg restriction. It would be ideal to repeat these experiments using BMDMs from WT and C1QBP KO mice to assess the potential contribution of C1QBP during IFNg restriction of T. gondii growth and survival.

    We agree that it would be preferable to use ΔC1QBP BMDMs for these experiments. However, knockout of C1QBP is lethal in mice (Yagi et al 2012, PMID: 22904065), therefore it is not possible to obtain primary ΔC1QBP BMDMs. We also attempted siRNA-mediated knockdown of C1QBP in wild-type primary BMDMs using a pool of three different siRNAs, but were only able to achieve

    Much of the data is analyzed with a paired two-sided t-test, but the authors used Bonferoni correction in some cases and Benjamini-Hochberg adjustment in other cases. It would be helpful to either consistently use the same correction or explain in a short section on stats in the methods the rationale for using different corrections.

    We have changed the manuscript to consistently use Benjamini-Hochberg correction for all tests. These correction methods represent different approaches to the multiple-testing problem: Bonferroni correction controls the family-wise error rate, while the Benjamini-Hochberg procedure controls the false-discovery rate. We favour the FDR-based approach as it is less dependent on the somewhat-arbitrary decision as to what constitutes a “family” of tests - for example: should tests in the RH and PRU strains be in the same family; should tests for number of parasites and number of vacuoles/host cells be in the same family; should the tests in BMDMs vs. THP-1s should be the same family. At the alpha level of 0.05, one in twenty significant results are false positives following Benjamini-Hochberg adjustment, as opposed to one in twenty of all tests carried out prior to adjustment. We find this appropriate for our study.

    The IFA images in Figure 2B appear to show considerable redistribution of ROP1 from the rhoptries to other parts of the parasite upon short Trition-X100 treatment of formaldehyde fixed samples. Inclusion of a low concentration of glutaraldehyde might help preserve the normal distribution of ROP1. Alternatively, or additionally, permeabilization with saponin or digitonin could help visualize ROP1 associated with the PVM. Improving the imaging is not critical to support the conclusions of the study, but would nevertheless be an asset.

    The ROP1 immunofluorescence staining within the parasites is likely from protein that is being trafficked through the ER or Golgi. This staining is more prominent with shorter Triton permeabilisation and in the complement lines. We have also observed PVM localisation of ROP1 using permeabilisation with 0.1% saponin for 15 min, but find that it is clearer and more consistent with the short Triton permeabilisation. We have added additional examples to the supplementary figure validating ROP1 knockout and complement.

    Reviewer #3

    The IFNg-dependent T. gondii control data as well as the Irgb6 recruitment data are extremely variable and preclude drawing any solid conclusions (Fig 2c-f, 3d, 4e-f, S3a-d, S8). The authors normalize the data by presenting the ratio of +IFNg/-IFNg in % of the measure they are analyzing. However, this does not "clean up" the data. The underlying cause for this problem are the extremely varied input, the time point analyzed and the nature of the microscopy experiment.

    We have found that parasite IFNγ-restriction assays are highly variable between biological replicates by all methods that we have tried. We thank Reviewer #1 for sharing their similar experience, as we have heard the same from several colleagues. As it becomes more common to plot individual data points rather than summary statistics, this is increasingly apparent in the literature (e.g. Wang et al 2020 Fig 2, Rinkenberger et al 2021). While we could select the three “best” replicates to publish or instead present only summary statistics to obscure the variation present, we do not feel that this would be in the best interest of the field or of open science. We address Reviewer #3’s specific concerns below.

    Presenting the data as a ratio/percentage of IFNγ-stimulated versus naive cells is not an attempt to “clean up” the data, it is standard practice in the field and reflects the hypothesis being tested. For example: Wang et al 2020 PMID: 33067458 (Saeij lab), Matta et al 2019 PMID: 31413201 (Sibley lab), Gay et al 2016 PMID: 27503074 (Hakimi lab), Bando et al 2018 PMID: 30283439 (Yamamoto lab), Fleckenstein et al 2012 PMID: 22802726 (Howard lab).

