Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by maintenance of cAMP level

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of
PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Reviewer #2 (Public Review):

Summary: In the manuscript, the authors have presented new mechanistic details to show how intracellular cAMP levels are maintained linked to the phosphodiesterase enzyme which in turn is controlled by PhoP. Later, they showed the physiological relevance linked to altered cAMP concentrations.

Strengths: Well thought out experiments. The authors carefully planned the experiments well to uncover the molecular aspects of it diligently.

Weaknesses: Some fresh queries were made based on the author's previous responses and hope to get satisfactory answers this time.

Author Response

The following is the authors’ response to the original reviews.

Public Review:

Summary:

This paper reports how mycobacterial cAMP level is increased under stressful conditions and that the increase is important in the survival of the bacterium in animal hosts.

Strengths:

The authors show that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of a PDE specific to cAMP is significant progress in understanding Mtb pathogenesis. An increase in cAMP apparently increases bacterial survival upon infection. On the practical side, the reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests.

We thank the reviewers for these extremely encouraging comments.

Weaknesses:

Repression of PDE promoter by binding of phosphorylated PhoP could have been shown at higher precision. The binding is now somewhere along a roughly 500 bp region. Although the regulation of PDE is shown to be by transcriptional repression only, it has been described as a homeostatic mechanism. The latter would have required a demonstration of both repression and activation by negative feedback.

We agree. We have now performed EMSA (Electrophoretic Mobility Shift Assay) experiments and included the data showing DNA binding of PhoP to the upstream regulatory region of rv0805 (rv0805up) as a supplemental figure (see Figure 2-figure supplement 1). The supplemental figure, figure caption, and the relevant results have been adjusted accordingly in the revised manuscript.

Further, as recommended by the reviewer we have now removed the term ‘homeostatic mechanism’ and rephrased it with ‘maintenance of cAMP level’ in the manuscript.

Response to Reviewers’ comments

Reviewer #1:

The authors have used homeostasis inappropriately. Homeostasis usually requires negative feedback (a clear example is the regulation of Lambda prm promoter). Here, there is no feedback from changes in PDE or cAMP level to their synthesis. Homeostasis does not belong to this paper anywhere.

As recommended by the reviewer, we have now removed “homeostasis” from the manuscript and mostly replaced it with “maintenance of cAMP level” in the revised manuscript.

The authors have frequently used adverbs at the beginning of a sentence, such as Notably (l.240, 272, 376), Importantly (l.66, 213), More importantly (l.134), Remarkably (l.264), Interestingly (l.115,301), Intriguingly (l.344), unambiguously (l.347), etc. The use of these words is generally counter-productive. The authors should scan the ms. to eliminate them as far as possible. The sentences would read more clearly and become more impactful.

Following reviewer’s recommendation, we have now eliminated most of the adverbs, mostly used at the beginning of sentences, in the revised manuscript.

Specific comments

(1) L.1: "maintenance of homeostasis" or increasing cAMP level.

As suggested by the reviewer, we have now replaced “maintenance of cAMP homeostasis” with “maintenance of cAMP level”.

(2) L.27: mechanism or reason; varying or various.

As recommended by the reviewer, we have now replaced “mechanism” with “reason” and the word “varying” is deleted while incorporating suggested changes in the abstract.

(3) L.28-29: The logic of connecting PhoP to cAMP doesn't follow well. The logic is much better in l.54, l.112-5 and l.130.

We thank the reviewer for this suggestion. We have now modified the statement within the ‘abstract’ in the revised manuscript (duplicated below):

“cAMP is one of the most widely used second messengers which impacts on a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-mycobacterial cAMP level could be controlled by the phoP locus since the major regulator plays a key role in bacterial response against numerous stress conditions.”

(4) L.30: discovers or reveals (?). Also, in l.101.

As recommended by the reviewer, we have now replaced ‘discovers’ with ‘reveals’ in the Abstract and ‘uncovered’ with ‘revealed’ in the Introduction section of the manuscript.

(5) L.31: Delete "The most - - derived". It is not obvious what most fundamental means here. I suggest: We find that PhoP-dependent ---involves specific binding of the regulator---PDE gene.

As recommended by the reviewer, we have modified the statement (duplicated below): “In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis leading to depletion of intra-mycobacterial cAMP level.”

