Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
The present study's main aim is to investigate the mechanism of how VirR controls the magnitude of MEV release in Mtb. The authors used various techniques, including genetics, transcriptomics, proteomics, and ultrastructural and biochemical methods. Several observations were made to link VirR-mediated vesiculogenesis with PG metabolism, lipid metabolism, and cell wall permeability. Finally, the authors presented evidence of a direct physical interaction of VirR with the LCP proteins involved in linking PG with AG, providing clues that VirR might act as a scaffold for LCP proteins and remodel the cell wall of Mtb. Since the Mtb cell wall provides a formidable anatomical barrier for the entry of antibiotics, targeting VirR might weaken the permeability of the pathogen along with the stimulation of the immune system due to enhanced vesiculogenesis. Therefore, VirR could be an excellent drug target. Overall, the study is an essential area of TB biology.
We thank the reviewer for the kind assessment of our paper.
Strengths:
The authors have done a commendable job of comprehensively examining the phenotypes associated with the VirR mutant using various techniques. Application of Cryo-EM technology confirmed increased thickness and altered arrangement of CM-L1 layer. The authors also confirmed that increased vesicle release in the mutant was not due to cell lysis, which contrasts with studies in other bacterial species.
Another strength of the manuscript is that biochemical experiments show altered permeability and PG turnover in the mutant, which fits with later experiments where authors provide evidence of a direct physical interaction of VirR with LCP proteins.
Transcriptomics and proteomics data were helpful in making connections with lipid metabolism, which the authors confirmed by analyzing the lipids and metabolites of the mutant.
Lastly, using three approaches, the authors confirm that VirR interacts with LCP proteins in Mtb via the LytR_C terminal domain.
Altogether, the work is comprehensive, experiments are designed well, and conclusions are made based on the data generated after verification using multiple complementary approaches.
We are glad that this reviewer finds our study of interest and well designed.
Weaknesses:
(1) The major weakness is that the mechanism of VirR-mediated EV release remains enigmatic. Most of the findings are observational and only associate enhanced vesiculogenesis observed in the VirR mutant with cell wall permeability and PG metabolism. The authors suggest that EV release occurs during cell division when PG is most fragile. However, this has yet to be tested in the manuscript - the AFM of the VirR mutant, which produces thicker PG with more pore density, displays enhanced vesiculogenesis. No evidence was presented to show that the PG of the mutant is fragile, and there are differences in cell division to explain increased vesiculogenesis. These observations, counterintuitive to the authors' hypothesis, need detailed experimental verification.
We concur with the reviewer that we do not have direct evidence showing a more fragile PG in the virR mutant and our statement is supported by a compendium of different results. However, this statement is framed in the discussion section as a possible scenario, acknowledging that more experiments are needed to make such connection. Nevertheless, we provide additional data on the molecular characterization of virRmut PG using MS to show a significant increase in the abundance of deacetylated muropeptides, a feature that has been linked to altered lysozyme sensitivity in other unrelated Gram-positive bacteria
(Fig 8 G,H).
(2.1) Transcriptomic data only adds a little substantial. Transcriptomic data do not correlate with the proteomics data. It remains unclear how VirR deregulates transcription.
We concur with the reviewer that information provided by transcriptomics and proteomics is a bit fragmented and, taking into consideration the low correlation between both datasets, it does not help to explain the phenotype observed in the mutant. This issue has also been raised by another reviewer so, we have paid special attention to that.
To refine the biological interpretation of the transcriptomic data we have integrated the complemented strain (virRmut-Comp) in our analyses. This led us to narrow down the virR-dependent transcriptomics signature to the sets of genes that appear simultaneously deregulated in virRmut with respect to both WT and complemented strain in either direction. Furthermore, to identify the transcription factors whose regulatory activity appear disrupted in the mutant strain, we have resorted to an external dataset (Minch et al. 2015) and found a set of 10 transcriptional regulators whose regulons appear significantly impacted in the virRmut strain. While admittedly these improvements do not fully address the question tackled by the reviewer, we found that they contribute to a more precise characterization of the VirR-dependent transcriptional signatures, as well as the regulons, in the genome-wide transcriptional regulatory network of the pathogen that appear altered because of virR disruption. We acknowledge that the lack of correlation between whole-cell lysates proteomics and transcriptomic data is something intriguing, albeit not uncommon in Mycobacterium tuberculosis. However, differences in the protein cargo of the vesicles from different strains share key pathways in common with the transcriptomic analyses, such as the enrichments in cell wall biogenesis and peptidoglycan biosynthesis that are observed both among genes that are downregulated in both cases in virRmut.