    - Varied input: looking at the Supplementary tables and calculating the input MOI in the -IFNg samples for the BMDM, for example, they range from 0.1-6. Most of the MOIs are in the 0.1-1 range, but this is still a huge variation and not the MOI of 0.3 the authors are aiming to add. Input host cells within one experiment often differ 5 fold between the T. gondii strains analyzed. Consistency in input is not achieved.

    The inputs of the restriction assays, as estimated by the parasite and host cell numbers in the unstimulated cells, are variable but not so extreme as Reviewer #3 says. Importantly, variability between strains within a biological replicate is far less than between biological replicates, and it is for this reason that we have analysed the results with paired tests. It is in fact a feature of microscopy-based assays that these sources of variability can be identified. We have attempted to be as open as possible with our data in reporting all of these measured parameters.

    MOI is the most appropriate measure of inter- and intra-replicate variability as it accounts for both host cell and vacuole number. We have now added estimated MOI as a column in the supplementary data files (S2B and S3B: MOI = “Vacuole - Minus IFNG Median”/”Host Nuclei - Minus IFNG Median”). Out of 49 data points, there are only three instances in the BMDM restriction assays where MOI >1 (1.26, 1.06, 1.42) and none in THP-1s. Because the MOIs are low, the range of MOI within each replicate is small. Furthermore, for MOI ≤1, we expect there will be little qualitative effect on restriction as the host cells will be infected with either 1 or 0 vacuoles. In our data, we do not find any correlation between MOI and restriction for any individual strain or for all strains combined.

    Regardless of the inter-replicate variability, the results for parasite restriction are statistically significant using an appropriate paired test. Our control parasite line PRUΔGRA12 behaves as expected (Fox et al 2019 PMID: 31266861) and differences between the RH and PRU strain in BMDMs dependent on the polymorphic effector ROP18 are apparent, although not tested here. While there is variation in the magnitude of restriction between replicates, there is a qualitative decrease in parasite survival in terms of total parasite number (Fig 2C and 2E) for ΔROP1 parasites compared to both ΔUPRT and the complemented lines in every replicate for both strains in BMDMs (7/7 replicates for each strain), and in all but one replicate in THP-1s (6/7 for RH, 7/7 for PRU). We consider that this restriction phenotype is highly robust.

    - Time point analyzed: the authors chose to analyze 24h p.i. IFNg-stimulated MEFs, BMDMs and THP-1s. All will have undergone a significant amount of cell death at this time point. The authors even present the varying host cell numbers in Fig S3: the +IFNg cell numbers vs -IFNg cell numbers in BMDMs range from 35-70% for example in RH; THP-1 seem slightly better but still there is a range of 70-120%.

    24 hpi or later is a standard timepoint at which to measure parasite restriction, as at earlier timepoints the dynamic range of the assays are reduced due to lower parasite numbers and therefore lower fluorescence, luminescence, or uracil uptake. For example, the following references all measure parasite restriction at at least 24 hpi: Wang et al 2020 Fig. 2: 24 hpi; Matta et al 2019 Fig. 3: 36-40 hpi; Gay et al 2016 Fig. 8: 36-48 hpi; Bando et al 2018: 24 hpi; Fleckenstein et al 2012 Fig. 4: 48 hpi.

    The Howard lab has shown that host cell death is directly proportional to MOI in murine cells (Zhao et al 2009 PMID: 19197351, Lilue et al 2013 PMID: 24175088). At low MOIs of

    - Nature of the experiment: Due to rampant host cell death at 24h p.i. analyzing the total parasite or vacuole number or even vacuole size is difficult in a microscopy experiment (dead cells will wash away even after fixation as they do not adhere anymore). Most laboratories (Howard, Yamamoto, Coers, Saeij, Steinfeldt etc), who study these mechanisms employ experimental systems that do not rely on washes/perturbations (plaque assays, plate reader T. gondii luciferase growth assays, uracil incorporation etc), or focus on the "number of parasites/vacuole" measure that are less dependent on host cell numbers (Saeij, Coers etc) or choose host cells that do not undergo IFNg-driven cell death (A549 cells, Sibley recent elife; only overexpression of the host factor induces cell death).