(6) L.36: --pathway decreases cAMP level, stress tolerance, and survival of the bacilli.

As recommended by the reviewer, we have now modified the statement (duplicated below): “Thus, genetic manipulation to inactivate PhoP-Rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacilli.

(7) L.41: 'keeps encountering" or encounters?

As suggested by the reviewer, we have replaced ‘keeps encountering’ with ‘encounters’ in the ‘Introduction’ section of the revised manuscript.

(8) L.61: responds, carries.

Our apologies for the embarrassing grammatical mistakes. We have rectified these errors in the revised manuscript.

(9) L.67: you mean burst in synthesis level, not burst of cAMP itself.

To improve clarity, we have now modified the statement in the revised manuscript (duplicated below): “Agarwal and colleagues had shown that burst in synthesis of bacterial cAMP upon infection of macrophages, improved bacterial survival by interfering with host signalling pathways (Agarwal et al., 2009)”

Reference

Agarwal N, Lamichhane G, Gupta R, Nolan S, Bishai WR (2009) Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase. Nature 460: 98-102

(10) L.77: Change Off to Of.

We are sorry for the inaccuracy. The suggested change has been made to the text.

(11) L.83: Did not discuss "degradation" earlier.

Following reviewer’s recommendation, we have now modified the statement in the revised manuscript (duplicated below).

“Together, these results strongly suggest that a balance between cAMP synthesis by adenylate cyclases and cAMP degradation by phosphodiesterases contributes to rapid adaptive response of mycobacteria in a hostile intracellular environment (Johnson and McDonough, 2018; McDonough and Rodriguez, 2011).”

Reference

Johnson RM, McDonough KA (2018) Cyclic nucleotide signaling in Mycobacterium tuberculosis: an expanding repertoire. Pathog Dis 76 (5)

McDonough KA, Rodriguez A (2011) The myriad roles of cyclic AMP in microbial pathogens: from signal to sword. Nature reviews Microbiology 10: 27-38

(12) L.95: Isn't PhoPR a two-component signal transduction system, the terminology that is more specific than a two-protein regulatory system?

As recommended by the reviewer, we have replaced “two protein regulatory system” with more specific “two-component signal transduction system” in the revised manuscript.

(13) L.124: check-point prevents things from happening. Here the mechanism you found allows growth and survival.

We agree. As recommended by the reviewer, we have now modified the sentence in the revised manuscript (duplicated below).

“Together, the newly identified mechanism of regulation of cAMP level allows intraphagosomal survival and growth program of mycobacteria.”

(14) L.132: why not say directly-"---under normal, and NO and acid stress conditions (Fig. 1A).

As recommended by the reviewer, we have now deleted the first part of the sentence and directly stated that “we compared cAMP levels………. under normal, NO and acidic stress conditions” (duplicated below).

“We compared cAMP levels of WT and phoPR-KO (lacking both phoP and phoR), grown under normal, NO stress and acid stress conditions (Fig. 1A).”

(15) L.134: The complementation is quite variable. Also true in Fig. 2A. If no simple answer, you can say- cAMP values increased in complemented cells, although to a variable extent, for reasons unknown.

We agree with the reviewer. We have now incorporated new text in the ‘Results’ section of the revised manuscript (duplicated below):

“A higher cAMP level in the complemented strain under NO stress is possibly attributable to reproducibly higher phoP expression in the complemented mutant under specific stress conditions (Khan et al., 2022).”

(16) L.154: You rather not say "conclude" and "most likely" at the same time. How about replacing "we conclude" with suggests? In that case, no need to say "most likely". Also, in l.306-7 & l.322-3.

We thank the reviewer for these suggestions. We have now modified the statements in the revised manuscript (duplicated below).

“We suggest that lower cAMP level of the mutant is not due to its higher efficacy of cAMP secretion.”

Following reviewer’s recommendation, we have incorporated similar changes in two other places of the ‘Results’ section of the revised manuscript.

(17) L.161: introduce both the acronyms here and not in l.162.

Following reviewer’s recommendation, we have made the suggested changes.

(18) L.164: Second, (to be in line with First).

We have made the suggested change.