(2.2) TLCs of lipids are not quantitative. For example, the TLC image of PDIM is poor; quantitative estimation needs metabolic labeling of lipids with radioactive precursors. Further, change in PDIMs is likely to affect other lipids (SL-1, PAT/DAT) that share a common precursor (propionyl- CoA).
We also agree with the reviewer that TLC, as it is, it is not quantitative. However, we do not have access to radioactive procedures. In the new version of the manuscript, we have run TLCs on all the strains tested to resolve SLs and PAT/DATs (Fig S8). Our results show a reduction in the pool of SL and DATs in the mutant, indicating that part of the methylmalonil pool is diverted to the synthesis of PDIMs.
(3) The connection of cholesterol with cell wall permeability is tenuous. Cholesterol will serve as a carbon source and contribute to the biosynthesis of methyl-branched lipids such as PDIM, SL-1, and PAD/DAT. Carbon sources also affect other aspects of physiology (redox, respiration, ATP), which can directly affect permeability and import/export of drugs. Authors should investigate whether restoration of the normal level of permeability and EV release is not due to the maintenance of cell wall lipid balance upon cholesterol exposure of the VirR mutant.
We concur with the reviewer that cholesterol as a sole carbon source is introducing many changes in Mtb cells beside permeability. Consequently, we investigated the virRmut lipid profile upon exposure to either cholesterol or TRZ (Fig S8). Both WT and virRmut-Comp strains were included in the analysis. Polar lipid analysis revealed that either cholesterol or TRZ exposure induced a marked reduction in PIMs and cardiolipin (DPG) levels in virRmut relative to WT or complemented strains (Fig S8A). Analysis of apolar lipids indicated that, relative to glycerol MM, virRmut cultured in the presence of cholesterol or TRZ showed reduced levels of TDM and DATs compared to WT and virRmut-Comp strains (Fig S8B). These results suggest a lack of correlation between modulation of cell permeability by cholesterol and TRZ and lipid levels in the absence of VirR.
Furthermore, about this section, we would like to mention that we have modified the reference used for the annotation of the DosR regulon: moving from the definition of the regulon used in the previous submission (coming from Rustad, el at. PLoS One 3(1), e1502 (2008). The enduring hypoxic response of Mycobacterium tuberculosis) to the more recent characterization of the regulon based on CHiPseq data, reported in Minch et al. 2015. This was done to ensure coherence with the transcriptomics analyses in the new figure 4.
(4) Finally, protein interaction data is based on experiments done once without statistical analysis. If the interaction between VirR and LCP protein is expected on the mycobacterial membrane, how the SPLIT_GFP system expressed in the cytoplasm is physiologically relevant. No explanation was provided as to why VirR interacts with the truncated version of LCP proteins and not with the full-length proteins.
We have repeated the experiments and applied statistics (Figure 9). As stated in the manuscript this assay has successfully been applied to interrogate interactions of domains of proteins embedded in the membrane of mycobacteria. Therefore, we believe that this assay is valid to interrogate interactions between Lcp proteins.
Reviewer #2 (Public Review):
Summary:
In this work, Vivian Salgueiro et al. have comprehensively investigated the role of VirR in the vesicle production process in Mtb using state-of-the-art omics, imaging, and several biochemical assays. From the present study, authors have drawn a positive correlation between cell membrane permeability and vesiculogenesis and implicated VirR in affecting membrane permeability, thereby impacting vesiculogenesis.
Strengths:
The authors have discovered a critical factor (i.e. membrane permeability) that affects vesicle production and release in Mycobacteria, which can broadly be applied to other bacteria and may be of significant interest to other scientists in the field. Through omics and multiple targeted assays such as targeted metabolomics, PG isolation, analysis of Diaminopimelic acid and glycosyl composition of the cell wall, and, importantly, molecular interactions with PG-AG ligating canonical LCP proteins, the authors have established that VirR is a central scaffold at the cell envelope remodelling process which is critical for MEV production.
