The Use of Disabled Insecticidal Proteins (DIPs) to Investigate the Interaction Between Aedes aegypti-Active Toxins from Bacillus thuringiensis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “The use of Disabled Insecticidal Proteins (DIPs) to investigate the interaction between Aedes aegypti-active toxins from Bacillus thuringiensis” is poorly written and is not definitive.
1. That “eight different toxins share a common mechanism of action” is not definitively demonstrated nor would such a phenomenon particularly “enhance our knowledge of host-pathogen interactions” in the A. aegypti system.
2. The so-called “shared common mechanism of action” is much more complicated than simply binding a cell surface receptor in the insect.  To which cell surface receptor do the toxins bind?  What happens downstream of the binding?
3. Please explain how the approach involving mutated toxins is relatively new.
4. Lines 81-85: The sentence structure is awkward rendering the statement highly confusing.
5. Toxin preparation presented in lines 94-99 is not rigorous.  SDS PAGE and densitometry does not prove purity of the toxins.  Was SDS-PAGE done under reducing or nonreducing conditions?
6. Line 165: What is meant by “efficiently go on”?
7. Lines 181-183: The authors state that in vitro binding assays are complemented using in vivo competition assays.  What about in situ analyses?
8. Lines 218-219: “may reflect an evolutionary history”?
9. The experimental results presented in this manuscript do not reveal that the multiple toxins used share the same receptor in A. aegypti.  The manuscript is highly speculative.

Comments on the Quality of English Language

The manuscript is poorly written.  English usage needs significant improvement.

Author Response

1. That “eight different toxins share a common mechanism of action” is not definitively demonstrated nor would such a phenomenon particularly “enhance our knowledge of host-pathogen interactions” in the A. aegypti system. The primary purpose of this manuscript is to apply the technique of in vivo competition between Bt Cry toxins to Aedes aegypti. As discussed in the introduction previous work involving a variety of different techniques (binding, RNAi, knockouts etc) have produced conflicting results concerning the mode of action (ie which receptors are used). Although other interpretations are possible, and are discussed in the manuscript, current thinking is that the existence of competition implies a shared receptor. We agree that this may not actually be the case and so have modified the summary text to say that the results “indicate” rather than “establish” a shared receptor, and have also revised the sentence to ensure that it doesn't imply that all of the toxins were active against this insect.
2. The so-called “shared common mechanism of action” is much more complicated than simply binding a cell surface receptor in the insect. To which cell surface receptor do the toxins bind? What happens downstream of the binding? The manuscript states that these are pore-forming toxins and that receptor binding is widely considered to be main determinant of specificity. Since this work is focused on the receptor, we don’t believe that there is a need to discuss other steps in the mechanism, such as the nature of the pore, in detail. If by complexity the reviewer is referring to the sequential binding and/or the signal transduction models then it should be noted that both of these models have now largely been discredited. In terms of what surface receptor is used then the introduction already discusses in detail what these might be.
3. Please explain how the approach involving mutated toxins is relatively new. Although mutants that affect toxicity, but not binding, have been described in the past the use of them in in vivo competition assays is a relatively new approach to studying the MoA of Cry toxins. The seminal paper is “Jerga, A.; Evdokimov, A.G.; Moshiri, F.; Haas, J.A.; Chen, M.; Clinton, W.; Fu, X.; Halls, C.; Jimenez-Juarez, N.; Kretzler, C.N.; et al. Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization. Insect Biochem Mol Biol 2019, 105, 79-88, doi:10.1016/j.ibmb.2018.12.006.” We would consider 2019 as being relatively new.
4. Lines 81-85: The sentence structure is awkward rendering the statement highly confusing. At some stage in the editing process the word “is” was deleted. This has now been reinstated making the sentence clearer.
5. Toxin preparation presented in lines 94-99 is not rigorous. SDS PAGE and densitometry does not prove purity of the toxins. Was SDS-PAGE done under reducing or nonreducing conditions? SDS PAGE is a perfectly suitable, and accepted, means of assessing the yield of Bt crystals. The aim was not to prove that the toxins were absolutely pure (there will always be some other bacterial proteins there) but to assess the concentration of the toxin. By default SDS PAGE samples are prepared under reducing conditions so there is no need to state this.
6. Line 165: What is meant by “efficiently go on”? Just because a toxin binds to a cell surface protein does not mean that it will go on (proceed) to form a pore. This is a perfectly acceptable term to use. With respect to the use of efficiently it has been demonstrated that toxins can, at high concentrations, form pores in lipid bilayers in the absence of receptors, however the formation of pores is much more efficient if a receptor is present.
7. Lines 181-183: The authors state that in vitro binding assays are complemented using in vivo competition assays. What about in situ analyses? In situ assays can be used to assess binding to the same region of the cell but do not have the resolution to say that two proteins are binding to the same receptor. In situ competition assays are therefore effectively just binding assays.
8. Lines 218-219: “may reflect an evolutionary history”? This is simply suggesting that ancestral Cry toxins may have bound to a particular receptor but that the accumulation of mutations (in toxin and/or receptor) may then have prevented it forming a pore (but not stopped binding) in a particular host.
9. The experimental results presented in this manuscript do not reveal that the multiple toxins used share the same receptor in A. aegypti. The manuscript is highly speculative. As discussed above the manuscript is describing the use of Disabled Insecticidal Proteins in in vivo competition assays where current thinking is that the presence of competition implies a shared receptor. Based on that understanding, which may be wrong, they do indicate a shared receptor. The data can be added to the other published literature from which more accurate models may one day be derived.