    High-content imaging has previously been used to characterise IFNγ-dependent restriction of T. gondii (Gay et al 2016 PMID: 27503074 Fig 8D, Rinkenberger et al 2021 PMID: 34871166, Fisch et al 2021 PMID: 34931666) and for anti-parasitic drug screening (Touquet et al 2018 PMID: 30157171). Certainly different methods have different strengths and weaknesses: for example, fixed imaging-based methods required washing which will remove dead host cells from the analysis, whereas e.g. luciferase or uracil incorporation assays do not; however, with imaging we can measure multiple potential phenotypes simultaneously and identify potential confounding factors that would be unknown in other assays. Acknowledging that every assay has specific weaknesses, we verify the role of ROP1 by orthogonal methods: in vivo CRISPR screen for growth in the mouse peritoneum, in vitro growth in IFNγ-stimulated macrophages, and (now added in revision) in vivo virulence studies. The results of all of these assays concur.

    If ROP1 alters the amount of host cell death induced in macrophages, host cell numbers in the +IFNg samples will vary in dependency of ROP1. Hence, in order to assess ROP1-dependent T. gondii restriction it is imperative to know if ROP1 alters host cell death. This can only be measured with cell death assays (total LDH or better a kinetic cell death assay) and not by looking at how many host cells remain after fixation in a microscopy experiment. To compare ROP1-driven parasite restriction, it is important to compare the same input MOI for each strain (-IFNg), determine a time point p.i. before host cell death has taken over and verify host cell numbers stay within a reasonable variation between strains. Input MOI consistency between T. gondii strains is typically measured by plaque viability assay.

    We have now carried out kinetic propidium iodide uptake assays to determine formally whether ROP1 affects host cell death in BMDMs, which have been added to the revised manuscript. At an MOI of 0.3, as targeted in our restriction assays, there is very little parasite-induced cell death and no significant differences between parasite strains. This is in line with the results from the Howard lab mentioned above (Zhao et al 2009 PMID: 19197351, Lilue et al 2013 PMID: 24175088), and confirms that the majority of host cell loss observed in the high-content imaging assays at an average MOI of 0.3 is not parasite-induced.

    We also carried out propidium iodide uptake assays at an MOI of 3, and found a slight but significant increase in host cell death for PRUΔROP1 in BMDMs of 10-15%. A small increase was also observed for RHΔROP1 compared to the complemented line, but was not significant compared to RHΔUPRT. This differential effect at low and high MOI is interesting, although we cannot rule out that a small phenotype is beyond the limit of detection at low MOI. We conclude that at low MOI host cell death does not have a detectable role in the restriction of ΔROP1 parasites, leaving the most likely mechanism as disruption of vacuoles. At high MOI, vacuole breakage appears to result in host cell death as host cytosolic sensors recognise parasite-derived PAMPs: parasite DNA is sensed by AIM2 in activated THP-1 macrophages leading to apoptosis (Fisch et al 2019 PMID: 31268602), while in murine cells vacuole breakage by the IRGs leads to necrotic cell death (Zhao et al 2009 PMID: 19197351). Increased cell death at high MOI is therefore also consistent with increased disruption of vacuoles.

    Fig 2C - Why are there datapoints with more than 100% T. gondii in +IFNg vs -IFNg samples? In these cases, no IFNg restriction was observed for the WT strain. This data is not reliable, as it has been demonstrated before that BMDMs control RH in an IFNg-dependent fashion.

    This is one replicate out of seven. Although removing this replicate would not affect the statistical significance of the results in terms of total parasite number or number of vacuoles, we do not consider it appropriate to remove data because it does not match our expectations. Published data also show survival of RH parasites in BMDMs at or close to 100% in some replicates as a result of biological and technical variability (e.g. Wang et al 2020 PMID: 33067458, Fig. 2), which is to be expected in primary cells derived from different donor mice. The average survival we determine for RH across replicates in ~75%, which is in line with published data.

