A universal stress protein is essential for the survival of Mycobacterium tuberculosis
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eLife assessment
This important study will be of interest to those working on mycobacterial signal transduction. A combination of experiments provides convincing evidence to show how universal stress proteins bind to cAMP and function by direct sequestration of the second messenger. Although the methods, data and analyses broadly support the conclusions, the main claims are only partially supported and can be strengthened through further analytic approaches.
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Abstract
Mycobacterium tuberculosis employs several signaling pathways to regulate its cellular physiology and survival within the host. Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways in which these bacteria utilize cAMP. We have earlier identified universal stress proteins (USP), Rv1636 and MSMEG_3811 in M. tuberculosis and M. smegmatis respectively, as abundantly expressed, novel cAMP-binding proteins. In this study, we show that these USPs may function to regulate cAMP signaling by direct sequestration of the second messenger. In slow-growing mycobacteria, concentrations of Rv1636 were equivalent to the amounts of cAMP present in the cell, and overexpression of Rv1636 in M. smegmatis increased levels of ‘bound’ cAMP. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. While msmeg_3811 could be readily deleted from the genome of M. smegmatis , we find that the rv1636 gene is essential for growth of M. tuberculosis , and this functionality depends on the cAMP-binding ability of Rv1636. This is the first evidence of a ‘sink’ for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intracellular ‘free’ pool of cAMP.
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eLife assessment
This important study will be of interest to those working on mycobacterial signal transduction. A combination of experiments provides convincing evidence to show how universal stress proteins bind to cAMP and function by direct sequestration of the second messenger. Although the methods, data and analyses broadly support the conclusions, the main claims are only partially supported and can be strengthened through further analytic approaches.
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Reviewer #1 (Public Review):
The essentiality of Rv1636 has previously been predicted in numerous genetic studies. Here, the authors provide evidence that Rv1636 is an essential protein in Mtb. The authors report that chromosomal deletion of the gene encoding Rv1636 is only possible when an additional copy of the wild type gene is provided at the L5 integration site in the chromosome. While this is a standard method of demonstrating gene/protein essentiality in this system, the manuscript only provides a PCR reaction with "no amplicon" as proof of a double crossover event in an engineered merodiploid strain (Fig 6C). The authors fail to provide definitive evidence for a double crossover mutation in the merodiploid using primers that amplify a double crossover-dependent amplicon or the authors should a provide a southern blot …
Reviewer #1 (Public Review):
The essentiality of Rv1636 has previously been predicted in numerous genetic studies. Here, the authors provide evidence that Rv1636 is an essential protein in Mtb. The authors report that chromosomal deletion of the gene encoding Rv1636 is only possible when an additional copy of the wild type gene is provided at the L5 integration site in the chromosome. While this is a standard method of demonstrating gene/protein essentiality in this system, the manuscript only provides a PCR reaction with "no amplicon" as proof of a double crossover event in an engineered merodiploid strain (Fig 6C). The authors fail to provide definitive evidence for a double crossover mutation in the merodiploid using primers that amplify a double crossover-dependent amplicon or the authors should a provide a southern blot demonstrating evidence for a bona fide double crossover event. The authors suggest that silencing the gene encoding Rv1636 with a CRISPRi system decreases viability of Mtb when a silencing guide RNA is expressed following Atc addition and spot plated onto agar. These studies lack a "no Atc control" and it is unclear how Mtb colonies appear after 6-7 days in these studies given the slow growth of this bacterium.
A sub-point of the manuscript describes the genetic organization around the gene that encodes Rv1636 in various Mycobacterial spp. Figure 1 also highlights the putative transcriptional start sites for the gene encoding Rv1636. The putative transcriptional start site information is just a summary of work from other groups and this information adds little to the main goals of this manuscript.
Another sub-point of this manuscript is that Rv1636 may be secreted by Mtb in a SecA2 dependent manner. The authors demonstrate that Rv1636 is not present in the culture filtrate of Mtb lacking SecA2 (Fig 2). However, these data are difficult to interpret without a secreted protein "loading control" which is typical for these types of experiments. The authors also report the development of a luciferase-based detection method for quantifying protein secretion in Mtb and use this to support their conclusion. This is a new tool that could be useful in detecting secreted proteins in Mtb. However, this method is not rigorously validated in these studies and do not present controls for cell lysis for example. Additionally, the authors fuse a ~19 kDA luciferase subunit to the C-terminus of CFP10 as a reporter for Esx1-dependent secretion. It is known that this region of CFP10 is critical for interactions with secretory components of the Esx1 system fractionation and it unclear if the CFP10 fusion protein is actually secreted.
The authors explore the idea that Rv1636 may potentially function as a "sink" for cAMP and quantify the molar amounts cAMP, ATP, and Rv1636 in Mtb. These studies demonstrate that the molar amounts of Rv1636 exceeds the levels of cAMP (free or protein-bound) in the cytosol of the Mtb. The authors conclude that the excess of Rv1636 may potentially be a "sink" for unbound cAMP but do not test this idea experimentally in Mtb due to the very low levels of cAMP in this bacteria.
