Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome
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Evaluation Summary:
Drug resistance in a problem in the control of many infections, including Mycobacterium tuberculosis. In mycobacteria, an error prone DNA polymerase facilitates DNA damage induced mutagenesis to increase the rate of generation of drug resistant strains. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. In this manuscript, the authors test their previously proposed model that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. This is of interest to a broad audience interested in microbiology, antibiotic resistance, and genome stability.
(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. The reviewers remained anonymous to the authors.)
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Abstract
A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded ‘mycobacterial mutasome’ – minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins – remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuB AAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB–β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB–β clamp complexes. These observations establish the essentiality of the ImuB–β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.
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Evaluation Summary:
Drug resistance in a problem in the control of many infections, including Mycobacterium tuberculosis. In mycobacteria, an error prone DNA polymerase facilitates DNA damage induced mutagenesis to increase the rate of generation of drug resistant strains. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. In this manuscript, the authors test their previously proposed model that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. This is of interest to a broad audience interested in microbiology, antibiotic resistance, and genome stability.
(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. The …
Evaluation Summary:
Drug resistance in a problem in the control of many infections, including Mycobacterium tuberculosis. In mycobacteria, an error prone DNA polymerase facilitates DNA damage induced mutagenesis to increase the rate of generation of drug resistant strains. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. In this manuscript, the authors test their previously proposed model that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. This is of interest to a broad audience interested in microbiology, antibiotic resistance, and genome stability.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, the authors test their previously proposed model (also presented in Figure 1A) that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. Although the exact function of ImuA' and ImuB is unknown, ImuB has long before been proposed to interact with DnaN via an interaction domain within ImuB that has already been identified. Since the experiments herein test and validate a well-establish model, the results are somewhat expected. However, all models should be tested experimentally, making this an important confirmation. The manuscript nicely makes use of both in vivo and in vitro approaches and the data is convincing for the most part, although the …
Reviewer #1 (Public Review):
In this manuscript, the authors test their previously proposed model (also presented in Figure 1A) that ImuB interacts with the DnaN DNA polymerase III β clamp to recruit DnaE2. The previously identified mutasome components ImuA', ImuB, and DnaE2 and essential for DNA-damage induced mutagenesis. Although the exact function of ImuA' and ImuB is unknown, ImuB has long before been proposed to interact with DnaN via an interaction domain within ImuB that has already been identified. Since the experiments herein test and validate a well-establish model, the results are somewhat expected. However, all models should be tested experimentally, making this an important confirmation. The manuscript nicely makes use of both in vivo and in vitro approaches and the data is convincing for the most part, although the inability of the fusion proteins used in this study to complement the knockout strains during exposure to the DNA damaging agent MMC does raise an important limitation of the tools used herein. The major concern is the limited new biological insight gained from the study.
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Reviewer #2 (Public Review):
The imuABC genes from Caulobacter crescentus were first described almost 20 years ago, yet very little biochemical analysis regarding their mechanism of action has been published. It is now appreciated that these imuABC genes are present in a large number of clinically important bacterial pathogens, underscoring their potential importance to adaptation and antibiotic resistance. The imuA gene in M. tuberculosis is somewhat diverged from imuA in C. crescentus (the first described ImuABC), and is thus named imuA' to reflect this. The goal of the work described in this report was to gain further insights into the function of the M. tuberculosis ImuA', ImuB, and ImuC proteins in DNA damage-induced mutagenesis. The authors used fluorescent fusions of the M. tuberculosis imuA'BC gene products to demonstrate that …
Reviewer #2 (Public Review):
The imuABC genes from Caulobacter crescentus were first described almost 20 years ago, yet very little biochemical analysis regarding their mechanism of action has been published. It is now appreciated that these imuABC genes are present in a large number of clinically important bacterial pathogens, underscoring their potential importance to adaptation and antibiotic resistance. The imuA gene in M. tuberculosis is somewhat diverged from imuA in C. crescentus (the first described ImuABC), and is thus named imuA' to reflect this. The goal of the work described in this report was to gain further insights into the function of the M. tuberculosis ImuA', ImuB, and ImuC proteins in DNA damage-induced mutagenesis. The authors used fluorescent fusions of the M. tuberculosis imuA'BC gene products to demonstrate that ImuA', ImuB, and ImuC each colocalized with the beta sliding clamp protein in live cells. The beta clamp helps to organize replication and repair proteins on the DNA at replication forks; thus their colocalization implies functional complexes. These in vivo results were correlated with biochemical results in which they used size exclusion chromatography to measure interactions between the purified beta sliding clamp protein and the ImuB protein or ImuA'-ImuB protein complex. These biochemical results support earlier published results describing these interactions using yeast-two-hybrid (Warner et al., 2010, PNAS). Importantly, the authors also demonstrate that both a mutant ImuB containing a disruption to its beta clamp binding motif, as well as griselimycin (GSR), an antimicrobial that binds the beta clamp at the same site required for its interaction with the clamp binding motif, both impaired ImuB-beta clamp interactions in vitro, and resulted in the loss of their colocalization in vivo. A potentially powerful conclusion of this work is that adjuvant therapies such as GSR may inhibit mutagenesis, limiting M. tuberculosis drug resistance, facilitating the ability of traditional antimicrobial therapies to treat the infection. While the work has several strengths, there are also some shortcomings.