    Fig 4C - The description of the IP in the legends and the materials is incomplete. In the materials it only states the loading of the IP fraction. Is the supernatant the post-IP fraction? 202200-HA is not described in the Figure legend. Why is the IP'ed version of C1QBP smaller than the supernatant version?

    We will clarify this in the figure legend and methods. The supernatant is the post-IP fraction. It was necessary to load as much material as possible to detect C1QBP in the post-IP supernatant of the RHΔKU80 and PRUΔKU80-infected samples, but as the protein concentration in the supernatant is far higher than in the IP fraction this appears to have caused slight retardation of protein migration in these lanes. This is only apparent for C1QBP as it is the only protein detected in both supernatant and IP.

    Fig 4E and F - There is almost no IFNg-dependent restriction in the WT MEFs for any of the parasite strains (4E). Some of the data even shows a dramatic increase of parasite load with IFNg versus without IFNg. Hence, no conclusion can be drawn about the function of C1QBP and making a ratio of KO vs WT (Fig 4F) is not justified as there was no IFNg restriction to begin with in WT cells.

    We included these data as we found statistically significant differences between the C1QBPflox/flox and C1QBP-/- MEFs that were dependent on the presence of ROP1 in the parasites. However, after the concerns raised by all three reviewers we agree that it is better to not to draw conclusions from these assays given the lack of parasite restriction that would otherwise be expected in MEFs (e.g. Niedelman et al 2012 PMID: 22761577). We will include these data only in the supplementary figures as a reference for other researchers working on the role of C1QBP in innate immunity who may use these previously published cell lines.

    Fig 3D - Something is wrong with the Irgb6 recruitment data. Many labs have shown that RH recruitment of Irgb6 is at most 10% due to the activity of ROP5 and ROP18. The authors get up to 40% recruitment for RH and again the data ranges from 5-42%, that's a huge variation!

    Recruitment of IRGB6 was determined by the ratio of the median fluorescence intensity in a 6 pixel radius around the parasites versus the median intensity in the rest of the infected cell cytoplasm. After carefully checking each step of this analysis pipeline, we found that the threshold for host cell cytoplasm segmentation based on CellMask staining was set too low and was including areas of empty space. This resulted in erroneously low median intensity in the host cell and so false-positive calling of IRGB6 recruitment to some vacuoles. We have corrected the cut-off threshold for host cell segmentation and reanalysed all the data with this corrected script. We now find average recruitment of ~5% for RHΔUPRT, ~15% for RHΔROP18, and ~60% for PRUΔUPRT (comparable to e.g. Fentress et al 2011, Fleckenstein et al 2012, Niedelman et al 2012, and Etheridge et al 2014), as well as less variability overall.

    Fig S3A and C: The authors conclude that ROP1 does not affect vacuole size. I disagree. The data for THP-1 is significant. However, due to the noisy input with such varied MOI and varied host cell numbers, at the moment, no solid conclusion can be drawn.

    Although there is a significant difference between RHΔUPRT and RHΔROP1, this is not rescued by complementation so we are hesitant to claim this as a phenotype of ROP1. There are no significant differences in the PRU strain. We highlight this in the text of the results section, as it may be an important strain- or host species-dependent mechanism.

    Fig S3B and D: The data clearly show huge variation in host cell numbers depending on IFNg and T. gondii infection. It is well-known that host cell death occurs in these experimental systems. In order to analyze whether ROP1 impacts host cell death a kinetic cell death assay is needed. Assessment of remaining host cell numbers in a 96 well plate microscopy experiment is not a quantitative assessment of host cell death, so the conclusion is not valid.

    Please refer to comments on host cell death above.