(19). Fig. 2C: There are no black and white bars. This is an important figure because the results appear in the abstract. The signal change from pH 7 to 4.5 is not much. An independent approach would have been desirable. If it were E. coli, I would have suggested beta-gal assay or in vivo footprints. Is a PhoP binding site recognizable in the promoter region of rv0805?

We apologize for the inaccuracy. We have corrected it in the revised manuscript. Also, we have now carried out DNA binding assays, and included the EMSA data of rv0805 upstream regulatory region binding to phosphorylated PhoP (P~PhoP) as a supplemental figure (Figure 2-figure supplement 1A-B). In this figure, we have also incorporated our results on the likely PhoP binding site within rv0805up. The new figure, figure caption and the relevant results have been adjusted accordingly in the revised manuscript.

(20) L.209: ORFs; also delete "of growth" from the sentence.

The suggested changes were made to the text.

(21) L.213: Delete Importantly and change "failed to" to 'did not' (since you did not motivate the expectation earlier, it is better to state the results in an unbiased way).

As recommended by the reviewer, both changes were included in the revised manuscript.

(22) L.217: The requirement of PhoR is a new result - why say "confirm". Change it to indicate. Also, delete "indeed" here and from L.233.

As recommended by the reviewer, both changes were included in the revised manuscript.

(23) L.224: Are the results in Fig 3-S1A under inducing conditions?

The results shown in Fig 3-S1A are not under inducing conditions of expression. For better clarity, we have modified the sentence describing Figure 3-figure supplement 1A (duplicated below).

“rv0805 ORF was cloned within the multicloning site of integrative pSTki (Parikh et al., 2013) between EcoRI and HindIII sites under the control of Pmyc1tetO promoter, and expression of rv0805 under non-inducing condition was verified by determining the mRNA level (Figure 3 - figure supplement 1A).

Reference:

Parikh et al (2013) Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria. Applied and environmental microbiology 79: 1718-1729

(24) L.225: ---cAMP level. Add (Fig. 3C) at the end of the next sentence.

As recommended by the reviewer, both the suggested changes were made to the revised text.

(25) L.231: Delete "Most importantly"- you didn't specify what are other less important results.

We agree. We have now deleted “most importantly” from the sentence in the revised text.

(26) L.243 & 254: Change homeostasis to level? Here you are showing mechanisms that can change cAMP level. Homeostasis here would mean how fluctuations in cAMP level are adjusted, usually requiring negative feedback.

As recommended by the reviewer, ‘homeostasis’ was replaced with ‘level’ in both places.

(27) L.256: stress response or stress? Also, in l.272

We are sorry for the inaccuracy. We have corrected these errors in the revised version of the manuscript.

(28) L.259: Change "maintenance of homeostasis" to 'repressing the rv0805 PDE gene'. It is safer to use a fact-based title. In this section, direct measurement of rv0805 mRNA, and/or cAMP levels in different genetic backgrounds seem desirable.

We agree. As recommended by the reviewer, we have modified the title of the ‘Results’ section in the revised manuscript (duplicated below).

“PhoP contributes to mycobacterial stress tolerance and intracellular survival by repressing the rv0805 PDE expression.”

Please note that direct measurements of rv0805 mRNA and cAMP levels are part of Fig. 3 and Figure 3- figure supplement 1A, respectively.

(29) Fig, 4A: White and grey symbols are not easily discriminated without zooming. Use color for phoPR-KO.

We agree. We have now indicated the phoPR-KO in blue in the revised Fig. 4.

(30) L.264: Delete remarkable or explain what is so remarkable. Aren't the results expected- the PDE level would go up in both cases. Direct measurement of PDE /cAMP levels would take the mystery out of the results.

As recommended by the reviewer, we have deleted ‘remarkably’ in the revised text. We have measured cAMP and PDE expression levels of the four strains in Fig. 3 and Figure 3-figure supplement 1.

(31) L.273: --suggesting a role of ---

We have modified this sentence in the revised version of the manuscript (duplicated below).

“A previous study had reported that phoP-deleted mutant strain was more sensitive to Cumene Hydrogen Peroxide (CHP), suggesting a role of PhoP in regulating mycobacterial stress response to oxidative stress (Walters et al., 2006).”

Reference:

Walters et al. (2006) The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol 60: 312-330

(32) L.275: Delete "transcriptome". CHP sensitivity alone doesn't speak for transcriptome.

