Deep mutational scanning of EccD 3 reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system

  1. Donovan D. Trinidad
  2. Christian B. Macdonald
  3. Oren S. Rosenberg
  4. James S. Fraser
  5. Willow Coyote-Maestas

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Abstract

Tuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus M. tuberculosis survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD 3 in the model organism M. smegmatis . We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD 3 . Our data combined with structural comparisons to ESX-5 complexes support a model where EccD 3 stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disruption. Our study is the first deep mutational scan in mycobacteria, which could help guide drug development toward novel treatment of tuberculosis. This study underscores the importance of context-specific mutational analyses for discovering essential protein interactions within mycobacterial systems.

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    Reviewer #1

    __Evidence, reproducibility and clarity __

    This is a well-written manuscript that describes a thorough study of the functionality of individual residues of a central component of the ESX-3 type VII secretion system of Mycobacterium smegmatis, EccD3, in the essential role of this protein transport system in iron acquisition. Using the powerful and unbiased approach of deep mutational scanning (DMS), the authors assessed the impact of different mutations on a large number of residues of this component. This carefully executed research highlights the importance of hydrophobic residues at the center the ubiquitin-like domain, specific residues of the linker domain that connects this domain with the transmembrane domains and specific residues that connect EccD3 with the MycP3 component.

    Major comments

    Since the LOF effects in the iron-sufficient and iron-deficient condition differ less than expected, the differences of the DMS results between these two conditions should be better presented, explained and discussed: 1. The authors discuss: "Of the 270 LOF mutations seen in the iron-deficient condition, 37 (13.7%) were tolerant in the iron sufficient condition, and 39 (14.44%) had strong LOF effects but weak LOF effects in the iron sufficient condition." Do the authors mean that 39 (14.44%) had strong LOF effects in the iron-deficient condition, but weak LOF effects in the iron-sufficient condition. In turn, does this mean that the remaining mutants (71.9%) had similar LOF effects in the two conditions?

    We thank this reviewer for their comment and for highlighting a lack of clarity. We have updated the main text to more effectively communicate our point - that 270 mutants had LOF effects in the iron-deficient media. 37 of these 270 mutants were tolerant in the iron-sufficient media. 39 of these 270 mutants had strong LOF effects in iron-deficient media, but were weak LOF in iron-sufficient media. The remaining 124/270 mutants had weak LOF effects in both conditions. The larger point is that removing iron leads to stronger selection - tolerant mutants become LOF, weak LOF become strong LOF. Removing iron pushes mutants at the bounds over the limit.

    __ The diagonal shape of the scatter plot in Fig. 2C, which shows the correlation of the Enrich2 scores of all mutants in the two conditions, indicates that the growth of most mutants is affected similarly in these conditions, but in Fig. 2D lower graph, which shows only the Enrich2 scores of missense mutants, there are clear differences between the two conditions. How can this be explained?__

    We apologize for any confusion created by this presentation of our data. We hoped to highlight that while effects are largely similar across conditions, there are some differences. As communicated in our first response, 270 out of our ~2700 missense mutations had LOF effects in the iron-deficient condition. 37 of these 270 mutants were tolerant in the iron-sufficient media. 39 of these 270 mutants had strong LOF effects in iron-deficient media, but were weak LOF in iron-sufficient media. The remaining 124 mutations had weak LOF effects in both conditions.

    While Figure 2C shows this difference, it is hard to see by nature of using a scatter plot. We have added contours to highlight how our data is distributed. Our density plots in Figure 2D are meant to try to highlight these differences, where the top plot is showing the effects of all missense mutations. Negatively scored mutations represent LOF effects, mutations with scores around 0 are considered tolerant, and the extremely rare scores with positive scores have GOF effects. Our bottom plot specifically zooms into the negatively scored mutations, to show the 270 LOF mutants we discussed. Specifically, we were hoping to highlight the 39 mutations that have strong LOF effects in iron-deficient media (so the purple line scores are more negative), but weak LOF effects in iron-sufficient media (the green line scores are less negative).

