Convergence of two global regulators to coordinate expression of essential virulence determinants of Mycobacterium tuberculosis
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This paper will be of broad interest to those working on the regulation of gene expression and mycobacteria as it deals with the collaboration of two important transcription regulators. A combination of experiments indicates how a complex of two regulators selectively turns on gene expression of a few genes in intracellular pathogen Mycobacterium tuberculosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Cyclic AMP (cAMP) is known to function as a global regulator of Mycobacterium tuberculosis gene expression. Sequence-based transcriptomic profiling identified the mycobacterial regulon controlled by the cAMP receptor protein, CRP. In this study, we identified a new subset of CRP-associated genes including virulence determinants which are also under the control of a major regulator, PhoP. Our results suggest that PhoP as a DNA binding transcription factor, impacts expression of these genes, and phosphorylated PhoP promotes CRP recruitment at the target promoters. Further, we uncover a distinct regulatory mechanism showing that activation of these genes requires direct recruitment of both PhoP and CRP at their target promoters. The most fundamental biological insight is derived from the inhibition of CRP binding at the regulatory regions in a PhoP-deleted strain owing to CRP-PhoP protein-protein interactions. Based on these results, a model is proposed suggesting how CRP and PhoP function as co-activators of the essential pathogenic determinants. Taken together, these results uncover a novel mode of regulation where a complex of two interacting virulence factors impact expression of virulence determinants. These results have significant implications on TB pathogenesis.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
Khan et al describe how two important transcription factors functionally cooperate to activate a few of the CRP-dependent genes in Mycobacterium tuberculosis. CRP is a global regulator in eubacteria needed to activate a number of genes while PhoP is an acid stress response regulator required for expression specific set of genes. The authors delineate the interaction between these two key regulators of the bacterial pathogen and show that in a subset of CRP-dependent promoters, PhoP binding recruits CRP to activate transcription.
The experiments are well designed and executed with a coalescent presentation of the manuscript. While the data is well organized and presented with clean images of phophorimages and blots to facilitate their easy understanding, interpretation could have been …
Author Response
Reviewer #1 (Public Review):
Khan et al describe how two important transcription factors functionally cooperate to activate a few of the CRP-dependent genes in Mycobacterium tuberculosis. CRP is a global regulator in eubacteria needed to activate a number of genes while PhoP is an acid stress response regulator required for expression specific set of genes. The authors delineate the interaction between these two key regulators of the bacterial pathogen and show that in a subset of CRP-dependent promoters, PhoP binding recruits CRP to activate transcription.
The experiments are well designed and executed with a coalescent presentation of the manuscript. While the data is well organized and presented with clean images of phophorimages and blots to facilitate their easy understanding, interpretation could have been more robust (see comments below).
We thank the reviewer for these extremely encouraging comments. We have now included substantial changes throughout the ‘Results’ section to improve interpretation of the results (please see below our responses).
Obviously, the strength of the paper is the description of hitherto unknown stress-specific cooperation between two well-studied transcription factors with most evidence supporting the claims. In E. coli (and in other bacteria) studies CRP mediated control of genes have led to the identification of different classes of CRP-dependent promoters with their own specific regulators. Such a description was lacking in M. tuberculosis and the PhoP - CRP collaboration described is likely to have implications on pathogenesis. The weakness (or possibly what remains to be explored) is that the precise mechanism of the cooperative transcription regulation is yet to be understood.
We agree with the reviewer’s comment that the precise mechanism of cooperative transcription regulation is yet to be fully understood. While we briefly mention it as the future scope of work in the concluding part of the ‘Discussion’ section, we have now included a new paragraph on the schematic model summarizing a possible mechanism of cooperative transcription regulation.