As suggested by the reviewer, we have deleted “transcriptome”. Also, please see our response to the previous comment (above).

(33) Fig. 4D and E: % Colocalization in the Merge panels is not much different among the four strains tested (to an untrained eye). Can the results be explained to readers not used to in vivo studies?

As recommended by the reviewer, we have now incorporated new text to explain the in vivo experiment (duplicated below).

“In this assay, WT-H37Rv inhibits phagosome maturation, whereas phagosomes with phoPR-KO mature into phagolysosomes (Anil Kumar et al., 2016).”

Further, for better clarity of the results shown in Fig. 4D, we have (a) increased size of the figure to highlight the difference in the ‘merge’ panel; (b) included “white arrowheads” in the merge panels of Fig. 4D to indicate auramine labeled mycobacteria, which either have inhibited or facilitated trafficking into lysosomes, and finally (c) incorporated method used to calculate percent co-localization in greater details in the ‘Material and Methods’ section of the revised manuscript.

Reference

Anil Kumar et al. (2016) EspR-dependent ESAT-6 secretion of Mycobacterium tuberculosis requires the presence of virulence regulator PhoP. J Biol Chem. 291, 19018-19030

(34) L.275-6: Delete "next" (also in l.347) and "Note that". In this paragraph, I was expecting some explanation on how phoPR-KO and WT-Rv0805 are behaving similarly. Even if the reason is not known, it should be mentioned.

The suggested changes have been made to the text. Also, as recommended by the reviewer, we have included the following text in the revised manuscript (duplicated below):

“Together, these results reveal similar behaviour of phoPR-KO, and WT-Rv0805 by demonstrating a comparably higher susceptibility of these strains to acidic pH and oxidative stress relative to WT bacteria and indicate a link between intra-mycobacterial cAMP level and bacterial stress response. Collectively, these data suggest that at least one of the mechanisms by which PhoP contributes to global stress response is attributable to maintenance of cAMP level.”

(35) L.281: ---WT and indicate a link between cAMP level and stress response in mycobacteria. (No mention of homeostasis).

The suggested change has been made to the revised text. Please see above our response to point # 34.

(36) L.288, 290: No Thus and no clearly.

Both the suggested changes have been made to the text.

(37) L.297: Can you be more direct and state --is due to reduced cAMP level?

As recommended by the reviewer, we have now modified the sentence to make it more direct in the revised manuscript (duplicated below):

“Together, our findings facilitate an integrated view of our results, suggesting that higher susceptibility of WT-Rv0805 to stress conditions, is attributable to its reduced cAMP level.”

(38) L.307: May delete "most likely----homeostasis". cAMP is not discussed here. The same deletion is desired in l.324.

We agree. As recommended by the reviewer, we have now modified the relevant texts in the revised manuscript. These are duplicated below.

“From these results, we suggest that ectopic expression of rv0805 impacts phagosome maturation arguing in favour of a role of PhoP in influencing phagosome-lysosome fusion in macrophages.”

“Thus, we suggest that one of the reasons which accounts for an attenuated phenotype of phoPR-KO in both cellular and animal models is attributable to PhoP-dependent repression of rv0805 PDE activity, which controls mycobacterial cAMP level.”

(39) L.342: cAMP level is regulated remains---

The suggested change has been made to the revised text (duplicated below):

“Although many bacterial pathogens modulate host cell cAMP level as a common strategy, the mechanism of regulation of mycobacterial cAMP level remains unknown.”

(40) L.373: tone down "most fundamental". It is not obvious what is so profound about a stress-response system that depends on PhoP also depends on PhoR. OR justify what is most fundamental about it.

We agree. Following reviewer’s recommendation, we have modified the text in the revised manuscript (duplicated below):

“In keeping with these results, we find that PhoP-dependent rv0805 expression requires PhoR (Figs. 3A-B), the cognate kinase which activates PhoP in a signal-dependent manner (Gupta et al., 2006; Singh et al., 2023).”

References:

Gupta et al. (2006) Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Letters 580, 5328-5338.

Singh et al. (2023) Dual functioning by the PhoR sensor is a key determinant to Mycobacterium tuberculosis virulence. PLoS Genetics 19(12): e1011070.

