Decision letter: Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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 imuBAAAAGG 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. Editor's evaluation This important study investigates the localization dynamics of the mycobacterial mutasome complex, comprised of ImuA', ImuB, and DnaE2. The mutasome complex has a key role in promoting mutagenic DNA replication during stress to increase the mutation rate and potential for selection of drug resistant mutations. The authors provide compelling evidence that ImuB localizes with the β-clamp upon damage exposure and that the clamp binding motif in ImuB is essential for its localization. These studies lay the ground for future work in this area and will be intriguing to a broad audience interested in bacterial physiology. https://doi.org/10.7554/eLife.75628.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), consistently ranks among the leading infectious killers worldwide (World Health Organization, 2021). The heavy burden imposed by TB on global public health is exacerbated by the emergence and spread of drug-resistant (DR) M. tuberculosis strains, with estimates indicating that DR-TB now accounts for approximately one-third of all deaths owing to antimicrobial resistance (Hasan et al., 2018). In the absence of a wholly protective vaccine, a continually replenishing pipeline of novel chemotherapeutics is required (Evans and Mizrahi, 2018) which, given the realities of modern antibiotic development (Nielsen et al., 2019), appears unsustainable. Therefore, alternative approaches must be explored including the identification of effective multidrug combinations (Cokol et al., 2017), the elucidation of ‘resistance-proof’ compounds (Kling et al., 2015), and the identification of so-called ‘anti-evolution’ drugs that might limit the development of drug resistance (Smith and Romesberg, 2007; Ragheb et al., 2019; Merrikh and Kohli, 2020). Whereas many bacterial pathogens accelerate their evolution by sampling the immediate environment – for example, via fratricide, natural competence, or conjugation (von Wintersdorff et al., 2016; Veening and Blokesch, 2017) – these mechanisms appear inaccessible to M. tuberculosis: the bacillus does not possess plasmids (Gray and Derbyshire, 2018) and there appears to be no role for horizontal gene transfer in the modern evolution of strains of the M. tuberculosis complex (Galagan, 2014; Boritsch and Brosch, 2016). Instead, genetic variation in M. tuberculosis results exclusively from chromosomal rearrangements and mutations, a feature reflecting its ecological isolation (an obligate pathogen, M. tuberculosis has no known host outside humans) and the natural bottlenecks that occur during transmission (Gagneux, 2018). A question which therefore arises is whether a specific molecular mechanism(s) drives M. tuberculosis mutagenesis – perhaps under stressful conditions – and, consequently, if the activity thereof might be inhibited pharmacologically. Multiple studies have investigated mycobacterial DNA replication and repair function in TB infection models (for recent reviews, Singh, 2017; Minias et al., 2018; Mittal et al., 2020). From these, the C-family DNA polymerase, DnaE2, has emerged as major contributor to mutagenesis under antibiotic treatment (Boshoff et al., 2003). A non-essential homolog of E. coli DNA Polymerase (Pol) IIIα (Timinskas et al., 2014), DnaE2 does not operate alone: the so-called ‘accessory factors’, imuA′ and imuB, are critical for DnaE2-dependent mutagenesis (Warner et al., 2010). Both proteins are of unknown function, however imuA′ and imuB are upregulated together with dnaE2 following exposure of mycobacteria to DNA damaging agents including mitomycin C (MMC). That observation prompted the proposal that the three proteins might represent a ‘mycobacterial mutasome’ – named according to its functional analogy with the E. coli DNA Pol V mutasome comprising UmuD′2C-RecA-ATP (Jiang et al., 2009; Erdem et al., 2014). Here, we apply live-cell fluorescence and time-lapse microscopy in characterizing a panel of mycobacterial reporter strains expressing fluorescent translational fusions of each of the known mutasome components. The results of these analyses, together with complementary in vitro biochemical assays utilizing purified mycobacterial proteins, support the inference that ImuB serves as a hub protein, interacting with the dnaN-encoded mycobacterial β clamp and ImuA′. They also reinforce the essentiality of the ImuB–β clamp protein–protein interaction for mutasome function. Notably, while a strong ImuA′–ImuB interaction is detected in vitro, our live-cell data indicate the dispensability of either ImuA′ or DnaE2 for ImuB localization – but not mutasome function – in bacilli exposed to genotoxic stress. Finally, using the β clamp-binding