We thank the reviewer for the kind assessment of the paper.
Weaknesses:
Throughout the study, the authors have utilized a CRISPR knockout of VirR. VirR is a non-essential gene for the growth of Mtb; a null mutant of VirR would have been a better choice for the study.
According to Tn mutant databases and CRISPR databases, virR is a non-essential gene. However, we have tried to interrupt this gene using the allelic exchange substitution approach via phages many times with no success. So far there is no precedent of a clean KO mutant in this gene. White et al., generated a virR mutant consisting of deletion of a large fragment of the c-terminal part of the protein, pretty much replicating the effect of the Tn insertion site in the virR Tn mutant. These precedents made us to switch to CRISPR technology.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) The authors monitored cell lysis by measuring the release of a cytoplasmic iron-responsive protein (IdeR). Since EV release is regulated by iron starvation, which is directly sensed by IdeR, another control (unrelated to iron) is needed. A much better approach would be to use hydrophobic/hydrophilic probes to measure changes in the cell wall envelope.
Does the VirR complemented strain have a faint IdeR band in the supernatant? The authors need to clarify. Also, it's unclear whether the complementation restored normal VirR levels or not.
We thank the reviewer for this recommendation. Consequently, we have complemented these studies by an alternative approach based on serially diluted cultures spotted on solid medium. These results align very well with that of western blot using IdeR levels in the supernatant as a surrogate of cell lysis.
We also noticed the presence of a faint IdeR band in the supernatant of the complemented strain and suggestive of a possible cell lysis. However, as shown in other section this was not translated into increased levels of vesiculation. As previously shown in a previous paper describing VirR as a genetic determinant of vesiculogenesis, VirR levels in the complemented strains are not just restored but increased considerably. This overexpression could explain the potential artifact of a leaky phenotype in the complemented strain. In addition to that previous study, the proteomic data included in this paper clearly shows a restoration of VirR levels relative to the WT strains.
(2) Figure 2C: The data are weak; I don't see any difference in incorporating FDAAs in MM media. Even in the 7H9 medium, differences appear only at the last time point (20 h). What happens at the time point after 20 h (e.g., 48 h)? How do we differentiate between defective permeability or anabolism leading to altered PG? No statistical analysis was performed.
We apologize for the incomplete assessment of the results in this figure. First, this figure just shows differential incorporation of FDAAs in the different strains in different media. As per previous studies (Kuru et al (2017) Nat. Protocols), these probes can freely enter into cells and may be incorporated into PG by at least three different mechanisms, depending on the species: through the cytoplasmic steps of PG biosynthesis and via two distinct transpeptidation reactions taking place in the periplasm. Consequently, the differential labeling observed in virRmut relative to WT strain may be a consequence of the enlarge PG observe din the mutant. We have repeated the experiment and created new data. First, we have cultured strains with a blue FDAA (HADA) for 48 to ensure full labeling. Then, we washed cells and cultured in the presence of a second FDAA, this time green (FDL) for 5 h. The differential incorporation of FDL relative to HADA was then measured under the fluorescence microscope. This experiment showed a virRmut incorporate more FDL that the other strains, suggesting an altered PG remodeling. modified the figure to make clearer the early and late time points of the time-course and applied statistics.
(3) Many genes (~ 1700) were deregulated in the mutant. Since these transcriptional changes do not correlate at the protein level in WCL, it's important to determine VirR-specificity. RNA-Seq of VirR complemented strain is important.
We think this was an extremely important point, and we thank the reviewer for pointing this out. Following their suggestion, we have analyzed and integrated data from the complemented strain, which we have added to the GEO submission, to conclude that, in fact, differences in expression between the complemented strain and either the WT, or virRmut are also common and highly significant. Albeit this is not completely unexpected, given the nature of our mutants and the fact that the complemented strains show significantly higher levels of expression of VirR -both at the RNA and protein levels- than the WT, it motivated us to narrow down our definition of VirR-dependent genes to adopt a combined criterium that integrated the complemented strain. Following this approach, we considered the set of genes upregulated (downregulated) in virRmut as those whose expression in that strain is, at the same time, significantly higher (lower) than in WT as well as in virRmut-Comp. Working with this integrated definition, the genes considered -399 upregulated and 502 downregulated genes- are those whose observed expression changes are more likely to be genuinely VirR-dependent rather than any non-specific consequence of the mutagenesis protocols. Despite the lower number of genes in these sets, the repetition of all our functional enrichment analyses based on this combined criterium leads us to conclusions that are largely compatible with those presented in the first version of the paper.
