NapR Regulates the Expression of Phosphoserine Aminotransferase SerC to Modulate Biofilm Formation and Resistance to Serine Stress of Mycobacteria
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis article explores the regulation of serine metabolism in mycobacteria by NapR. Using advanced molecular tools like CRISPRi, the authors demonstrate that NapR regulates SerC. Additionally, proteomic analyses shed light on the role of NapR as an important regulator of the global physiology of mycobacteria.
While the importance of SerC in serine biosynthesis is acknowledged, its relevance for the survival of Mycobacterium tuberculosis within macrophages requires further discussion.
Previous studies have indicated that SerC in M. tuberculosis contributes to adaptation to hypoxia and antibiotic resistance (Bai et al., 2011; Geeraerts et al., 2021). Elaborating on this aspect would reinforce the findings presented in the article.
The section discussing biofilm formation notes that suppressing SerC in M. smegmatis leads to reduced biofilm formation; however, it fails to clarify how this suppression impacts the composition of the extracellular matrix.
Furthermore, there is insufficient evidence linking NapR to c-di-GMP production, a critical regulator of biofilm formation in other bacterial species.
Although the study reports various differentially expressed proteins, a more detailed biological interpretation and experimental validation of the affected metabolic pathways are needed.
Additionally, validating some key genes identified in the proteomic analysis using RT-qPCR could significantly strengthen the results.
Some figures in the study have insufficient resolution, making it difficult to interpret crucial aspects. Moreover, the labeling of axes in several graphs needs improvement to enhance clarity and understanding of the data.
In Figure 2, even when SerC regulation is emphasized, it remains unclear whether the intensities of the EMSA bands accurately reflect variations in NapR binding affinity.
In Figure 5 growth curves at different serine concentrations are demonstrated, however the text does not offer an adequate analysis of the biological significance of these data, which is essential for interpreting their implications.
Author Response
We thank the reviewer for his/her positive comments as well as critics on our manuscript! We have revised the manuscript and addressed all the Point raised by the reviewer point-by-point. For convenience, the changes in the revised manuscript were highlighted in red. Please see the responses below.
Point1: While the importance of SerC in serine biosynthesis is acknowledged, its relevance for the survival of Mycobacterium tuberculosis within macrophages requires further discussion.
Response1: We thank the reviewer for bringing out this proposal. Existing studies have shown that serine can stimulate macrophages to produce IL-1β, which is crucial for macrophage resistance to Mycobacterium tuberculosis infection. SerC is an essential component of de novo serine synthesis in Mycobacterium, and inhibition of SerC expression may reduce intracellular serine levels in macrophages infected with M. tuberculosis, thereby suppressing IL-1β secretion.
Point2: Previous studies have indicated that SerC in M. tuberculosis contributes to adaptation to hypoxia and antibiotic resistance. Elaborating on this aspect would reinforce the findings presented in the article.
Response2: We appreciate this constructive comment raised by the reviewer. As suggested, the elaboration about SerC in adaptation to hypoxia and antibiotic resistance was added as “Isoniazid, rifampin, and H2O2 induce the upregulation of serC expression in M. smegmatis, while inhibiting serC expression enhances the bactericidal effect of these agents and H2O2 on M. smegmatis”. (please see the page 2 , line 87-90)
Point3: The section discussing biofilm formation notes that suppressing SerC in M. smegmatis leads to reduced biofilm formation; however, it fails to clarify how this suppression impacts the composition of the extracellular matrix.
Response3: We thank the reviewer for pointing this out. SerC has a minimal effect on the growth of Mycobacterium smegmatis in liquid culture (Figure S3) but significantly impacts biofilm formation at the air-liquid interface (Figure 2E). This indicates that the effect on biofilm formation is not due to growth inhibition caused by SerC suppression. However, our study could not determine which specific EPS synthesis pathway is affected by SerC suppression, requiring further investigation in future studies.
Point4: Furthermore, there is insufficient evidence linking NapR to c-di-GMP production, a critical regulator of biofilm formation in other bacterial species.
Response4: This is a great question. The molecular mechanism of c-di-GMP regulating biofilm formation in Pseudomonas aeruginosa has been well documented. However, in Mycobacterium tuberculosis, only Lsr2 has been reported to be regulated by c-di-GMP, thereby affecting biofilm formation. NapR is a potential target of c-di-GMP, which requires further investigation.
Point5: Although the study reports various differentially expressed proteins, a more detailed biological interpretation and experimental validation of the affected metabolic pathways are needed.
Response5: We thank the reviewer for bringing out this proposal. The affected metabolic pathways were identified through enrichment analysis based on proteomics data using the KEGG or GO databases. Further detailed biological interpretation of these metabolic pathways have been added in the text. Please see the page 9 , line 289-290 and page 10 , line 294-299. For experimental validation, the key genes glyA and trpA were detected, and the results were described in Figure X and in the text. Please see the page 10 , line 319-321.
Point6: Additionally, validating some key genes identified in the proteomic analysis using RT-qPCR could significantly strengthen the results.
Response6: We appreciate this constructive comment raised by the reviewer. The RT-qPCR analysis for glyA and trpA have been presented in Figure S8D and the corresponding results were discussed in the text. Please see the page 10 , line 319-321.
Point7: Some figures in the study have insufficient resolution, making it difficult to interpret crucial aspects. Moreover, the labeling of axes in several graphs needs improvement to enhance clarity and understanding of the data.
Response7:We apologize for the insufficient resolution and labeling. According to the reviewer's Point, Figures 4C, 4D, and 4E, as well as Figures S6 and S7, have been replaced with higher-resolution versions. The axis annotations in Figures 2B, 2F, 4B, 5A, 5B, S2C, and S3A have been improved.