antibiotic, griselimycin (GRS) (Kling et al., 2015), we demonstrate in biochemical assays and in live mycobacteria the capacity to inhibit mutasome function through the pharmacological disruption of ImuB–β focus formation. These observations suggest that, through its inhibition of β clamp binding, GRS might naturally limit the capacity for induced mutagenesis. As well as revealing a built-in mechanism protecting against auto-induced mutations to GRS resistance, our results therefore imply the potential utility of ‘anti-evolution’ antibiotics for TB. Results ImuB forms distinct subcellular foci under DNA damaging conditions Our previous genetic evidence (Warner et al., 2010) informed a tentative model in which the presumed catalytically inactive Y family Pol homolog, ImuB, functioned as an adapter protein. According to the model, DnaE2 gains access to the repair site by interacting with ImuB, which similarly interacts with ImuA′ and the dnaN-encoded β clamp subunit. To investigate the subcellular localizations of each of the mutasome proteins in live bacilli, we constructed reporter alleles in which the M. smegmatis mutasome proteins were labeled by N-terminal translational attachment of either Enhanced Green (EGFP) or Venus Fluorescent Protein (VFP) tags. The reporter alleles were introduced into each of three individual M. smegmatis mutasome gene deletion mutants – ΔdnaE2, ΔimuA′, and ΔimuB (Warner et al., 2010) – to yield the fluorescently tagged complemented strains, ΔdnaE2 attB::egfp-dnaE2 (strain designated G-DnaE2, carrying G-dnaE2 allele), ΔimuB attB::egfp-imuB (G-ImuB), and ΔimuA′ attB::vfp-imuA′ (V-ImuA′) (Figure 1—figure supplement 1A). The mycobacterial DNA damage response was induced by exposing the strains to the natural product antibiotic, MMC, an alkylating agent that causes monofunctional DNA adducts and inter- and intra-strand cross-links (Bargonetti et al., 2010). Following exposure of G-ImuB to MMC for 4 hr, distinct EGFP-ImuB foci were observed (Figure 1A). In contrast, a yellow fluorescence signal was observable throughout V-ImuAʹ cells, suggesting diffuse distribution of the VFP-ImuA′ protein in the mycobacterial cytoplasm (Figure 1B). Although less distinct than G-ImuB, EGFP-DnaE2 produced similar evidence of focus formation in G-DnaE2 cells (Figure 1C). Notably, the significant increase in signal detectable in V-ImuA′, G-ImuB, and G-DnaE2 cells following MMC exposure (Figure 1—figure supplement 1B) confirmed that expression of the respective fluorescence reporter alleles was DNA damage dependent in all three complemented mutants. Figure 1 with 1 supplement see all Download asset Open asset Visualization of the mycobacterial mutasome components. Representative stills from fluorescence microscopy experiments of M. smegmatis expressing translational reporters of the different mutasome components in their respective knockout backgrounds. Phase-contrast and fluorescence images of M. smegmatis expressing (i) G-imuB, (ii) V-imuA′, and (iii) G-dnaE2 alleles are represented following 4 hr exposure to ultra-violet (UV) and 1× minimun inhibitory concentration (MIC) mitomycin C (MMC). White boxes indicate zoomed-in regions shown in the panels at right. The far right-hand panels indicate the fluorescence intensity determined along the longitudinal axis of a representative cell from each reporter mutant; the specific cell analyzed is outlined in the corresponding image to the left of the graph. Fluorescence microscopy experiments were repeated two to four times. Scale bars, 5 µm. Source data are available in Figure1.zip which can be accessed at http://doi.org/10.5061/dryad.76hdr7szc. To ascertain if these observations were true for other types of DNA damage, the three reporter mutants were subjected to ultra-violet (UV) light exposure. Equivalent fluorescence phenotypes were observed for each of the three reporter alleles under both DNA damaging treatments (Figure 1). As UV exposure causes cyclobutane pyrimidine dimers or pyrimidine–pyrimidone (6–4) photoproducts (Boshoff et al., 2003), while MMC generates inter-strand DNA cross-links at CpG sites (Tomasz, 1995), these results indicated that expression and localization (recruitment) of the mutasome components might be independent of the nature of the genotoxic stress applied. N-terminal fluorescent reporters retain wild-type mutagenic function but are deficient in DNA damage tolerance The addition of bulky fluorescent tags can disrupt the function of DNA replication and repair proteins (Renzette et al., 2005). To determine if any of the tagged mutasome proteins was affected, the functionalities of the egfp-imuB, vfp-imuA′, and egfp-dnaE2 alleles were assessed in two standard assays (Boshoff et al., 2003; Warner et al., 2010): the first investigated DNA damage-induced mutagenesis by measuring the frequency of rifampicin (RIF) resistance following exposure to genotoxic stress, and the second tested DNA damage tolerance by spotting serial dilutions of each strain on media containing a DNA damaging agent. As observed previously (Boshoff et al., 2003; Warner et al., 2010), exposure of the wild-type parental M. smegmatis mc2155 to a sub-lethal dose of UV irradiation increased the frequency of RIF resistance 50- to 100-fold, as determined from enumeration of colony-forming units (CFU) on RIF-containing solid growth medium. In contrast, induced mutagenesis was greatly reduced in the ΔimuA′, ΔimuB, and ΔdnaE2 deletion mutants, with mutation frequencies for these ‘mutasome-deficient’ strains approximately 20-fold lower than wild-type (Figure 2A). Notably, complementation with the cognate fluorescent reporter allele in V-ImuA′, G-ImuB, and G-DnaE2 restored the UV-induced mutation frequencies of the three respective knockout mutants to near wild-type levels, establishing that each of the fluorescence reporter alleles retained function in UV-induced mutagenesis assays. In assays utilizing MMC instead of UV, a similar 20-fold reduction in MMC-induced mutagenesis was observed in each of the three single knockout strains compared to wild-type, and this defect was restored when complemented with the respective fluorescent reporters (Figure 2B). In combination, these results confirmed the preservation of wild-type mutagenic function in the fluorescently tagged fusion proteins, irrespective of DNA damaging agent applied. Figure 2 with 1 supplement see all Download asset Open asset Functional validation of translational reporters. (A) N-terminally tagged fluorescence reporter mutants of M. smegmatis ImuA′, ImuB, and DnaE2 retain function in DNA damage-induced mutagenesis. Cultures of M. smegmatis deletion mutants and complemented derivatives were exposed to 25 mJ/cm2 of 254 nm ultra-violet (UV) light and allowed to recover for 3 hr before selection of rifampicin (RIF)-resistant mutants on RIF-containing 7H10 solid agar plates. (i) Mutation frequencies were calculated as a fraction of the CFU/ml of each culture prior to exposure to UV irradiation. Complementation with the corresponding fluorescence reporter alleles restored the resistance frequencies of the three mutasome knockout mutants (ΔimuA′, ΔimuB, and ΔdnaE2) to levels observed in wild-type M. smegmatis. (ii) Representative RIF-containing plates with RIF-resistant mutants. (B) The same strains were exposed to 0.5× MIC mitomycin C (MMC) for 6 hr before plating on RIF-containing 7H10 solid plates. (i) Mutation frequencies were calculated as a fraction of the CFU/ml of each culture prior to exposure to MMC. As for the UV-induced mutagenesis assay, the fluorescence reporter alleles restored mutation frequencies to wild-type levels. (ii) Representative images of the RIF-containing plates with RIF-resistant mutants. (C) Serial dilutions of M. smegmatis deletion mutants and complemented strains were spotted on standard 7H10 and MMC-containing 7H10 plates. Results represent a minimum of three replicates for each strain. Source data are available in Figure2.zip which can be accessed at http://doi.org/10.5061/dryad.76hdr7szc. Surprisingly, the DNA damage tolerance assay – in which CFU-forming ability was tested during continuous exposure to MMC in solid growth media – produced contrasting results (Figure 2C): whereas the damage hypersusceptibility of the dnaE2 knockout was reversed in the G-DnaE2 strain, complementation of either ΔimuA′ or ΔimuB with its corresponding fluorescent reporter allele failed to restore a wild-type phenotype. The reason for these discrepant observations – restoration of both UV- and MMC-induced mutagenesis but not MMC-induced DNA damage tolerance – in the V-ImuA′ and G-imuB strains is not clear. Although mutasome components are expressed in response to genotoxic stress arising from a variety of different sources, it is possible the different types and/or extent of DNA damage induced in the two separate assays used here (induced mutagenesis vs. DNA damage tolerance) might require distinct interactions with a different partner protein(s) and, further, that one/more of these might have been disrupted by the presence of the fluorescent tag(s). It is also plausible that, in the DNA damage survival assay, extended incubation in the presence of MMC (a clastogen with multiple effects on DNA integrity) might exacerbate the suboptimal operation of the mutasome owing to the presence of the bulky fluorophore – which differs significantly from the very brief exposure to the genotoxins in the induced mutagenesis assays. Consistent with the proposed impact of treatment duration on the functionality of the fluorescently tagged mutasome fusions, both V-ImuA′ and G-ImuB mutants phenocopied wild-type in a UV damage sensitivity assay (Figure 2—figure supplement 