    T. gondii "type" is always spelt with a lowercase "t" by convention.

    We have corrected this.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors identify 22 putative T. gondii virulence factors in an in vivo CRISPR screen and aim to analyze ROP1 as one of the hits for its contribution and mechanism in IFNg-driven parasite control mostly in murine and human macrophages. Somewhat disappointingly, most of the data are too variable to draw conclusions. The underlying cause for the variability in the data are the divergent input host cell numbers within the same experiment, variable MOI and most importantly, probably the loss of host cells due to cell death (the infected ones wash away more easily). The authors try to remedy this by pooling and normalizing data, and while there may be a trend to ROP1 counteracting IFNg-driven T. gondii control (Fig 2), the result overall is not convincing. Even if more robust data would confirm the role of ROP1 in innate immune evasion, all mechanistic "tries" the authors undertake in this study show no phenotype (Fig 3 and S6) or are unreliable (Fig 4, see comments below).

    Major concern:

    The IFNg-dependent T. gondii control data as well as the Irgb6 recruitment data are extremely variable and preclude drawing any solid conclusions (Fig 2c-f, 3d, 4e-f, S3a-d, S8). The authors normalize the data by presenting the ratio of +IFNg/-IFNg in % of the measure they are analyzing. However, this does not "clean up" the data. The underlying cause for this problem are the extremely varied input, the time point analyzed and the nature of the microscopy experiment.

    • Varied input: looking at the Supplementary tables and calculating the input MOI in the -IFNg samples for the BMDM, for example, they range from 0.1-6. Most of the MOIs are in the 0.1-1 range, but this is still a huge variation and not the MOI of 0.3 the authors are aiming to add. Input host cells within one experiment often differ 5 fold between the T. gondii strains analyzed. Consistency in input is not achieved.
    • Time point analyzed: the authors chose to analyze 24h p.i. IFNg-stimulated MEFs, BMDMs and THP-1s. All will have undergone a significant amount of cell death at this time point. The authors even present the varying host cell numbers in Fig S3: the +IFNg cell numbers vs -IFNg cell numbers in BMDMs range from 35-70% for example in RH; THP-1 seem slightly better but still there is a range of 70-120%.
    • Nature of the experiment: Due to rampant host cell death at 24h p.i. analyzing the total parasite or vacuole number or even vacuole size is difficult in a microscopy experiment (dead cells will wash away even after fixation as they do not adhere anymore). Most laboratories (Howard, Yamamoto, Coers, Saeij, Steinfeldt etc), who study these mechanisms employ experimental systems that do not rely on washes/perturbations (plaque assays, plate reader T. gondii luciferase growth assays, uracil incorporation etc), or focus on the "number of parasites/vacuole" measure that are less dependent on host cell numbers (Saeij, Coers etc) or choose host cells that do not undergo IFNg-driven cell death (A549 cells, Sibley recent elife; only overexpression of the host factor induces cell death). If ROP1 alters the amount of host cell death induced in macrophages, host cell numbers in the +IFNg samples will vary in dependency of ROP1. Hence, in order to assess ROP1-dependent T. gondii restriction it is imperative to know if ROP1 alters host cell death. This can only be measured with cell death assays (total LDH or better a kinetic cell death assay) and not by looking at how many host cells remain after fixation in a microscopy experiment. To compare ROP1-driven parasite restriction, it is important to compare the same input MOI for each strain (-IFNg), determine a time point p.i. before host cell death has taken over and verify host cell numbers stay within a reasonable variation between strains. Input MOI consistency between T. gondii strains is typically measured by plaque viability assay.

    Other specific major comments:

    Fig 2C - Why are there datapoints with more than 100% T. gondii in +IFNg vs -IFNg samples? In these cases, no IFNg restriction was observed for the WT strain. This data is not reliable, as it has been demonstrated before that BMDMs control RH in an IFNg-dependent fashion.

    Fig 4C - The description of the IP in the legends and the materials is incomplete. In the materials it only states the loading of the IP fraction. Is the supernatant the post-IP fraction? 202200-HA is not described in the Figure legend. Why is the IP'ed version of C1QBP smaller than the supernatant version?

    Fig 4E and F - There is almost no IFNg-dependent restriction in the WT MEFs for any of the parasite strains (4E). Some of the data even shows a dramatic increase of parasite load with IFNg versus without IFNg. Hence, no conclusion can be drawn about the function of C1QBP and making a ratio of KO vs WT (Fig 4F) is not justified as there was no IFNg restriction to begin with in WT cells.

