1. Author Response:

    Reviewer #2:

    Recommendations:

    A) In the section 'Electrophysiological characterization of TcMscS', the authors present compelling evidence that TcMscS gates in response to tension in the membrane. However, it is unclear, both in the text and the caption, if the trace shown in Figure 2 panel C was collected under tension. If it was, please include the applied pressure value in either the text or caption. Additionally, within this section the applied pressure to the patch is frequently unclear. One way to clear this up would be to 1- add the applied pressure to each trace or to 2- add the applied pressure for each patch to the figure caption. -In Panel E: can you comment on the conductance of the channels in the three traces? Why do you see channels that are approximately 1/2 the size of the first trace in the second two traces?

    Figure 2 has been corrected, the pressure value for Panel A is denoted below the panel and only one scale bar is now shown in Panel E. The legend for the figure was re-written to assign the pressure ramp to panels C and D.

    B) In the section 'TcMscS gene targeting by CRISPR-Cas9' the authors utilized CRISPR to KO TcMscS to determine its function, based on the immunofluorescence and qPCR TcMscS has been successfully knocked out. In lines 251-264, the authors complemented the KO with an overexpression vector in an attempt to confirm the role of TcMscS. In this section, it is very unclear what strains C1 and C2 are and how they are different from one another. Neither of these constructs successfully restores the growth rate. The authors can clarify the differences between the two constructs or they can remove this section from the manuscript, particularly Figure 5 supplement 3. The manuscript is strong and compelling without this panel.

    We agree with the reviewer that, in this case, the complementation did not fully revert the KO phenotype, but instead had a detrimental effect, probably due to the overexpression of functional channels. We decided to include the data to explain why we could not verify the reversion of the phenotype. We believe is important to share this information with the community, since often negative results are excluded from manuscripts and, in this case, could be useful for other groups to have a precedent on the toxic effect of overexpressing channels in T. cruzi.

    C1 and C2 correspond to two different clones of the complementation with TcMscS-myc tagged. We show both clones to illustrate the consistency of the phenotype of the complemented strains. A clarification of C1 and C2 clonal nature has been indicated in the manuscript (lines 267-268).

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  2. Reviewer #3 (Public Review):

    This manuscript investigates the structure, electrophysiological and biological functions of a novel mechanosensitive channel from the parasitic protist Trypanosoma cruzi. The channel was identified bioinformatically as being significantly related in sequence to known mechanosensitive channels of the McsS superfamily. Studies on channel proteins in pathogenic protists such as trypanosomes are limited, and this investigation focuses on a previously unstudied mechanosensitive channel which is of potential interest to parasite biology, because trypanosomes are subjected to various mechanical stress forces during their life cycles.

    Analysis of the sequence of the TcMscS protein by bioinformatics and structural prediction concludes that it is relatively divergent from previously studied members of the family from other microorganisms. Of particular interest, the divergent C-terminal domain contains a proposed novel cytoplasmic gate that could filter solutes on the cytosolic side. These observations are predictive rather than data driven. The recombinant protein was expressed in E. coli giant spheroplasts and studied by cell-detached patch clamp electrophysiology. The authors have clearly demonstrated that the channel is activated by pressure steps and fluxes K+, Cl-, Ca2+, and they speculate that it may be able to transport osmolytes such as amino acids. Other well supported conclusions are that the channel is located primarily in the contractive vacuole complex (CVC) in insect vector stage epimastigotes, an organelle that expels water from the parasite, but it occupies a wider range of subcellular sites in infective metacyclic and bloodstream trypomastigotes, and it localizes primarily to the plasma membrane of amastigotes. Thus, the channel changes its location during the life cycle. A MscS knockout line was generated and demonstrably shown to have impaired cell volume regulatory responses when subjected to changes in extracellular osmolarity, decreased motility, reduced ability to transform into infective stages such as metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes, and altered Ca2+ homeostasis. Hence, the biological impacts of this channel are broad and significant.

    A strength of the manuscript is that it is well-executed study and examines many aspects of channel function and biology. Precisely how the channel mediates the biological functions is less clear and will require future investigations. For instance, whether the channel has functions related to shear stresses encountered by the parasites when they enter host cells or extravasate through vasculature is currently rather speculative, albeit of considerable potential interest. How the channel mediates volume changes or affects motility is also unclear. In addition, the manuscript would benefit from some editing to make several points or interpretations clearer to the readers.

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  3. Reviewer #2 (Public Review):

    In the manuscript 'A novel mechanosensitive channel controls osmoregulation, differentiation and infectivity in Trypanosoma cruzi' the authors show conclusive evidence that TcMscS is a mechanosensitive channel. They also show that TcMscS has additional roles outside of mechanosensation, likely playing a role in the infectivity of T. cruzi. This manuscript is well written with data that clearly supports the authors hypothesis. The evidence provided in the manuscript clearly shows that TcMscS gates in response to tension and that when knocked out of the genome there is a reduction in infectivity. This work will be impactful to both all researchers studying mechanosensation as it shows that mechanosensitive channels have roles outside of tension sensation.

    Recommendations:

    A) In the section 'Electrophysiological characterization of TcMscS', the authors present compelling evidence that TcMscS gates in response to tension in the membrane. However, it is unclear, both in the text and the caption, if the trace shown in Figure 2 panel C was collected under tension. If it was, please include the applied pressure value in either the text or caption. Additionally, within this section the applied pressure to the patch is frequently unclear. One way to clear this up would be to 1- add the applied pressure to each trace or to 2- add the applied pressure for each patch to the figure caption. -In Panel E: can you comment on the conductance of the channels in the three traces? Why do you see channels that are approximately 1/2 the size of the first trace in the second two traces?

    B) In the section 'TcMscS gene targeting by CRISPR-Cas9' the authors utilized CRISPR to KO TcMscS to determine its function, based on the immunofluorescence and qPCR TcMscS has been successfully knocked out. In lines 251-264, the authors complemented the KO with an overexpression vector in an attempt to confirm the role of TcMscS. In this section, it is very unclear what strains C1 and C2 are and how they are different from one another. Neither of these constructs successfully restores the growth rate. The authors can clarify the differences between the two constructs or they can remove this section from the manuscript, particularly Figure 5 supplement 3. The manuscript is strong and compelling without this panel.

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  4. Reviewer #1 (Public Review):

    The authors found a mechanosensitive channel gene in T. cruzi, and aimed to characterize the functions. The strength of this manuscript is that the channel has been examined from various aspects: the modelled molecular structures; expression and localization during development; electrophysiological characteristics; cell motility; responses to osmotic stress; regulation of Ca2+ homeostasis; infectivity to host cells. The weakness of this study is the assessment of motility and the nature of the recombinant protein. To conclude, this study provides data sufficient for reporting the discovery and initial characterization of the novel mechanosensitive in T. cruzi. The most significant finding is the correlation with infection, but the involvement in the response to osmotic stress is also interesting in the field of cell biology.

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  5. Evaluation Summary:

    The authors found a mechanosensitive channel gene in T. cruzi, and aimed to characterize its functions. The authors provide conclusive evidence that TcMscS is a mechanosensitive channel. They also show that TcMscS has additional roles outside of mechanosensation, likely playing a role in the infectivity of T. cruzi.

    (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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