1); however, these explanations are speculative and require further investigation. Given the inferred functionality of the fluorescence-tagged alleles in DNA damage-induced mutagenesis, we deemed them useful to investigate mutasome recruitment in live mycobacterial cells. ImuB localizes with the dnaN-encoded β clamp following DNA damage We previously inferred that a putative interaction between ImuB and the dnaN-encoded β clamp was essential for mutasome function (Warner et al., 2010). To investigate the predicted interaction of ImuB and the β clamp in live bacilli, each of the three mutasome reporter alleles was introduced separately into an M. smegmatis mutant encoding an mCherry-tagged β clamp, mCherry-DnaN (Santi et al., 2013). The mCherry-DnaN reporter was chosen as background strain owing to its previous validation in single-cell, time-lapse fluorescence microscopy analyses of M. smegmatis replisome location (Santi et al., 2013; Santi and McKinney, 2015). For the time-lapse experiments, the resulting M. smegmatis dual reporter strains were grown in standard 7H9/OADC medium for 12 hr, following which the cells were exposed to MMC for 4.5 hr before switching back to 7H9/OADC for post-treatment recovery (Figure 3; Videos 1–3). At 4 hr post MMC treatment, distinct EGFP-ImuB foci were observed which, when overlaid with the mCherry-DnaN fluorescence signal, showed considerable overlap, suggesting association of the β clamp with ImuB (Figure 3A, D; Video 1). In addition to G-ImuB, the number of mCherry-DnaN foci also increased upon DNA damage (Figure 3; Figure 3—figure supplement 1A). In MMC-treated cells, the EGFP-ImuB signal was mostly detected in very close proximity to mCherry-DnaN foci (>50% of cells contained mCherry-DnaN and G-ImuB located within 0.3 μm of each other); almost the same frequency of association of mCherry-DnaN and G-ImuB foci was observed in bacilli exposed to UV, though the proportion of cells containing mCherry-DnaN foci alone was greater (Figure 3A, D; Figure 3—figure supplement 1B). In combination, these results are consistent with the direct physical interaction of ImuB and the β clamp suggested previously by yeast two-hybrid and site-directed mutagenesis studies (Warner et al., 2010). Figure 3 with 2 supplements see all Download asset Open asset Representative time-lapse series of single cells of M. smegmatis expressing the mutasome reporters in combination with mCherry-DnaN. (A) G-ImuB (green) and mCherry-DnaN (magenta), (B) V-ImuA′ (green) and mCherry-DnaN (magenta), and (C) G-DnaE2 (green) and mCherry-DnaN (magenta). Overlapping signals are viewed as white. The cells were exposed to 0.5× MIC MMC from time 0 hr until 4.5 hr, after which the medium was switched back to standard 7H9/OADC medium. Up to 80 XY points were imaged at 10-min intervals on fluorescence and phase channels for up to 36 hr. The experiments were repeated two to four times. Numbers indicate hours elapsed; scale bars, 5 μm. 7H9, Middlebrook 7H9 medium; MMC, mitomycin C. (D) Population-scale analysis of cells with both mCherry-DnaN foci and G-ImuB foci showed distinct overlap in location suggesting co-occurrence of the respective proteins. Source data are available in Figure3.zip which can be accessed at http://doi.org/10.5061/dryad.76hdr7szc. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse microscopy of G-ImuB and mCherry-DnaN dual reporter. Representative time-lapse movie of the reporter strain expressing G-ImuB and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment with MMC (100 was at hr. This was repeated times. Numbers indicate the hours in the time-lapse 7H9, Middlebrook MMC, mitomycin C. Scale 5 μm. G-ImuB, white. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse microscopy of V-ImuA′ and mCherry-DnaN dual reporter. Representative time-lapse movie of the reporter strain expressing and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment with MMC (100 was at hr. This was repeated three times. Numbers indicate the hours in the time-lapse 7H9, Middlebrook MMC, mitomycin C. Scale 5 μm. white. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse microscopy of G-DnaE2 and mCherry-DnaN dual reporter. Representative time-lapse movie of the reporter strain expressing G-DnaE2 and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment with MMC (100 was at hr. This was repeated three times. Numbers indicate the hours in the time-lapse 7H9, Middlebrook MMC, mitomycin C. Scale 5 μm. G-DnaE2, white. For V-ImuA′, a diffuse fluorescence signal was detected throughout the cells (Figure Video any the potential recruitment of ImuA′ to β clamp foci. In contrast, the results for DnaE2 were overlap of fluorescence signals from EGFP-DnaE2 and mCherry-DnaN proteins was detected (Figure and was within post of MMC from the Although not as consistent as the ImuB–β clamp the co-occurrence