    __ Regarding the authors' explanation for the observed LOF effects in the permissive condition, "This speaks to the sensitivity of next-generation sequencing compared to the strong differences observed between conditions in phenotypic growth curves." But this sensitivity does not explain the observed large LOF effects but no growth difference in the permissive condition, unless the analysis is less quantitative than expected? Could it be that there is local iron depletion in this mixed culture, causing selection pressure even in the iron-sufficient condition? Moreover, the severity of the growth defect at the time of sampling, i.e., after 24 hours of growth, is unclear. Indeed, the growth curve in Fig. 1 shows that the growth of the double mutant in iron-deficient conditions is significantly impaired at that timepoint. In the growth curve in Fig. 2B (and also slightly in Fig. 2F), however, the growth defect is less pronounced: the double mutant has a similar OD600 as the WT strain, although the error bar is larger. Is this variability between replicates also seen in the DMS analysis? In general, no statistics are shown for the DMS analysis and there is no information on the significance of the observed LOF effects. In addition, the legend should explain how many replicates the DMS data are based on.__

    We thank this reviewer for their comment and for highlighting a point of confusion. In addition to increased sensitivity in next generation sequencing compared to our growth curve experiments, our data analysis and variant scoring was performed by comparing growth rates of our mutant strains to our wild type strain. So, any effect on viability or growth rates seen by expression mutant variants will be more notable in our DMS scoring, as they are relative to wild type. In contrast, our growth curves are plotted as the raw OD600 values of each strain. We believe this difference underlies the difference seen in our heatmaps and growth rates.

    It is also a relevant and important point that our libraries are grown as mixed cultures, where there is competition over the limited iron in their growth media, as we highlight in our discussion.

    While the double mutant does show a stark growth defect at 24 hours in Figure 1 compared to the WT and complement, it grows just as well as those strains in Figure 2B. The growth defect becomes notable after 24 hours. Within this experiment, we observed variability in growth at the 24hr timepoint for the negative control strain, but also selection when compared to the positive control and library growth at later time points. We analyzed our DMS data in accordance with typical methods used in the field (see: https://doi.org/10.1186/s13059-017-1272-5). We include statistics for the DMS analysis as supplemental Figure 1. We apologize for any confusion regarding the figure caption, however in our manuscript we do point out that our library growth in Figure 2B was repeated in triplicate in the figure caption, and the samples collected during that experiment were the ones used to generate the DMS data.

    Minor comments

    1. Line and page numbering should be added to the manuscript to facilitate the reviewing process.

    We have updated our manuscript to include line and page numbering.

    __ "Knockout of the entire ESX-3 operon leads to inhibited M. smegmatis growth in a low-iron environment. When individual components of the ESX-3 system are deleted, growth is only available under impaired if the additional siderophore exochelin formyltransferase fxbA is also knocked out20." First, a reference should be added to the first sentence. Second, Siegrist et al. did not exactly show this. They showed that the fxbA/eccC3 double mutant grows slower that the fxbA single mutant. To my knowledge there is no publication showing that single esx-3 component mutants grow as WT in iron-deficient conditions. Do the authors have data demonstrating this? If true, it is surprising that mutating EccD3 has a milder phenotype compared the complete region deletion, as it is a crucial ESX-3 component.__

    We apologize for any confusion. We had the relevant reference two lines prior, and have since added it to that sentence as well.

    The reviewer is correct that Siegrest et al did not show the effects of just ESX-3 component single deletions. However, Siegrest et al. 2009 demonstrated that deleting the entire ESX-3 operon results in growth similar to the wild type strain in low-iron media. In contrast, the fxbA single knockout exhibits a notable growth defect, and the fxbA/ESX-3 double knockout has an even more severe growth defect. Following the logic that a double knockout is needed to observe a growth defect in low-iron media, Siegrest et al. 2014 demonstrated this also extends to single ESX-3 component knockouts, such as the fxbA/eccD3 double knockout strain. To ensure clarity and accuracy, I will edit the sentence to say "When individual components of the ESX-3 system are deleted, growth is significantly impaired when the additional siderophore exochelin formyltransferase fxbA is also knocked out."

    __ Reference to Table 1, should be a reference to Table S1.__

    We have updated our manuscript to correct this reference.

    __ "Our heatmaps surprisingly reveal residues where substitutions are deleterious specifically in the iron-sufficient condition" Refer here to Fig. S2.__

    We have updated our manuscript to include this reference.

    __ "In the iron-deficient condition, 6/551 (1.08%) missense mutations have a weak LOF effect, and 0 have strong effects." More clearly explain this refers to the residues of the transmembrane region.__

    We have updated our manuscript to provide more clarity.