From the data presented it is apparent that PhoP binds to whiB up promoter own efficiently. It is also evident that CRP is recruited to its site as a result of PhoP binding. This is reminiscent of the bacteriophage Lamba paradigm of positive cooperativity. Thus, it is not reciprocal synergy (as stated in the paper in one place). It is PhoP mediated recruitment as claimed elsewhere. Indeed, PhoP null mutants nicely support the latter interpretation
The reviewer raises an important and interesting point on positive cooperativity resembling bacteriophage lambda paradigm. We agree. We have now modified text of the ‘Results’ section to establish clarity on this matter.
A discussion on why and how CRP binds on its own in other CRP-dependent promoters would help better appreciate the need for PhoP sites next to CRP sites for their cooperative interaction in these promoter subsets. CRP sites could be at a varied distance with respect to the promoter as seen in E. coli.
Again, this is an interesting point. We thank the reviewer for bringing this point to our attention. As recommended by the reviewer, we have now included the following text in the ‘Discussion’ section of the revised manuscript.
“Notably, the subset of genes which undergo differential expression in Δcrp-H37Rv conforms a pattern largely resembling canonical CRP regulon of E. coli with CRP binding sites either proximal to transcription start sites, leading to repression or distal to transcription start sites, leading to promoter activation, respectively (Kahramanoglou et al., 2014). It is noteworthy that CRP has been suggested to function as a general chromosomal organizer (Grainger et al., 2005). In this study, we uncover that strikingly PhoP binding sites are present next to CRP binding sites, located only distal upstream of promoters, and therefore, associated with activation. We propose that in case of these co-regulated promoters, the additional stability of the transcription initiation complex is derived from protein-protein interaction between CRP and PhoP. These two interacting proteins remain bound to their cognate sites away from the start site, and contribute to stability of the transcription initiation complex, providing access for mycobacterial RNA polymerase (RNAP) to bind and transcribe genes. A schematic model is shown in Fig. 6C. Together, these molecular events mitigate stress by controlling expression of numerous genes and perhaps contribute to better survival of the bacilli in cellular and animal models.”
Reviewer #2 (Public Review):
In this manuscript by Khan et al., the authors set out to characterize how the cAMP receptor protein, CRP, and PhoP function to coregulate a subset of virulence genes in Mycobacterium tuberculosis. To this end, the authors use a wide variety of molecular techniques to monitor gene regulation, DNA-binding activity, and protein-protein interactions between phosphorylated PhoP and CRP. The authors conclude that phosphorylated PhoP functions to recruit CRP to promoter regions, where together the two regulators function synergistically to control gene expression. In general, the conclusions of the manuscript appear to be justified by the data, however, the text is difficult to follow. The current version of the paper is likely of interest to scientists within the field of mycobacterial signal transduction.
The major strength of the paper is that the authors test their hypothesis using a variety of complementary approaches. The authors demonstrate a genetic interaction between CRP and PhoP in vivo and reconstitute the phenomenon in vitro, providing compelling evidence that the coregulation by these well-studied regulators does take place. The major weakness is that the logic of the manuscript is difficult to follow as a reader, at times making an evaluation of results and interpretations difficult. The majority of the experimentation involves the whiB1 promoter while conclusions are extrapolated broadly.
We would like to thank the reviewer for her/his constructive comments and suggestions. In the revised manuscript, we have now included numerous changes throughout the ‘Results’ and ‘Discussion’ sections to improve logic of the manuscript and interpretation of the results (please see below our responses). Also, we have included experiments as requested by the reviewers and provided additional data and explanations that address their concerns.
-
Evaluation Summary:
This paper will be of broad interest to those working on the regulation of gene expression and mycobacteria as it deals with the collaboration of two important transcription regulators. A combination of experiments indicates how a complex of two regulators selectively turns on gene expression of a few genes in intracellular pathogen Mycobacterium tuberculosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
Khan et al describe how two important transcription factors functionally cooperate to activate a few of the CRP-dependent genes in Mycobacterium tuberculosis. CRP is a global regulator in eubacteria needed to activate a number of genes while PhoP is an acid stress response regulator required for expression specific set of genes. The authors delineate the interaction between these two key regulators of the bacterial pathogen and show that in a subset of CRP-dependent promoters, PhoP binding recruits CRP to activate transcription.