The manuscript is poorly written.  English usage needs significant improvement. The manuscript was written by a native English speaker. Could the reviewer please point out specific examples where the grammar needs improvement.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates receptor sharing among multiple Cry proteins using in vivo competition assays. The study addresses an interesting and relevant question; however, the current data presentation and experimental validation limit the strength of the conclusions. The results rely on in vivo competition assays to infer receptor sharing, but it is unclear whether the observed inhibition truly reflects receptor competition or is influenced by variability in toxin concentrations, the relative excess of DIPs, or differences in larval feeding and susceptibility. Additional controls, quantitative analyses, and functional validation of the DIPs are necessary to support the claims and strengthen the conclusions.

Major Comments:

1. Evidence should be provided to demonstrate that the Cry2Aa and Cry1Ca DIPs are properly folded and retain receptor-binding capability, as inactivity alone is not sufficient to confirm functional integrity.
2. The manuscript would benefit from full dose-response data, statistical analyses, and a clearly defined quantitative threshold (e.g., percent inhibition at a specific competitor ratio) to distinguish “partial” versus “complete” inhibition for the results presented in Figure 1 and Table 1. The reproducibility of these assays across independent replicates should also be reported.
3. To rule out non-specific effects, particularly for inhibition observed with Cry1Aa/Cry1Ac, control experiments using an irrelevant protein (e.g., BSA) at equivalent concentrations should be included. Potential differences in DIP stability or degradation in the larval gut should also be considered as factors that may influence the observed inhibition patterns.
4. The table should be clarified by explicitly labeling rows as the “Active Toxin” and columns as the “Competitor” protein. Protein nomenclature should be consistent throughout the manuscript; for example, the competitor should be labeled Cry11Aa-E100A to match the text description. A graphical representation of inhibition at multiple competitor ratios would further clarify the distinction between partial and complete inhibition.

Minor Comments:

1. Line 49: Correct the spelling of “chikununya” to “chikungunya.”
2. Experimental details and reproducibility should be reported, including the number of independent replicates for each competition assay and the variability between experiments. It should also be clarified whether larvae from the same developmental stage and genetic background were used consistently in all assays.

Author Response

This manuscript investigates receptor sharing among multiple Cry proteins using in vivo competition assays. The study addresses an interesting and relevant question; however, the current data presentation and experimental validation limit the strength of the conclusions. The results rely on in vivo competition assays to infer receptor sharing, but it is unclear whether the observed inhibition truly reflects receptor competition or is influenced by variability in toxin concentrations, the relative excess of DIPs, or differences in larval feeding and susceptibility. Additional controls, quantitative analyses, and functional validation of the DIPs are necessary to support the claims and strengthen the conclusions.