    __ "The MycP transmembrane helix has been hypothesized to be required for ESX complex specificity, targeting MycP to associate with the correct ESX homologue." I miss a reference here. And I thought that the transmembrane domain of MycP was required for complex stability not for specificity?__

    We thank the reviewer for pointing out our missing citation, and asking us to clarify our point. I believe the literature suggests that both the protease and transmembrane domains of MycP are required for both complex stability and specificity. van Winden et al. 2016 https://doi.org/10.1128/mbio.01471-16 show that MycP5 needs to be present for secretion. The protease activity can be abolished and the ESX-5 complex can still secrete and be pulled down, as seen by BN-PAGE. van Winden et al. 2019 https://doi.org/10.1074/jbc.RA118.007090 show that truncated mutants missing either the protease domain or the transmembrane domain cannot rescue ESX-5 secretion or complex stability in a MycP knockout strain. More relevant, they attempted to rescue MycP1 and MycP5 mutants by creating chimeric proteins that either had the MycP1 protease domain and MycP5 transmembrane domain, or the MycP5 protease domain and MycP1 transmembrane domain. If the protease and transmembrane domains were required for complex stability and NOT specificity, we would see MycP5 rescue ESX-1 secretion in the MycP1 mutant strains and vice versa. We would also see the chimera proteins rescue both ESX-1 and ESX-5 secretion and complex stability. Instead, we see that neither chimera rescued ESX-1 nor ESX-5 secretion or complex stability, implying that both MycP domains are necessary.

    We will amend our paper text to reference MycP's role in complex stability instead of specificity, and soften the language: "The MycP transmembrane helix has been shown to be required for ESX complex stability, as MycP knockouts and truncated mutants abolish ESX secretion and pulldowns of the entire complex."

    __ "....role in ESX function relating to EccB3 and EccC3. In the transmembrane, ..... we" Insert "region" after "transmembrane"__

    We have updated our manuscript to include this update.

    Significance

    The study provides insight into individual residues of a central component of the ESX-3 type VII secretion system for functionality, which is useful for those studying the functioning of mycobacterial type VII secretion systems. Moreover, because this system is essential for the growth of the important pathogen M. tuberculosis, this knowledge can be used to design new anti-tuberculosis compounds that block the ESX-3 system. Although the results mainly confirm previous observations (highlighting specific residues important for the stability of ubiquitin and residues of other parts of EccD important for protein-protein interactions within the ESX-3/ESX-5 membrane complex), to my knowledge this is the first time DMS has been applied to mycobacteria. This study is therefore of interest to mycobacteriologists.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

    1. The authors engineered an M. smegmatis knockout strain with deletions of fxbA and eccD3. Deletion of fxbA renders the exocholin iron uptake system non-functional, forcing the bacteria to rely on siderophore-mediated iron uptake under iron-limiting conditions. This process, in turn, depends on ESX-3 secretion activity, as PPE4, a known ESX-3 substrate, has been previously implicated in iron utilization in M. tuberculosis (Tufariello et al., 2016). This experimental setup links EccD3 function to a growth phenotype under iron-limiting conditions, as mutations impairing ESX-3 secretion disrupt iron utilization and mycobacterial growth.
    2. By complementing the knockout strain with a library of EccD3 mutant variants, the authors systematically identify residues essential for protein-protein interactions within the ESX-3 core complex. Structural analysis corroborates the functional relevance of these residues, specifically those mediating interactions between EccD3 and other ESX-3 components, or those disrupting the hydrophobic core of the EccD3 ubiquitin-like (Ubl) domain.
    3. Structural comparisons with the MycP5-bound ESX-5 complex allow the authors to predict residues within EccD3 that may interact with MycP3 during ESX-3 core complex assembly. Furthermore, comparisons with the ESX-5 hexamer suggest residues that may stabilize or drive oligomerization of the ESX-3 dimer into its putative hexameric state. These insights are significant and provide testable hypotheses for future studies.
    4. The methodology is limited to ESX-3. The authors exploit the essentiality of ESX-3 for siderophore-dependent growth under iron-limiting conditions. However, this functional readout cannot be directly transferred to other ESX systems (ESX-1, ESX-2, ESX-4, ESX-5), which have distinct substrates, biological roles, and regulatory mechanisms.

    Significance

    This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

    Thank you for your thoughtful and supportive feedback. We appreciate your time and effort in reviewing our study.