Instead, the authors continue exploring the idea that specific proteins can serve as a cAMP "sink" using M. smegmatis (Msm) since this bacterium produces more cAMP (~25x) in the cytosol compared to Mtb. The authors present data that over expression of Rv1636 in Msm increases the amount of protein-bound cAMP. It is presumed here that the protein-bound cAMP is bound to Rv1636. Alternatively, deleting the Rv1636 homolog in Msm (MSMEG_3811) results in an increase in the amount of "free cAMP". Again, it is presumed that deleting the cAMP binding protein MSMEG_3811 is responsible for the increase in the amount of "free cAMP" in the cell.
Lastly, the authors use two small molecule compounds that may bind Rv1636 and demonstrate some level of bacterial inhibition using a spot plating method. No evidence is provided to demonstrate that these compounds are specifically binding/inhibiting Rv1636. These studies are lacking rigorous demonstration of "on target" inhibition and add very little to the reliable conclusions in this paper.
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Reviewer #2 (Public Review):
In this paper by Banerjee et al., the authors described the potential role of two universal stress proteins in M. smegmatis and M. tuberculosis in regulating intracellular free cAMP concentration, which was a unique observation. The experiments were logically designed to prove the expression and interactions; it would have been worthwhile to explore beyond to gain an insight into how the changing levels of free cAMP could modulate any key phenotypes in the bacteria such as virulence, antibiotic resistance, etc. in the content of knockout/knockdown and overexpression of MSMEG_3811 and Rv1636 in individual organisms. The preliminary data of natural inhibitor STOCKIN43384 impacting the survival of M. smegmatis was interesting, but authors need to prove the MOA by using knockdown and overexpression strains of …
Reviewer #2 (Public Review):
In this paper by Banerjee et al., the authors described the potential role of two universal stress proteins in M. smegmatis and M. tuberculosis in regulating intracellular free cAMP concentration, which was a unique observation. The experiments were logically designed to prove the expression and interactions; it would have been worthwhile to explore beyond to gain an insight into how the changing levels of free cAMP could modulate any key phenotypes in the bacteria such as virulence, antibiotic resistance, etc. in the content of knockout/knockdown and overexpression of MSMEG_3811 and Rv1636 in individual organisms. The preliminary data of natural inhibitor STOCKIN43384 impacting the survival of M. smegmatis was interesting, but authors need to prove the MOA by using knockdown and overexpression strains of Rv1636.
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Reviewer #3 (Public Review):
This paper describes fundamental work which attempts to understand how universal stress proteins Rv1636/Msmeg3811 function as a sink allowing mycobacteria to use intra-bacterial cAMP. Because cAMP is a major second messenger, Rv1636 remains essential to mycobacteria. A compound that inhibits cAMP binding of Rv1636 also can effectively inhibit mycobacterial growth. The major strength of the manuscript is that the authors probed their hypothesis by different approaches. In general, the conclusions from the results are largely justified. However, I find the manuscript quite difficult to follow. Also, the results and functional analyses are inadequate as they rely on a limited set of experiments, thereby making the evidences less than compelling.
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Author Response:
We thank the reviewers for their insightful comments and will resubmit a revised version where we address most of the issues raised. At this time, our immediate responses are as follows.
1. We have data to confirm the presence of the merodiploid strain by PCR but did not show the data in the original version for brevity. We will show that data in the revised version.
2. We also have, of course, a no ATC control in our CRISPRi experiments and will also show that data in the resubmission.
3. As a loading control for the SecA2 strains, we will show PknG blots (a protein secreted by SecA2;PMID: 29709019) that we have with us.
4. In the nanoluc assays, the construct we made that was fused to CFP10 was generated so that there was a long linker between the C-terminus of CFP10 and nanoluc. We also have other controls in that …
Author Response:
We thank the reviewers for their insightful comments and will resubmit a revised version where we address most of the issues raised. At this time, our immediate responses are as follows.
1. We have data to confirm the presence of the merodiploid strain by PCR but did not show the data in the original version for brevity. We will show that data in the revised version.
2. We also have, of course, a no ATC control in our CRISPRi experiments and will also show that data in the resubmission.
3. As a loading control for the SecA2 strains, we will show PknG blots (a protein secreted by SecA2;PMID: 29709019) that we have with us.
4. In the nanoluc assays, the construct we made that was fused to CFP10 was generated so that there was a long linker between the C-terminus of CFP10 and nanoluc. We also have other controls in that experiment to show that the CFP10-nanoluc protein was secreted in the ΔRD10 strain and not in the ΔSecA2 strain. We will attempt to show fusion protein secretion using CFP10 antibodies in the revised version of the manuscript.
5. We will perform experiments with the inhibitor using the merodiploid strain and in partial knockdown strains to confirm that the inhibitor does indeed specifically act on Rv1636.
6. We will modify the discussion to talk more about the role and processes of cAMP synthesis and degradation in the revised version of the paper. Further, the manuscript will be checked for spelling and grammatical errors before resubmission, and the arrangement of data modified as suggested by the reviewers.
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