(4) Transcriptome data provide no clues about how VirR could mediate expression deregulation. Is there an overlap with the regulations/regulons of any Mtb transcription factors? One clue is DosR; however, DosR only regulates 50-60 genes in Mtb.
Again, we would like to thank the reviewer for this recommendation, which we have followed accordingly to generate a new section in the results named “VirR-dependent genes intersect the regulons of key transcriptional regulators of the responses to stress, dormancy, and cell wall remodeling”. As we explain in this new section, we resorted to the regulon annotations reported in (Minch et al. 2015), where ChIP-seq data is collected on binding events between a panel of 143 transcription factors (TFs) and DNA genome-wide. The dataset includes 7248 binding events between regulators and DNA motifs in the vicinity of targets’ promoters. After completing enrichment analyses with the resulting regulons, we identified 10 transcription factors whose intersections with the sets of up and downregulated genes in virRmut were larger than expected by chance (One tailed Fisher exact test, OR>2, FDR<0.1). Those regulators -which, as guessed by the referee, included DevR-, control key pathways related with cell wall remodeling, stress responses, and transition to dormancy.
(5) How many proteins that are enriched or depleted in the EVs of the VirR mutant also affected transcriptionally in the mutant? How does VirR regulate the abundance and transport of protein in EVs?
While the intersection between genes and proteins that appear upregulated in the virRmut strain both at transcriptional and vesicular protein levels (N=21) was found larger than expected by chance (OR=2.0 p=7.0E-3), downregulated genes and proteins in virRmut (N=14) were not enriched in each other. These results, indicated, at most, a scarce correlation between RNA and protein levels (a phenomenon nonetheless previously observed in Mycobacterium tuberculosis, among other organisms, see Cortés et al. 2013). Admittedly, the compilation of these omics data is insufficient, by itself to pinpoint the specific regulatory mechanisms through which the absence of VirR impacts protein abundance in EVs. For the sake of transparency, this has been acknowledged in the discussion section of the resubmitted version of the manuscript.
(6) The assumption that a depleted pool of methylmalonyl CoA is due to increased utilization for PDIM biosynthesis is problematic. Without flux-based measurement, we don't know if MMCoA is consumed more or produced less, more so because Acc is repressed in the VirR mutant EVs. Further, MMCoA feeds into the TCA cycle and other methyl-branched lipids. Without data on other lipids and metabolism, the depletion of MMCoA is difficult to explain.
The differential expression statistics compiled suggest that both effects may be at place, since we observed, at the same time, a downregulation of enzymes controlling methylmalonyl synthesis from propionyl-CoA (i.e. Acc, at the protein level), as well as an upregulation of enzymes related with its incorporation into DIM/PDIMs (i.e. pps genes). Both effects, combined, would favor an increased rate of methylmalonyl production, and a slower depletion rate, thus contributing to the higher levels observed. We however concur with the reviewer that fluxomics analyses will contribute to shed light on this question in a more decisive manner, and we have acknowledged this in the discussion section too.
(7) Figure 5: Deregulation of rubredoxins and copper indicates impaired redox balance and respiration in the mutant. The data is complex to connect with permeability as TRZ is mycobactericidal and also known to affect the respiratory chain. The authors need to investigate if, in addition to permeability, the presence of VirR is essential for maintaining bioenergetics.
The data related to rubredoxins and copper has been modified after reanalyzing transcriptomic data including the complemented strain. Nevertheless, we found that some features of the response to stresses may be impaired in the mutant, including the one to oxidative stress. In this regard, we found the enhanced sensitivity of the mutant to H2O2 relative to WT and complemented strains. This piece of data is now included as Fig S3 in the new version of the manuscript.