    Reviewer #3

    __Evidence, reproducibility and clarity __

    The manuscript by Trinidad et al. provides a deep mutational scanning (DMS) analysis to investigate the functional roles of residues from the EccD3 subunit of the Type VII ESX-3 secretion apparatus from M. smegmatis. A previously published structure of ESX-3 from M. smegmatis by the Rosenberg group (Oren Rosenberg is also an author of this paper) is used as basis for structural interpretation of the DMS data presented in this contribution. A shortcoming of the previous structure, despite being very rich in terms of structural details, was in the lack of hexameric pore formation, which has been established more recently by structures of the related ESX-5 system.

    Technically, DMS is state-of-the art and a powerful approach to systematically scan residues of potential functional interest. Therefore, the data presented here, provide a remarkable repository for further interpretation in this contribution and by other future investigations. The experimental data have been deposited in Github enabling access by others in the future.

    Overall, the paper would benefit from an improved overall organisation. I found in part hard to extract some of the main points from the way the data are presented. In essence, two separate screens were performed, the first one focusing on the EccD3 Ubl domain and adjacent linker regions and a second one on the EccD3 TM region. I think the paper could be better structured accordingly. Tables of residues with strong effects in iron-deficient and iron-sufficient media, together with their structural annotation, would facilitate extracting main messages from this manuscript. Without going too much in detail, there is also scope for improvement of most of the structural figures. More consistency in terms of color coding with the previous paper by Powileit et al. (2019) would also help navigation.

    A potential weakness of the paper is in the limited scope of interpretation of the data in the context of the dimeric ESX-3 assembly, which is actually acknowledged by the authors. Computational AI-based methods should allow generating a complete pore model of ESX-3, which would allow interpretation of some of the data in a more functional relevant context. This would enhance the validity of the current interpretations presented.

    We acknowledge the lack of a hexameric ESX-3 structure, and would love to base our analysis on such a structure. Unfortunately, experimentally purifying and determining such a structure is beyond the scope of this manuscript. While AI-based methods are certainly exciting and helpful to make sense of mutational data, they are not able to computationally predict such large structures. The AlphaFold3 server website is commonly used for these purposes and allows predictions of up to 5000 tokens (or amino acids). An ESX-3 hexamer would be composed of 6x EccB proteins (519 AA each), 6x EccC proteins (1326 AA each), 12x EccD proteins (476 AA each), and 6x EccE proteins (310 AA each). Together, this complex would be made up of 18,642 amino acids.

    We tried using alphafold to predict an ESX-5 dimer complex, as well as reproduce the ESX-3 dimer complex, and were unable to produce these structures. Each ESX protomer is assembled correctly, as each protein within the complex makes appropriate contacts with each other. We see the EccD-dimers still form the membrane vestibule within each ESX complex. The issue is the ESX dimer complex has not assembled correctly: the EccC transmembrane helix 1 of a protomer should interact with the EccB transmembrane helix of the neighboring protomer; and, the N-terminus of EccB in one protomer should interact with the loop between the EccD transmembrane helices 10 and 11 in the neighboring protomer. Instead, Alphafold creates contacts along the EccD proteins from both complexes. We have included a "top-down" view of the ESX-5 dimer, where the periplasmic domains of EccB have been cleaved off for clarity.

    A side view:

    Here we have the ESX-3 dimer structure published by Poweleit et al. side-by-side with the ESX-3 dimer predicted by alphafold, visualized in Pmyol. The alphafold structure largely has each proteins' domains and folds properly predicted, including even the EccD3 dimer found in each ESX protomer. However, the protomers are not assembled into a dimer properly as compared to the purified ESX-3 dimer from PDB: 6umm. We included a "front" and "side view", as well as a "top down" view where the cytoplasmic domains have been hidden for visual clarity.

    The use of full names and acronyms needs to be more consistent. As an example, the terms "ubiquitin-like" and ubiquitin-like (Ubl) and UBl are used in parallel throughout the manuscript. The percentages given in various places of the paper could be reduced to integers, as they generally relate to relatively small data sets. Please express numbers with a precision, reasonable matching expected statistical significance.

    We apologize for the lack of consistency in how we referred to the ubiquitin-like domain. I originally wrote "ubiquitin-like (Ubl)" once per section (intro, results, discussion). I have edited these all to just "Ubl" after the introduction, except for figure and section titles. We have also reduced our percentages to integers.

    Some of the DMS experiments have been repeated three-fold, which should be a minimal number to allow extracting statistical significance, other experiments have only been repeated two-fold. Could this be clarified, please?