of DnaE2 and β clamp signals was observed in multiple cells and different ImuA′ and DnaE2 are not required for ImuB focus formation We showed previously that deletion of imuA′ phenocopied of either imuB or dnaE2 in the MMC sensitivity assay (Warner et al., 2010) and, consistent with the that all three components are essential for mutasome this phenotype was not exacerbated in a knockout strain. with yeast two-hybrid data which indicated a direct interaction between ImuB and ImuA′ (Warner et al., 2010), this observation the that a in ImuA′ might ImuB protein localization. To this the allele was introduced into the ΔimuA′ deletion a ΔimuA′ attB::egfp-imuB reporter strain. the absence of ImuA′ in this EGFP-ImuB foci were observed following treatment with MMC (Figure 3—figure supplement 2A). the absence of functional DnaE2 no impact on ImuB focus formation in either the site-directed attB::egfp-imuB strain (Figure 3—figure supplement or the ΔdnaE2 attB::egfp-imuB mutant (Figure 3—figure supplement In combination, these results appear to a role for either ImuA′ or DnaE2 in ImuB instead the critical of the ImuB–β clamp interaction for mutasome mutasome proteins in biochemical assays in vitro inference from this and previous work the composition of the mycobacterial mutasome has been from assays. To this we expressed and purified M. smegmatis mutasome proteins for biochemical in E. coli of ImuB alone of protein that was to while to ImuA′ alone failed to protein. In contrast, of ImuB with ImuA′ both proteins in a (Figure the complex be via a in This confirmed that ImuA′ and ImuB in vitro, a complex at protein as as (Figure supplement previous yeast two-hybrid results (Warner et al., 2010). In E. of DnaE2 in protein, while DnaE2 in M. smegmatis to be with cell following with the expression very were and not be in culture Figure 4 with 1 supplement see all Download asset Open asset ImuB and ImuA′–ImuB with and these interactions are disrupted by griselimycin (A) of M. smegmatis (i) and (ii) in the absence or presence of For these experiments, 5 was to of (i) or (ii) The of the individual proteins and or complex are shown for and all were for (B) analysis of of the are in the same as the corresponding shown in Source data are available in which can be accessed at http://doi.org/10.5061/dryad.76hdr7szc. we analyzed the interaction of the dnaN-encoded β clamp with ImuB or the complex (Figure of the M. smegmatis β clamp with (Figure panel or ImuB (Figure panel were into an and subjected to the β clamp and at and of the β clamp with either ImuB or a in the to of complex formation. This was confirmed by which indicated of the β clamp with ImuB and (Figure EGFP-ImuB and VFP-ImuA′ a complex Our assays discrepant complementation phenotypes for the induced mutagenesis DNA damage tolerance assay (Figure the that the fluorescent tags in the mutants might disrupt a protein–protein essential for DNA damage We therefore investigated the capacity of the fluorescently labeled EGFP-ImuB and VFP-ImuA′ proteins to a To this was with in E. coli and the complex analyzed in three (Figure supplement 1B). the cell was a to the complex via the in the containing the complex were on a to the complex via the on Finally, the complex was a all EGFP-ImuB and VFP-ImuA′ were as a complex, as indicated by analysis and fluorescent of EGFP-ImuB and VFP-ImuA′ in the same In combination, these observations suggest that the fluorescent tags not disrupt ImuA′–ImuB complex formation in vitro – a which that the absence in live cells of a ImuA′ phenotype was not to the presence of N-terminal of ImuB–β clamp-binding focus formation work that the β clamp-binding of ImuB was essential for mutasome mutant strains carrying either a allele the in the ImuB or a imuBAAAAGG allele which the wild-type β clamp-binding is with the phenocopied imuB deletion (Warner et al., 2010). Therefore, to the that the recruitment of EGFP-ImuB and mCherry-DnaN into foci was dependent on the ImuB–β clamp protein–protein we introduced an allele into the ΔimuB In to the wild-type reporter (G-ImuB), the β clamp-binding motif mutant no foci in any cell imaged following exposure to MMC (Figure Instead, the fluorescence was detectable throughout the cell as a diffuse This the inferred essentiality of the physical interaction between ImuB and β for ImuB localization and, that of ImuB–β foci a for functional mutasome formation. Figure 5 with 1 supplement see all Download asset Open asset the ImuB–β clamp (A) Representative images of G-ImuB exposed to MIC mitomycin C (MMC) for 4 hr or MIC MMC griselimycin (GRS) for 4 hr and the mutant exposed to MIC MMC for 4 hr Scale bars, 5 μm. (B) of M. smegmatis β clamp with (i) (ii) ImuB, or (iii) the DNA The interaction of the β clamp with GRS is represented by the of the complex interactions with ImuB and are from the respective