(41) L.395: delete correspondingly (?)

The suggested change has been made to the text.

(42) L.396: Delete "appear to" and "somewhat". The uncertainty is already implied in "suggest". The evidence that ectopic expression of rv0805 is functionally equivalent to phoP deletion is quite clear in this paper and not saying that clearly is confusing.

We agree with the reviewer. The suggested changes have been made to the revised text (duplicated below):

“Thus, our results suggest that ectopic expression of rv0805 is functionally equivalent to deletion of the phoP locus.”

(43) L.401: --over-expressing bacilli, induction level of rv0805 expression was significantly different in Matange et al and our studies. The next sentence is also very wordy.

We have made changes to the text to address the reviewer’s concern. Also, the next sentence has been rewritten (duplicated below).

“Although both studies were performed with rv0805 over-expressing bacilli, the fact that important differences in the expression of PDEs, in this study (Matange et al., 2013) and in our assays - yielding significantly different levels of rv0805 expression - most likely account for this discrepancy. While we cannot rule out the possibility of cleavage of other cyclic nucleotides by Rv0805 (Keppetipola & Shuman, 2008; Shenoy et al., 2007; Shenoy et al., 2005), consistent with a previous study our results correlate rv0805 expression with intra-mycobacterial cAMP level (Agarwal et al., 2009).”

References:

Matange et al. (2013) Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response. Tuberculosis (Edinb) 93: 492-500.

Keppetipola N, Shuman S (2008) A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. J Biol Chem 283: 30942-30949

Shenoy et al. (2007) Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis. Journal of molecular biology 365: 211-225

Shenoy et al. (2005) The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis. Biochemistry 44: 15695-15704

Agarwal N, Lamichhane G, Gupta R, Nolan S, Bishai WR (2009) Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase. Nature 460: 98-102

(44) L.409: To avoid saying "conclude" and "most likely" at the same time, can you start the sentence thus: 'We infer that Pho-----rv0805 is a---.

We agree. We have made suggested changes to the text. The modified sentence is duplicated below:

“We infer that PhoP-dependent regulation of Rv0805 is a critical regulator of intra-mycobacterial cAMP level.”

(45) L.424. Delete "According to this model". In the preceding sentence, the subject is results, not model. This whole paragraph needs to be rewritten in fewer lines. The shorter the summary statement, the greater would be its impact (less is more here). I would delete the red circles from the figure- it appears that in the repressed state, you are making more products. Replace the circles with an arrow. The legend could be "Increased cAMP level and effective stress response" and "Decreased cAMP---and reduced---.

We thank the reviewer for these suggestions. Following reviewer’s recommendations, we have made numerous changes and rewritten the paragraph in the revised manuscript (duplicated below):

“In summary, upon sensing low acidic pH as a signal PhoR activates PhoP, P~PhoP binds to rv0805 upstream regulatory region and functions as a specific repressor of Rv0805. Therefore, we observed (a) a reproducibly lower level of cAMP in phoPR-KO relative to WT-H37Rv, (b) a significantly reduced expression of rv0805 in WT-H37Rv, grown under acidic pH relative to normal conditions, and (c) comparable cAMP levels in phoPR-KO and WT-Rv0805. This is why the two strains remain ineffective to mount an appropriate stress response, most likely due to their inability to coordinate regulation of gene expression because of dysregulation of intra-mycobacterial cAMP level. However, without uncoupling regulatory control of PhoPR and rv0805 expression, we cannot confirm that dysregulation of cAMP level accounts for virulence attenuation of phoPR-KO. Given the fact that rv0805-depleted M. tuberculosis is growth attenuated in vivo (McDowell et al., 2023), paradoxically ectopic expression of rv0805 leads to dysregulated metabolic adaptation, thereby resulting in reduced stress tolerance and intracellular survival.”

Also, the suggested changes have been incorporated in Fig. 6 and the figure caption.

Reference

McDowell JR, Bai G, Lasek-Nesselquist E, Eisele LE, Wu Y, Hurteau G, Johnson R, Bai Y, Chen Y, Chan J et al (2023) Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification. Mol Microbiol 119: 401-422

(46) L.458 & 500: ---was used to transform.

Following reviewer’s recommendation, the suggested changes were made to the text in the Materials and Methods section of the revised manuscript.