Major Comments:

1. Evidence should be provided to demonstrate that the Cry2Aa and Cry1Ca DIPs are properly folded and retain receptor-binding capability, as inactivity alone is not sufficient to confirm functional integrity. All mutant proteins that we produce are tested for misfolding in the following way: i) the size and the shape of the crystals produced in Bt are compared to the wild-type, ii) the ability of the crystal to release protoxin upon treatment with alkali is confirmed, iii) the ability of the solubilized toxin to resist degradation by 1mg/ml trypsin is confirmed. Only if these criteria are met do we use the protein. Text has been added to the manuscript M&M section to detail this. In addition: i) the mutants that we created have all been used in other published papers and shown to behave normally, ii) in a different context we have published experiments involving Cry1CaDIP showing that the toxin behaves normally and indeed binds to its target ( https://doi.org/10.3390/biom14070795).
2. The manuscript would benefit from full dose-response data, statistical analyses, and a clearly defined quantitative threshold (e.g., percent inhibition at a specific competitor ratio) to distinguish “partial” versus “complete” inhibition for the results presented in Figure 1 and Table 1. The reproducibility of these assays across independent replicates should also be reported. All assays were of course repeated to ensure consistency. Repeat assays however often used different fold excesses of competitor in order to get a more detailed dose-response curve. We are thus happy with the reported trends even if statistical analyses have not been performed at individual fold excesses. We feel that showing individual graphs for each assay would be less informative for the reader than the provided table which is why we opted to show the data for one assay but summarise the others.

 

3. To rule out non-specific effects, particularly for inhibition observed with Cry1Aa/Cry1Ac, control experiments using an irrelevant protein (e.g., BSA) at equivalent concentrations should be included. Potential differences in DIP stability or degradation in the larval gut should also be considered as factors that may influence the observed inhibition patterns. Experiments were performed with BSA at 100 fold excess concentration (no effect on toxin activity was found). This control has now been added to the manuscript. The fact that there was a consistent difference between the inhibition of Cry2Aa and the other toxins (except with Cry2Aa and Cry2AaDIP) also suggests the lack of a non-specific effect. In the manuscript mentioned in our response to query 1 above we did find, in that system, some competitors that did not affect the activity of the toxin, again indicating the lack of a non-specific effect. In the manuscript discussion we do state that there might be other explanations for the observed inhibition. As mentioned above there is no evidence of differences in stability. A main focus of this paper is to investigate the usefulness of in vivo competition assays. Current thinking is that the presence of competition implies a shared receptor. Based on that understanding, which may be wrong, they do indicate a shared receptor. The data can be added to the other published literature from which more accurate models may one day be derived.

 

4. The table should be clarified by explicitly labeling rows as the “Active Toxin” and columns as the “Competitor” protein. Protein nomenclature should be consistent throughout the manuscript; for example, the competitor should be labeled Cry11Aa-E100A to match the text description. A graphical representation of inhibition at multiple competitor ratios would further clarify the distinction between partial and complete inhibition. As suggested the table layout has been amended. We felt it better to refer to all the mutants simply with the DIP descriptor after the toxin name. Listing the actual mutations for each mutant on each occasion feels excessive, especially for Cry2Aa DIP with its four separate mutations. The Cry11Aa mutation was listed once to describe which mutation was made but thereafter referred to as Cry11AaDIP.

Minor Comments:

1. Line 49: Correct the spelling of “chikununya” to “chikungunya.” Corrected
2. Experimental details and reproducibility should be reported, including the number of independent replicates for each competition assay and the variability between experiments. It should also be clarified whether larvae from the same developmental stage and genetic background were used consistently in all assays. The manuscript states the stage of larvae used (3^rd instar) and the source of the insects (Infravec2).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revisions have satisfactorily addressed all previous concerns, and I have no additional questions or comments. For clarity, however, I suggest specifying the meanings of “complete,” “partial,” and “ND” in Table 1, for example, in a table legend or footnote.