The experiments are well designed and executed with a coalescent presentation of the manuscript. While the data is well organized and presented with clean images of phophorimages and blots to facilitate their easy understanding, interpretation could have been more robust (see …
Reviewer #1 (Public Review):
Khan et al describe how two important transcription factors functionally cooperate to activate a few of the CRP-dependent genes in Mycobacterium tuberculosis. CRP is a global regulator in eubacteria needed to activate a number of genes while PhoP is an acid stress response regulator required for expression specific set of genes. The authors delineate the interaction between these two key regulators of the bacterial pathogen and show that in a subset of CRP-dependent promoters, PhoP binding recruits CRP to activate transcription.
The experiments are well designed and executed with a coalescent presentation of the manuscript. While the data is well organized and presented with clean images of phophorimages and blots to facilitate their easy understanding, interpretation could have been more robust (see comments below).
Obviously, the strength of the paper is the description of hitherto unknown stress-specific cooperation between two well-studied transcription factors with most evidence supporting the claims. In E.coli (and in other bacteria) studies CRP mediated control of genes have lead to the identification of different classes of CRP-dependent promoters with their own specific regulators. Such a description was lacking in M.tuberculosis and the PhoP - CRP collaboration described is likely to have implications on pathogenesis. The weakness (or possibly what remains to be explored) is that the precise mechanism of the cooperative transcription regulation is yet to be understood.
From the data presented it is apparent that PhoP binds to whiB up promoter own efficiently. It is also evident that CRP is recruited to its site as a result of PhoP binding. This is reminiscent of the bacteriophage Lamba paradigm of positive cooperativity. Thus, it is not reciprocal synergy (as stated in the paper in one place). It is Phop mediated recruitment as claimed elsewhere. Indeed, PhoP null mutants nicely support the latter interpretation
A discussion on why and how CRP binds on its own in other CRP-dependent promoters would help better appreciate the need for PhoP sites next to CRP sites for their cooperative interaction in these promoter subsets. CRP sites could be at a varied distance with respect to the promoter as seen in E.coli.
-
Reviewer #2 (Public Review):
In this manuscript by Khan et al., the authors set out to characterize how the cAMP receptor protein, CRP, and PhoP function to coregulate a subset of virulence genes in Mycobacterium tuberculosis. To this end, the authors use a wide variety of molecular techniques to monitor gene regulation, DNA-binding activity, and protein-protein interactions between phosphorylated PhoP and CRP. The authors conclude that phosphorylated PhoP functions to recruit CRP to promoter regions, where together the two regulators function synergistically to control gene expression. In general, the conclusions of the manuscript appear to be justified by the data, however, the text is difficult to follow. The current version of the paper is likely of interest to scientists within the field of mycobacterial signal transduction.
The …
Reviewer #2 (Public Review):
In this manuscript by Khan et al., the authors set out to characterize how the cAMP receptor protein, CRP, and PhoP function to coregulate a subset of virulence genes in Mycobacterium tuberculosis. To this end, the authors use a wide variety of molecular techniques to monitor gene regulation, DNA-binding activity, and protein-protein interactions between phosphorylated PhoP and CRP. The authors conclude that phosphorylated PhoP functions to recruit CRP to promoter regions, where together the two regulators function synergistically to control gene expression. In general, the conclusions of the manuscript appear to be justified by the data, however, the text is difficult to follow. The current version of the paper is likely of interest to scientists within the field of mycobacterial signal transduction.
The major strength of the paper is that the authors test their hypothesis using a variety of complementary approaches. The authors demonstrate a genetic interaction between CRP and PhoP in vivo and reconstitute the phenomenon in vitro, providing compelling evidence that the coregulation by these well-studied regulators does take place. The major weakness is that the logic of the manuscript is difficult to follow as a reader, at times making an evaluation of results and interpretations difficult. The majority of the experimentation involves the whiB1 promoter while conclusions are extrapolated broadly.
-