(47) L.460: --- antibiotics plates.

Both suggested changes were made to the text.

(48) L.466-7: --they were transferred-pH 4.5) and grown for further-

We thank the reviewer for these suggestions. The suggested changes were made to the text.

(49) L.486: ---full-length ORFs of interest were---

The suggested changes were incorporated in the revised manuscript.

(50) L.497: The RNAs were 20 nt long and complementary---

As recommended by the reviewer, we have modified the text in the revised manuscript (duplicated below).

“The RNAs were 20 nt long and complementary to the non-template strand of the target gene.”

Reviewer #2:

(1) Rephrase this sentence in the abstract: “Because growing evidence connects PhoP with varying stress response, we hypothesized that the level of 3’,5’ cAMP, one of the most widely used second messengers, was regulated by the phoP locus, linking numerous stress responses with cAMP production”.

As recommended by the reviewer, we have now rewritten the sentence. The modified text is incorporated in the revised manuscript (duplicated below):

“cAMP is one of the most widely used second messengers, which impacts on a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-mycobacterial cAMP level could be controlled by the phoP locus since the major regulator plays a key role in bacterial responses against numerous stress conditions.”

Also, please see our response to specific comments #1-3 of Reviewer 1.

(2) Line 134: please describe the complementation strain features as it is mentioned for the first time (plasmid, copy number, promoter etc.) in the manuscript. Especially under NO stress what could be the authors' justification regarding the high cAMP concentration in the complementation strain?

As recommended by the reviewer, the details of construction of the complemented strain have been incorporated in the ‘Materials and Methods’ section of the revised manuscript (duplicated below):

“To complement phoPR expression, pSM607 containing a 3.6- kb DNA fragment of M. tuberculosis phoPR including 200-bp phoP promoter region, a hygromycin resistance cassette, attP site and the gene encoding phage L5 integrase, as detailed earlier (Walters et al., 2006) was used to transform phoPR mutant to integrate at the L5 attB site.”

To address the reviewer’s other concern, we have now included the following sentence in the ‘Results’ section of the revised manuscript (duplicated below):

“A higher cAMP level in the complemented strain under NO stress is possibly attributable to reproducibly higher phoP expression in the complemented mutant under specific stress condition (Khan et al., 2022).”

Reference:

Khan et al. (2022) Convergence of two global regulators to coordinate expression of essential virulence determinants of Mycobacterium tuberculosis. eLife 2022, 11:e80965.

(3) In Figure 1C, it is a bit confusing to see the numbers 1,2,3 and 4 and nothing is referred to these numbers in the figure legend so it's better to remove them.

We agree with the reviewer. We have now removed the lane numbers from the figure (Fig. 1C) in the revised manuscript.

(4) Line 852: rephrase it "insignificantly different".

The suggested change has been made to the text. The modified text is incorporated in the manuscript (duplicated below):

“Note that the difference in expression levels of rv0805 between WT and phoPR-KO was significant (p<0.01), whereas the fold difference in mRNA level between WT and the complemented mutant (Compl.) remains nonsignificant (not indicated).”

(5) Line198-200: There are no open/black bars, they all are coloured bars. Correct the same. The significance test should be done for the same gene (suppose rv0805 up) in different pH conditions. Right now, it is not revealing anything and misleading.

We apologize for the inaccuracy. We have now rectified the error. As recommended by the reviewer, Fig. 4C was modified, and the significance tests were carried out between samples involving identical promoter enrichments under different pH conditions. The modified figure, figure legend, and the relevant results have been adjusted accordingly in the revised manuscript.

(6) Line 213: Is there any difference between this complementation strain (phoPR-KO:: phoPphoR with the one used in Figure 1A, 1B, and 2A? If yes, then please describe it.

The same complemented mutant strain, which has been described in the ‘Materials and Methods’ section of the revised manuscript, was used in the experiments described in Fig. 1A, Fig.1B and Fig. 2A.

(7) Line 223: Please mention the copy number and promoter of the vector construct.

As recommended by the reviewer, we have now mentioned the promoter of the vector and incorporated new text with regard to copy number of the expression vector in the revised manuscript (duplicated below).

