Author Response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
The paper by Lee and Ouellette explores the role of cyclic-d-AMP in chlamydial developmental progression. The manuscript uses a collection of different recombinant plasmids to up- and down-regulate cdAMP production, and then uses classical molecular and microbiological approaches to examine the effects of expression induction in each of the transformed strains.
Strengths:
This laboratory is a leader in the use of molecular genetic manipulation in Chlamydia trachomatis and their efforts to make such efforts mainstream is commendable. Overall, the model described and defended by these investigators is thorough and significant.
Thank you for these comments.
Weaknesses:
The biggest weakness in the document is their reliance on quantitative data that is statistically not significant, in the interpretation of results. These challenges can be addressed in a revision by the authors.
Thank you for these comments. We point out that, while certain RT-qPCR data may not be statistically significant, our RNAseq data indicate late genes are, as a group, statistically significantly increased when increasing c-di-AMP levels and decreased when decreasing c-di-AMP levels. We do not believe running additional experiments to “achieve” statistical significance in the RT-qPCR data is worthwhile. We hope the reviewer agrees with this assessment.
We have also included new data in this revised manuscript, which we believe further strengthens aspects of the conclusions linked to individual expression of full-length DacA isoforms. We have also quantified inclusion areas and bacterial sizes for critical strains.
Reviewer #2 (Public review):
Summary:
This manuscript describes the role of the production of c-di-AMP on the chlamydial developmental cycle. Chlamydia are obligate intracellular bacterial pathogens that rely on eukaryotic host cells for growth. The chlamydial life cycle depends on a cell form developmental cycle that produces phenotypically distinct cell forms with specific roles during the infectious cycle. The RB cell form replicates amplifying chlamydia numbers while the EB cell form mediates entry into new host cells disseminating the infection to new hosts. Regulation of cell form development is a critical question in chlamydia biology and pathogenesis. Chlamydia must balance amplification (RB numbers) and dissemination (EB numbers) to maximize survival in its infection niche. The main findings In this manuscript show that overexpression of the dacA-ybbR operon results in increased production of c-di-AMP and early expression of the transitionary gene hctA and late gene omcB. The authors also knocked down the expression of the dacA-ybbR operon and reported a reduction in the expression of both hctA and omcB. The authors conclude with a model suggesting the amount of c-di-AMP determines the fate of the RB, continued replication, or EB conversion. Overall, this is a very intriguing study with important implications however the data is very preliminary and the model is very rudimentary and is not well supported by the data.
Thank you for your comments. Chlamydia is not an easy experimental system, but we have done our best to address the reviewer’s concerns in this revised submission.
Describing the significance of the findings:
The findings are important and point to very exciting new avenues to explore the important questions in chlamydial cell form development. The authors present a model that is not quantified and does not match the data well.
Describing the strength of evidence:
The evidence presented is incomplete. The authors do a nice job of showing that overexpression of the dacA-ybbR operon increases c-di-AMP and that knockdown or overexpression of the catalytically dead DacA protein decreases the c-di-AMP levels. However, the effects on the developmental cycle and how they fit the proposed model are less well supported.
dacA-ybbR ectopic expression:
For the dacA-ybbR ectopic expression experiments they show that hctA is induced early but there is no significant change in OmcB gene expression. This is problematic as when RBs are treated with Pen (this paper) and (DOI 10.1128/MSYSTEMS.00689-20) hctA is expressed in the aberrant cell forms but these forms do not go on to express the late genes suggesting stress events can result in changes in the developmental expression kinetic profile. The RNA-seq data are a little reassuring as many of the EB/Late genes were shown to be upregulated by dacA-ybbR ectopic expression in this assay.
As the reviewer notes, we also generated RNAseq data, which validates that late gene transcripts (including sigma28 and sigma54 regulated genes) are statistically significantly increased earlier in the developmental cycle in parallel to increased c-di-AMP levels. The lack of statistical significance in the RT-qPCR data for omcB, which shows a trend of higher transcripts, is less concerning given the statistically significantly RNAseq dataset. We have reported the data from three replicates for the RT-qPCR and do not think it would be worthwhile to attempt more replicates in an attempt to “achieve” statistical significance.
We recognize that hctA may also increase during stress as noted by the Grieshaber Lab. In re-evaluating these data, we decided to remove the Penicillin-linked studies from the manuscript since they detract from the focus of the story we are trying to tell given the potential caveat the reviewer mentions.
The authors also demonstrate that this ectopic expression reduces the overall growth rate but produces EBs earlier in the cycle but overall fewer EBs late in the cycle. This observation matches their model well as when RBs convert early there is less amplification of cell numbers.
dacA knockdown and dacA(mut)
The authors showed that dacA knockdown and ectopic expression of the dacA mutant both reduced the amount of c-di-AMP. The authors show that for both of these conditions, hctA and omcB expression is reduced at 24 hpi. This was also partially supported by the RNA-seq data for the dacA knockdown as many of the late genes were downregulated. However, a shift to an increase in RB-only genes was not readily evident. This is maybe not surprising as the chlamydial inclusion would just have an increase in RB forms and changes in cell form ratios would need more time points.
Thank you for this comment. We agree that it is not surprising given the shift in cell forms. The reduction in hctA transcripts argues against a stress state as noted above by the reviewer, and the RNAseq data from dacA-KD conditions indicates at least that secondary differentiation has been delayed. We agree that more time points would help address the reviewer’s point, but the time and cost to perform such studies is prohibitive with an obligate intracellular bacterium.