    Fig 3D - Something is wrong with the Irgb6 recruitment data. Many labs have shown that RH recruitment of Irgb6 is at most 10% due to the activity of ROP5 and ROP18. The authors get up to 40% recruitment for RH and again the data ranges from 5-42%, that's a huge variation!

    Fig S3A and C: The authors conclude that ROP1 does not affect vacuole size. I disagree. The data for THP-1 is significant. However, due to the noisy input with such varied MOI and varied host cell numbers, at the moment, no solid conclusion can be drawn.

    Fig S3B and D: The data clearly show huge variation in host cell numbers depending on IFNg and T. gondii infection. It is well-known that host cell death occurs in these experimental systems. In order to analyze whether ROP1 impacts host cell death a kinetic cell death assay is needed. Assessment of remaining host cell numbers in a 96 well plate microscopy experiment is not a quantitative assessment of host cell death, so the conclusion is not valid.

    Minor comments:

    T. gondii "type" is always spelt with a lower case "t" by convention.

    Significance

    T. gondii protein ROP1 was identified by the authors in an in vivo CRISPR screen as a potential effector protein mediating parasite resistance to innate immunity. The function of ROP1 is currently unknown. The data presented in this manuscript does not deliver conclusive evidence that ROP1 counteracts IFNg-driven immunity and a potential mechanism has not been uncovered.

    Referees cross-commenting

    This section contains comments of all reviewers

    Reviewer 1

    I agree with the comments of review #2 in regard to additional experiments needed for validation. I also agree with reviewer #3 about the variable data with Irgb6 recruitment and IFNg control- we have also done these assays and struggled with the variability. The suggestions for how to reduce variability with modified protocols are good - but is our role really to instruct them how to do the experiments? It seems you are asking them to start over, and I am not sure the situation is that bleak. I would be more inclined to allow them to claim only features that are clearly supported within the variability of the current assays. Perhaps that weakens the conclusions they can make and hence ultimate decision - but I feel that this choice (of fixing it or compromising) should be up to the authors.

    Reviewer 3

    Thanks for your points, yes I agree, statements can be made about the role of ROP1 in innate immune defence (i.e. it probably contributes to control of vacuole numbers and size in both mouse and human), but the authors should more carefully place them in the framework of their assays.

    Rev 2, point 1 is very good (title suggestion). Rev 1, point 1, I agree very much (validate ROP1 phenotype in vivo). Rev 2, minor point 1 (explain your stats) and Rev 1, point 3 (depict absolute numbers in comparisons). This is my point also, the authors try to normalise the data and apply varied statistics to smooth over the variability. All reviewers pointed out the problems with the C1QBP data, specifically the >100% data of +IFNg vs -IFNg and additionally the potential for an in vitro artefact (Rev 1).

    My main points remain:

    • statements on ROP1 and its impact on host cell death without conducting any host cell death assay cannot be made.
    • the Irgb6 recruitment data is puzzling and contradicts all the many published data on Irgb6 recruitment levels.
    • C1QBP data is not interpretable with data where IFNg does not restriction WT parasites.

    In the end, maybe suggesting in vivo validation of ROP1, remove impact on host cell death claim and the C1QBP data (or conduct further experiments to understand role)?

    Reviewer 1

    Yes I agree with the points and this slightly reworded final assessment:

    Request in vivo validation of ROP1 Clarify discrepancies in Irgb6 data Remove impact on host cell death claim Soften the conclusions about C1QBP data (or conduct further experiments to understand its role).

    Reviewer 2

    Thanks for leading the discussion R1 and R3. It looks like we are in good agreement. The study has some merit but will require more in vitro and in vivo experiments to support the conclusions and to substantiate the relationship between ROP1 and C1QBP.