Strengths:
The authors describe fluorescent fusions of the M. tuberculosis ImuA', ImuB, and ImuC proteins that support ImuABC function in UV-induced mutagenesis. Using these fusions, they demonstrate colocalization of the different Imu proteins and the beta clamp in live cells following treatment with MMC.
This is the first report of purified ImuA' and ImuB, which allowed the authors to biochemically test for their interactions with each other, as well as interaction of ImuB with the beta clamp, both of which were previously reported based on results of yeast-two-hybrid experiments (Warner et al., 2010, PNAS). This, together with the live cell imaging work, goes a long way towards testing and refining the model for ImuA'BC 'mutasome' function first proposed in 2010 (Warner et al., PNAS).
The authors provide in vitro results suggesting GSR, in addition to its known role in blocking replication, may also inhibit mutagenesis in M. tuberculosis. This is an exciting possibility, and provides support for the view that anti-evolution therapies may be possible.
Shortcomings:
Colocalization studies were performed using ImuA' and ImuB fluorescent fusions that failed to complement MMS sensitivity. Given their in vivo inactivity of these fusions, what does their colocalization with the beta clamp actually mean? Since these same fusions supported UV mutagenesis, it seems UV may be a superior means of analyzing colocalization of these fluorescently tagged proteins.
The methods used to analyze the colocalization is not explained for non-experts. A more complete description of how colocalization was established along the z-axis is needed. Likewise, a more thorough discussion of exactly what the foci consist of is needed.
ImuC could not be purified for in vitro analysis. This is unfortunate since it is thought to harbor the polymerase activity involved in mutagenesis. While not the fault of the authors, this significantly limits the scope of the in vitro work regarding ImuA'BC function in mutagenesis.
The in vitro analysis of ImuA'-ImuB, ImuB-beta clamp, and ImuA'-ImuB-beta clamp interactions lack quantitative descriptions, and a complete analysis of the possible interactions among these proteins was not explored, limiting our understanding of their possible function in mutagenesis.
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Reviewer #3 (Public Review):
In this study, Gessner et al., characterize the localization dynamics of the mycobacterial mutasome complex, comprising ImuA', ImuB and DnaE2, in order to understand the molecular composition and mechanism of action of this complex in living cells. For this, they construct semi-functional fluorescent fusion constructs of ImuB (as well as ImuA' and DnaE2) in M. smegmatis, a non-pathogenic model system used to study several pathways present in M. tuberculosis. They find that ImuB localizes with the beta-clamp upon damage exposure. They further show that the clamp binding motif in ImuB is essential for its localization as well as in vitro beta-clamp interaction. Finally, they treat cells with the beta-clamp targeting antibiotic griselimycin and find that it abrogates ImuB interaction with the clamp. They …
Reviewer #3 (Public Review):
In this study, Gessner et al., characterize the localization dynamics of the mycobacterial mutasome complex, comprising ImuA', ImuB and DnaE2, in order to understand the molecular composition and mechanism of action of this complex in living cells. For this, they construct semi-functional fluorescent fusion constructs of ImuB (as well as ImuA' and DnaE2) in M. smegmatis, a non-pathogenic model system used to study several pathways present in M. tuberculosis. They find that ImuB localizes with the beta-clamp upon damage exposure. They further show that the clamp binding motif in ImuB is essential for its localization as well as in vitro beta-clamp interaction. Finally, they treat cells with the beta-clamp targeting antibiotic griselimycin and find that it abrogates ImuB interaction with the clamp. They suggest that ImuB localization (or its disruption) can serve as a reliable proxy to screen for mutasome-inhibiting antibacterial drugs.
Strengths:
The in vivo dynamics of the ImuA'-ImuB-DnaE2 complex is not well-studied, when compared with its E. coli counterpart UmuDC-RecA. In this direction, the authors generate a set of tools that can be utilized to understand the molecular mechanism of action of this complex in detail. The distinct localization dynamics of ImuA' and ImuB is of particular interest, given the several unresolved questions associated with ImuA' function in induced mutagenesis.
Limitations:
The strength of the manuscript lies in the imaging data, which is rich with information with regards to the dynamics of localizations over time for clamp, ImuB and DnaE2, under damage. In the current form, the authors do not utlize this to provide insights into any "real-time" dynamics of the mutasome. The results with regards to the clamp interaction are well-done, but largely confirmatory. Importantly, it remains unclear whether the localization read-out is reliably indicative of mutasome function, given the discrepancy in performance of the constructs in UV vs MMC-induced damage. The authors primarily rely on MMC for all their in vivo read-outs and the tags do not perform as wild type in this damage condition. This needs to be resolved.
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