“Although copy number of episomal vectors with pAl5000 origin of replication (oriM) have been reported to be 3 by Southern hybridization (Ranes et al, 1990), in this case wild-type and mutant Rv0805 proteins were expressed from single-copy chromosomal integrants (Parikh et al., 2013).”

References

Ranes et al., (1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector. J Bacteriol 172: 2793-2797

Parikh et al., (2013) Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria. Applied and environmental microbiology 79: 1718-1729

(8) Figure 3 - Figure Supplement 1: not sure why the authors measured mRNA levels of rv1357 and rv2387? These genes were not overexpressed!

The mRNA levels of rv1357 and rv2387 were measured to show that overexpression of either the wild-type or mutant Rv0805 did not influence expression of other PDEs like Rv1357 and Rv2387. We have now mentioned it explicitly in the revised manuscript (duplicated below).

“In contrast, other PDE encoding genes (rv1357 and rv2387), under identical conditions, demonstrate comparable expression levels in WT-H37Rv and rv0805 over-expressing strains.”

(9) Line 234: Wrong interpretation it should be PDE mRNA levels in WT-Rv0805 and WT-Rv0805M.

As recommended by the reviewer, we have now modified the statement to improve clarity (duplicated below).

“The corresponding mRNA levels of PDEs (wild-type and the mutant) are over-expressed approximately 4.5-6 -fold relative to the genomic rv0805 level of WT-H37Rv (Figure 3-figure supplement 1A).”

(10) Line 237: Remove the sentence "Thus, we conclude......identical expression strategy", you have already talked about why phosphodiesterase activity is crucial for cAMP concentration and it is well understood.

Following reviewer’s recommendation, we have now removed the sentence from the revised manuscript.

(11) Figure 3E: Authors should comment on why the cAMP concentration is not significantly changed even though the mRNA level changes are drastic (~90%). How do you correlate that? Is it because of other PDEs?

We agree. As suggested by the reviewer, we have now incorporated new text in the revised manuscript (duplicated below).

“We speculate that effective knocking down of phoP or rv0805 is not truly reflected in the extent of variation of cAMP levels possibly due to the presence of numerous other mycobacterial PDEs.”

(12) Line 505,506: Is it the translation start site or the transcription start site? Because mRNA level changes are reported.

It is the translational start sites, and gene-specific small guide RNAs were designed to inhibit mRNA expression.

(13) Line 292: There is a difference between red and green bars. Authors should do statistical analysis and then comment on whether overexpression of WT and mutant pde are different or similar, to me they are different; also, explain why the WT-Rv0805 strain is different than the phoPR-KO strain in the context of cell wall metabolism.

As recommended by the reviewer, we have now included statistical significance of the data in the revised version, and modified the text accordingly in the manuscript.

Also, we included text explaining why WT-Rv0805 is different compared to phoPR-KO strain in the context of cell wall metabolism (duplicated below).

“Together, these results suggest that both strains expressing wild type or mutant PDEs share a largely similar cell-wall properties and are consistent with (a) a recent study reporting no significant effect of cAMP dysregulation on mycobacterial cell wall structure/permeability (Wong et al., 2023), and (b) role of PhoP in cell wall composition and complex lipid biosynthesis (Walters et al., 2006; Asensio et al., 2006; Goyal et al., 2011).”

References:

Wong et al. (2023) Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis. eLife 2023; 12: e81177

Walters et al. (2006) The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol 60: 312-330

Asensio et al. (2006) The Virulence-associated Two-component PhoP-PhoR System Controls the Biosynthesis of Polyketide-derived Lipids in Mycobacterium tuberculosis. J Biol Chem 281: 1313-1316.

Goyal et al. (2011) Phosphorylation of PhoP protein plays direct regulatory role in lipid biosynthesis of Mycobacterium tuberculosis. J Biol Chem 286: 45197-45208

(14) Line 299-303: Authors should explain how the colocalization % are calculated. Also, in the figure 4D merge panel please highlight the difference.

As suggested by the reviewer, we have now explained the methodology used to calculate percent colocalization in greater details. Also, we have modified Figure 4D to highlight the difference between samples shown in merge panel. Please see our response to comment # 33 from the Reviewer 1.

(15) General comment: There are multiple instances where writing needs to be improved.

We are sorry for the inaccuracies. We have now done thorough editing of the manuscript and made numerous corrections throughout.