Point8:In Figure 2, even when SerC regulation is emphasized, it remains unclear whether the intensities of the EMSA bands accurately reflect variations in NapR binding affinity.
Response8: NapR of Mycobacterium smegmatis and BCG Vaccine exhibits different binding affinities to the serC promoters. In Figure 2A, as the concentration of NapRMsm increased, the amount of bound serCMsm p increased, while the amount of unbound DNA decreased. However, even at a concentration of 0.5 μM, NapRMsm can cannot completely bind to serCMsm p, but can fully bind to serCBCG p (Figure S9A). Similarly, 0.5 μM NapRBCG fully binds to serCBCG p (Figure 4A) but fails to fully bind to serCMsm p (Figure S9A), indicating that the binding affinity between Nap and serCMsm p might be nonspecific. This affinity difference has been also addressed in the discussion section. Please see the page 11 , line 342-359.
Point9: In Figure 5 growth curves at different serine concentrations are demonstrated, however the text does not offer an adequate analysis of the biological significance of these data, which is essential for interpreting their implications.
Response9: We thank the reviewer for the suggestive Point. The serine-induced growth inhibition observed in BCG Vaccine is an intriguing phenomenon. In our study, Mycobacterium smegmatis did not experience serine-induced growth inhibition (Figure S3), whereas BCG Vaccine showed significant growth inhibition under the same conditions (Figure 5). This suggests that serine stress in BCG Vaccine may be linked to its pathogenic nature. While our study does not explain the underlying mechanism, we hypothesize that high serine concentrations may serve as a signal for impending IL-1β secretion, as elevated serine levels can induce macrophages to secrete IL-1β. This signaling may trigger Mycobacterium tuberculosis to enter a growth-inhibition state in preparation for potential stress. This affinity difference has been also addressed in the discussion section. Please see the page 12 , line386-416.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study advances our understanding of the regulation of biofilm formation by transcription factors. It demonstrated that NapR regulates serC by directly binding to its promoter. This study is well-designed and clearly presented. The paper could be accepted for publication after minor revisions.
NapR functions as a transcriptional activator in the nonpathogenic strain M. smegmatis, and as a transcriptional repressor in pathogenic BCG vaccine strain. How about in M. tuberculosis? Does NapR act as an activator or a repressor?
Author Response
We thank the reviewer for his/her positive comments as well as critics on our manuscript! We have revised the manuscript and addressed all the Point raised by the reviewer point-by-point. For convenience, the changes in the revised manuscript were highlighted in red. Please see the responses below.
Point1：NapR functions as a transcriptional activator in the nonpathogenic strain M. smegmatis, and as a transcriptional repressor in pathogenic BCG vaccine strain. How about in M. tuberculosis? Does NapR act as an activator or a repressor?
Response1: We thank the reviewer for the valuable questions. Mycobacterium tuberculosis is a pathogen transmitted through the respiratory route and can cause severe diseases in humans. Research on M. tuberculosis must be conducted in biosafety level 3 (BSL-3) laboratories, and this study did not include experiments under such conditions. BCG vaccine, a pathogen with high homology to M. tuberculosis, shares 100% homology between NapRBCG and NapRMtb (the intracellular NapR of M. tuberculosis), 100% homology between SerCBCG and SerCMtb, and 100% homology between serCBCG p and serCMtb p. Therefore, we hypothesize that NapRMtb represses the transcription of serCMtb. The inference about the function of NapR in M. tuberculosis was addressed in discussion section. Please see the page 11 , line 352-355.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis is a big work on the on the expression on serC. It is a nice work done in this article.
The introduduction was maybe a little too basic!
in order to be able to make a replicate of this work, the material and methods section needs to have supplier names, city and country added. And the cultivation platforme was used for the 7H9 is not mentioned.
I am still missing an explenation of why the NapR has different regulation on serCmsm and serCbcg. It's like there is a missing link in this mechanisme.
Author Response
We thank the reviewer for his/her positive comments as well as critics on our manuscript! We have revised the manuscript and addressed all the Point raised by the reviewer point-by-point. For convenience, the changes in the revised manuscript were highlighted in red. Please see the responses below.
Point1: The introduduction was maybe a little too basic!
Response1: We thank the reviewer for bringing out this proposal. According to the reviewer's Point, the introduction has been revised to simplify basic content and include some cutting-edge research advancements.
Point2: in order to be able to make a replicate of this work, the material and methods section needs to have supplier names, city and country added. And the cultivation platforme was used for the 7H9 is not mentioned.
Response2: According to the reviewer's Point, the material and methods section has been improved with the updated manufacturers and countries of the reagents used, as well as the companies responsible for specific experiments. The Middlebrook 7H9 Broth used in this study was commercially pre-prepared and obtained from BD Difco .
Point3: I am still missing an explenation of why the NapR has different regulation on serCmsm and serCbcg. It's like there is a missing link in this mechanisme.
Response3: In our study, NapR exhibited different binding abilities to the serC promoters of BCG Vaccine and Mycobacterium smegmatis. At a concentration of 0.5 μM, NapRMsm fully binds to serCBCG p (Figure S9A) but cannot completely bind to serCMsm p (Figure 2A). Similarly, 0.5 μM NapRBCG fully binds to serCBCG p (Figure 4A) but fails to completely bind to serCMsm p (Figure S9A). Therefore, we hypothesize that the differential transcriptional regulation of serC by NapR in M. smegmatis and BCG Vaccine is due to differences in the serC promoters. Validating this hypothesis will require further research. Please see the page 11 , line 342-359.