Interestingly, the overall growth rate appears to differ in these two conditions, growth is unaffected by dacA knockdown but is significantly affected by the expression of the mutant. In both cases, EB production is repressed. The overall model they present does not support this data well as if RBs were blocked from converting into EBs then the growth rate should increase as the RB cell form replicates while the EB cell form does not. This should shift the population to replicating cells.
We agree that it seems that perturbing c-di-AMP production by knockdown or overexpressing the mutant DacA(D164N) has different impacts on chlamydial growth. We have generated new data, which we believe addresses this. Overexpressing membrane-localized DacA isoforms is clearly detrimental to chlamydiae as noted in the manuscript. However, when we removed the transmembrane domain and expressed N-terminal truncations of these isoforms, we observed no effects of overexpression on chlamydial morphology or growth. Importantly, for the wild-type full-length or truncated isoforms, overexpressing each resulted in the same level of c-di-AMP production, further supporting that the negative effect of overexpressing the wild-type full-length is linked to its membrane localization and not c-di-AMP levels. These data have been included as new Figure 3. These data indicate that too much DacA in the membrane is disruptive and suggest that the balance of DacA to YbbR is important since overexpression of both did not result in the same phenotype. This is further described in the Discussion.
As it relates to knockdown of dacA-ybbR, we have essentially removed/reduced the amount of these proteins from the membrane and have blocked the production of c-di-AMP. This is fundamentally different from overexpression.
Overall this is a very intriguing finding that will require more gene expression data, phenotypic characterization of cell forms, and better quantitative models to fully interpret these findings.
Reviewer #1 (Recommendations for the authors):
There is a generally consistent set of experiments conducted with each of the mutant strains, allowing a straightforward examination of the effects of each transformant. There are a few general and specific things that need to be addressed for both the benefit of the reader and the accuracy of interpretation. The following is a list of items that need to be addressed in the document, with an overall goal of making it more readable and making the interpretations more quantitatively defended.
Specific comments:
(1) The manuscript overall is wordy and there are quite a few examples of text in the results that should be in the discussion (examples include lines 224-225, 248-262, 282-288, 304-308) the manuscript overall could use a careful editing for verbosity.
Thank you for this comment. We have removed some of the indicated sentences. However, to maintain the flow and logic of the manuscript, some statements may have been preserved to help transition between sections. As far as verbosity, we have tried to be as clear as possible in our descriptions of the results to minimize ambiguity. Others who read our manuscript appreciated the thoroughness of our descriptions.
(2) There is also a trend in the document to base fact statements on qualitative and quantitative differences that do not approach statistical significance. Examples of this include the following: lines 156-158, 190-192, 198-199, 230-232, 239-242, 292-293). This is something the authors need to be careful about, as these different statistically insignificant differences may tend to multiply a degree of uncertainty across the entire manuscript.
We have quantified inclusion areas and tried to remove instances of qualitative assessments as noted by the reviewer. In regards to some of the transcripts, we can only report the data as they are. In some cases, there are trends that are not statistically significant, but it would seem to be inaccurate to state that they were unchanged. In other cases, a two-fold or less difference in transcript levels may be statistically significant but biologically insignificant. A reader can and should make their own conclusions.
(3) Any description of inclusion or RB size being modestly different needs to be defended with microscopic quantification.
We have quantified inclusion areas and RB sizes and tried to remove instances of qualitative assessments as noted by the reviewer.
(4) It would be very helpful to reviewers if there was a figure number added to each figure in the reviewer-delivered text.
Added.
(5) Figure 1A: This should indicate that the genes indicated beneath each developmental form are on high (I think that is what that means).
We have reorganized Figure 1 to better improve the flow.
(6) Figure 1B is exactly the same as the three images in Figure 8B. I would delete this in Figure 1. This relates to comment 9.
We presented this intentionally to clearly illustrate to the reader, who may not be knowledgeable in this area, what we propose is happening in the various strains. As such, we respectfully disagree and have left this aspect of the figure unchanged.
(7) Figure 1D: It is not clear if the period in E.V has any meaning. I think this is just a typo. Also, the color coding needs to be indicated here. What do the gray bars represent? The labeling for the gene schematic for dacA-KDcom should not be directly below the first graph in D. This makes the reader think this is a label for the graph. This can be accomplished if the image in panel B is removed and the first graph in panel D is moved into B. This will make a better figure.
We have reorganized Figure 1 to better improve the flow.
(8) Figure 2 C, G: The utility of these panels is not clear. For them to have any value, they need to be expressed in genome copies. If they are truly just a measure of chlamydia genomic DNA, they have minimal utility to the reader. There are similar panels in several other figures.
We have reported genome copies as suggested in lieu of ng gDNA for these measurements. Importantly, it does not alter any interpretations.
(9) I am not sure about the overall utility of Figure 8. Granted, a summary of their model is useful, but the cartoons in the figure are identical or very nearly identical to model figures shown in two other publications from the same group (PMID: 39576108, 39464112) These are referenced at least tangentially in the current manuscript (Jensen paper- now published- and ref 53). Because the model has been published before, if they are to be included, there needs to be a direct comparison of the results in each of these three papers, as they basically describe the same developmental process. The model images should also be referenced directly to the first of the other papers.
This was intentional so that readers familiar with our work will see the similarities between these systems. We have added additional comments in the Discussion related to our newly published work. As an aside, Dr. Lee generated the first version of the figure that was adapted by others in the lab. It is perhaps unlucky that those other studies have been published before his work.