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    Referee #2

    Evidence, reproducibility and clarity

    Toxoplasma gondii secretes a variety of effector proteins that manipulate many aspects of host cell physiology including several that facilitate evasion of cell autonomous immunity due to activation by interferon gamma. The current study features a new CRISPR gRNA library targeting genes encoding proteins that are released from two types of secretory organelles that have previously been strongly implicated in immune evasion, namely rhoptries and dense granules. The authors use the library to identify parasite genes that are more important to in vivo infection than they are suring in vitro culture. That the screen identified several known immune evasion genes suggests that the experiment worked as intended. Among the somewhat marginal hits, the authors identified ROP1, which they pursued further since the function of this protein has remained unknown despite being the first identified ROP protein. As rigorous component of the study, the authors disrupt and restore expression of ROP1 in two different strains (RH and Pru) and identify ROP1 knockout parasite susceptibility to IFNg stimulation in two different types of macrophages (murine BMDMs and human THP-1). Two functional assays were used to indicate that rhoptry secretion is not impaired in parasites lacking ROP1. Co-immunoprecipitation assays suggest an interaction between ROP1 and a multifunctional host protein, C1QBP, inspiring the hypothesis that ROP1 targets C1QBP to evade cell autonomous immunity. However, host cells lacking C1QBP appear to be more restrictive to growth of WT parasites and neutral to ROP1 deficient parasites, implying that C1QBP contributes to T. gondii survival in a manner that is dependent on ROP1. The authors are appropriately cautious when discussing the extent to which the function of ROP1 is mechanistically linked to C1QBP. Overall, the study provides some new evidence for a role of ROP1 in limiting IFNg dependent clearance of T. gondii and identifies some clues to potential mechanism without being able to nail down this aspect.

    Main comments

    1. The title indicates a positive role for ROP1 in subverting innate immune restriction, but the data indicate that a deficiency in ROP1 causes susceptibility to innate immune restriction. This might seem like a subtle discord, but in the absence of identifying the mechanism of ROP1 subversion of innate immune restriction it seems more appropriate to provide a title the better reflects the findings i.e., that ROP1 deficient parasites are more susceptible to innate immune restriction.
    2. The reference strains and complement strains lack UPRT, but from the available information it appears the KO strains have UPRT. If this is the case, it is necessary to rule out that the presence of UPRT doesn't render parasites more susceptible to IFNg mediated killing by performing additional experiments comparing RH∆ku80 with RH∆ku80∆uprt and Pru∆ku80 with Pru∆ku80∆uprt.
    3. Although it is understandable why the authors chose MEFs to test the role of C1QBP because MEFs were used for the Co-IP, the MEFs do not appear to be responding to IFNg for parasite growth restriction (-/+ IFNg % survival is at or above 100% for WT MEFs). As such, the authors are potentially blind to the role of C1QBP in the context of IFNg restriction. It would be ideal to repeat these experiments using BMDMs from WT and C1QBP KO mice to assess the potential contribution of C1QBP during IFNg restriction of T. gondii growth and survival.

    Minor comments

    1. Much of the data is analyzed with a paired two-sided t-test, but the authors used Bonferonni correction in some cases and Benjamini-Hochberg adjustment in other cases. It would be helpful to either consistently use the same correction or explain in a short section on stats in the methods the rationale for using different corrections.
    2. The IFA images in Figure 2B appear to show considerable redistribution of ROP1 from the rhoptries to other parts of the parasite upon short Trition-X100 treatment of formaldehyde fixed samples. Inclusion of a low concentration of glutaraldehyde might help preserve the normal distribution of ROP1. Alternatively, or additionally, permeabilization with saponin or digitonin could help visualize ROP1 associated with the PVM. Improving the imaging is not critical to support the conclusions of the study, but would nevertheless be an asset.