(8) Differential regulation of DoS regulon and cholesterol growth could also be linked to differences in metabolism, redox, and respiration. What is the phenotype of VirR mutants in terms of growth and respiration in the presence of cholesterol/TRZ?
We thank the reviewer for this suggestion. Consequently, we have added a new section to Results that suggest that other aspects of mycobacterial physiology may be affected in the virR mutant when cultured in the presence of cholesterol or TRZ:
“Modulation of EV levels and permeability in virRmut by cholesterol and TRZ. We next wondered about the effect of culturing virRmut on both cholesterol or TRZ could have on cell growth, permeability and EV production. In the case of cholesterol, it has also been shown to affect other aspects of physiology (redox, respiration, ATP), which can directly affect permeability (Lu et al., 2017). We monitored virRmut growth cultured in MM supplemented with either glycerol, cholesterol as a sole carbon source, and TRZ at 3 ug ml-1 for 20 days. While cholesterol significantly enhanced the growth virRmut after 5 days relative to glycerol medium, supplementation of glycerol medium with TRZ restricted growth during the whole time-course (Fig S5A). The study of cell permeability in the same conditions indicated that the enhanced cell permeability observed in glycerol MM was reduced when virRmut when cultured with cholesterol as sole carbon source. Conversely, the presence of TRZ increased cell permeability relative to the medium containing solely glycerol (Fig S5C). As we have previously observed for the WT strain, either condition (Chol or TRZ) also modified vesiculation levels in the mutant accordingly (Fig S5B). These results strongly indicates that other aspects of mycobacterial physiology besides permeability are also affected in the virR mutant and may contribute to the observed enhanced vesiculation.
(9) PDIM TLC is not evident; both DimA and DImB should be clearly shown. It will also be necessary to show other methyl-branched lipids, such as SL-1 and PAT/DAT, because the increase in PDIM can take away methyl malonyl CoA from the biosynthesis of SL-1 and PAT/DAT. Studies have shown that SLI-, PAT/DAT, and PDIM are tightly regulated, where an increase in one lipid pool can affect the abundance of other lipids. Quantitative assays using 14C acetate/propionate are most appropriate for these experiments.
We apologize for the fact that TLC analysis is not performed in a radioactive fashion. However, we do not have access to this approach. To answer reviewer question about the fact that other methyl-branched lipids may explain the altered flux of methyl malonyl CoA, we have run TLCs on all the strains tested to resolve SLs and PAT/DATs (Fig S8). Notably, we observed a reduction in the level of these lipids (SL1 or PAT/DAT) in virRmut cultured in glycerol relative to WT and complemented strains, suggesting that the excess of PDIM synthesis can take away methyl malonyl CoA from the biosynthesis of SL-1 and PAT/DAT in the absence of VirR (Fig S8B).
(10) Figure 8: Interaction between VirR and Lcp proteins. Since these interactions are happening in the membrane, using a split GFP system where proteins are expressed in the cytoplasm is unlikely to be relevant.
Also, experiments on Figure 8C are performed once, and representation needs to be clarified; split GFP needs a positive control, and negative control (CtpC) is not indicated in the figure.
We have repeated the experiments and applied statistics (Figure 9). As stated in the manuscript this assay has successfully been applied to interrogate interactions of domains of proteins embedded in the membrane of mycobacteria. Therefore, we believe that this assay is valid to interrogate interactions between Lcp proteins.
Reviewer #2 (Recommendations For The Authors):
(1) Authors should consider making more effort to mine the omics data and integrate them. Given the amount of data that is generated with the omics, they need to be looked at together to find out threads that connect all of them.
In the resubmitted version of the paper, we have followed reviewer´s recommendation by incorporating new analyses that integrated the virRmut-C strain, and tried to provide context to the differences found in the context of broader transcriptional regulatory networks (new figure 4), as well as in the context of metabolic pathways related with PDIM biosynthesis from methylmalonyl (figure 6I, already present in the first submission). We consider that these additions contribute to a deeper interpretation of the omics data in the line of what was suggested by the reviewer.
(2) The interpretation given by authors in lines 387-390 is an interpretation that does not have sufficient support and, hence should be moved into discussion.
We thank the reviewer for this recommendation. We believe that these new analyses and integration studies now support the above statement.