    Significance

    Immune evasion is critical to the survival of pathogens including Toxoplasma gondii, yet the effector proteins responsible for such evasion remain incompletely identified or understood. This study identifies a role for ROP1 in parasite survival in interferon gamma stimulated host cells, thus addressing a long standing question of this protein's function during infection. The study appears suited to a microbiology or infection themed journal. My areas of expertise are T. gondii pathogenesis, virulence, secretion, invasion, egress, resource acquisition, and persistence.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Butterworth et al identify a novel function of Toxoplasma ROP1 being involved in subverting host IFN-g restriction of parasite growth. The authors executed a CRISPR-Cas9 sgRNA screen in a type II strain parasite targeting a sub-pool of genes characterized with respect to their localization in rhoptries and dense-granules. They adopted an approach of sequentially sequencing the survivors following growth in vitro in human foreskin fibroblasts followed by in vivo expansion in mouse peritoneum. Measurement of relative depletion of sgRNA sequences produced significant hits of factors involved in parasite survival. They identified many parasite proteins previously known to be involved in different aspects of parasite virulence including those involved in countering murine IFN-g mediated anti-toxoplasma response. They then focused on identifying the function of ROP1 in augmenting virulence and survival of the parasite in mice. Lastly, using immunoprecipitation in mouse cells they identified host C1QBP as a protein that may be involved in facilitating ROP1 mediated parasite resistance against the IFN-g response.

    Major Comments:

    1. The authors identified ROP1 as a significant hit from their in vivo screen. However, they have not done any validation experiments using rop1 KO parasites in mice. Previous studies have shown no virulence defect in mice for rop1 KO in the type I background (PMID: 8719248). The result could be different in the type II strain used here, but this needs to be tested and shown.
    2. The data in Fig. 2 and S3 do support that reduced parasitemia was due to decrease in number of vacuoles rather than their size or host cell death. However, it is important to control for invasion and/or egress differences of rop1 KO parasites in IFN-g activated cells.
    3. It is important and informative to depict absolute parasite/size when making multiple comparisons. For example, the data in Fig. 4E shows C1QBP-/- MEFs can clear both RH and PRU better than the WT. However the authors do not comment on what is the meaning of > 100% parasite numbers in IFN-g treated MEFs with respect to untreated in WT. Since the data re normalized, it is difficult o appreciate what the actual differences are. Additionally, C1QBP-/- MEFs show close to equal survival in control and IFN-g treated condition (approximately 100%). Is it correct to infer that C1QBP has no effect on parasite survival? This should be considered in light of the comment below on colocalisation of C1BQ.
    4. The authors observed good restriction of both RH and PRU in IFN-g activated THP1s without cell death (Fig S1D). It is important to incorporate this information into the main result and discuss their implications in contrast to a previous report from 2019 (Fisch et al, PMID: 31268602).
    5. The authors should consider conducting C1QBP functional assays to explore potential roles in parasite survival/growth within host cells. For example, it would be informative to measure the extent of autophagy or transcriptomic profile of the KO to deduce or suggest possible mechanism of restriction.

    Minor Comments:

    1. The authors state in their text that "C1QBP localised primarily to the mitochondria (Figure S7A) and therefore did not see any co-localisation with ROP1". The authors should discuss in more detail as this finding seems to contradict the interaction studies. Is there any independent evidence to corroborate the interaction studies and show they are not simply an in vitro artifact?
    2. The authors state in their text that "Enhanced restriction of Δrop1 parasites is primarily mediated through increased vacuole destruction". Their data is more suggestive of growth restriction. The authors should provide more direct data for destruction of the vacuoles or change the wording to indicate it is due to growth restriction.
    3. The authors should provide high resolution IFA images and decrease their size to an equivalent size of other sub-figures. Empty white space between sub-figures can be minimized. Font size of the figure labels/axis/titles should be matched and increased slightly.

    Significance

    The work has significance with respect to host-parasite interaction in the Toxoplasma field. The report aims to assign a function to the first identified rhoptry protein in Toxoplasma. It employs CRISPR-Cas9 screens in Toxoplasma to study and identify the function of proteins involved in parasite virulence. The study may be of less interest to groups working in the field of host-pathogen interaction, innate immunity, as the genes studied here are not widely conserved nor do they provide obvious parallels to other systems.

  5. Version published to 10.1101/2022.03.21.485090v1 on bioRxiv