    We apologize for this oversight, and thank the reviewer for pointing this out. All experiments were done in triplicate, the exception being the site-directed mutant growth curves, which were performed in duplicate. We have repeated this experiment in triplicate in response to this point. As we repeated this experiment, mutant R134A dropped out due to technical reasons, and so we did not include it in the updated growth curves.

    Specific comments on text and figures:

    Figure 1: The EM densities shown considerably deviate from those that were shown in the original publication by Poweleit et al (2019). If there is an aim is to reinterpret the data this needs to be described in sufficient technical detail. There may be a case for this, in light of recent advances in computational AI-vased structural biology.

    We acknowledge this may be confusing and we apologize for that, as the EM density I have shown in this manuscript uses the same map we used to create the one seen in the original publication Poweleit et al 2019. There are existing crystal structures of EccB1 and the ATPase domains of EccC1 that we used to create homology models of EccB3 and EccC3 using the structure-prediction software RaptorX for the 2019 publication. These homology models were then combined with a low resolution EM density to create the model seen in the 2019 eLife paper. I did not include those homology models in this manuscript, as I did not believe those predictions were relevant to this study. I wanted to include the highest resolution and thus most accurate depiction of our ESX-3 structure.

    Introduction, statement "We made comparisons to a prior DMS on ubiquitin to increase signal-to-noise in our interpretation of the Ubl domain mutagenesis data." Could this be further explained please? I could not find anything in addition in the Methods section and elsewhere.

    __ __We apologize for the confusion!

    EccD3 Ubl domain and ubiquitin DMS dataset comparisons

    To compare the DMS data of EccD3 Ubl with that of ubiquitin, we first identified homologous residues in each structure. This was achieved by aligning the EccD3 Ubl domain with ubiquitin (PDB: 1ubq) using PyMOL and assessing the positional correspondence of side chains (e.g., ubiquitin residue I3 aligned with EccD3 residue V12). Next, we referenced missense mutation datasets to calculate the average DMS score for each residue position in both proteins. We then generated a scatter plot to compare the average missense scores for ubiquitin and EccD3 Ubl using ggplot2. Data points were color-coded according to the functional roles assigned to ubiquitin, with residues forming the hydrophobic patch and core highlighted, while all other residues were represented in grey.

    Description of "vestibule" as a core feature of the ESX-3 structure. As mentioned above, this is very much a result of the presented dimeric arrangement. In the context of a complete pore model, these features may change or even disappear.

    While we would certainly welcome an ESX-3 hexamer model to definitively determine whether this feature persists, such a model is not currently available. However, the highly homologous ESX-5 complex retains these EccD vestibules, and there is no reason to believe these features would change or disappear. Therefore, based on our interpretation of the ESX-3 dimer and ESX-5 hexamer we believe that the EccD membrane vestibule is not just an artifact of the ESX-3 dimer complex.

    It is possible that the reviewer misunderstood what we were referring to as the vestibule. We updated the language in the text to improve clarity. However the vestibule is not a consequence of ESX-3 complex dimer formation. It is an inherent feature of the ESX monomer complexes, where two EccD proteins dimerize to form said vestibule. Furthermore, there is no evidence to suggest that this feature would be lost in a hexameric state.

    Structurally, the ESX-3 dimer consists of two ESX-3 monomer complexes, each containing one EccB, one EccC, one EccE, and two EccD proteins. Therefore, each ESX-3 monomer inherently includes an EccD dimer. The presence of the EccD dimer is not exclusive to the ESX-3 dimer but is a fundamental component of each ESX-3 complex. Similarly, the ESX-5 hexamer retains the EccD dimer within each ESX-5 complex, further supporting the idea that this structural feature is conserved.

    Figure 2, panel B: Isn't right that "positive" and "negative" need to exchanged? Perhaps, there is something I misunderstood.

    We apologize for the confusion, and appreciate the reviewer pointing out this inconsistency. We have updated the manuscript to correct this.

    Figure 2, panel F: it is hard to extract the assignments from the overlaid curves.

    We apologize for a lack of clarity in how this growth curve was presented. We have included labels at the end point to show where each sample is.

    Figure 3, caption "from low (red) to white (tolerant)": for the sake of consistency, please either put the color in parentheses, or functional description. Does this statement relate to panel A or B? "All other residues are colored white". I can't see this.

    We apologize for the inconsistency, and have updated this label. We hope we have clarified the fact that the entire structure is white except for the residues we colored red.

    Results text "In contrast to ubiquitin, all hydrophobic core residues in the EccD3 Ubl domain are equally intolerant to charged residue swaps. Unsurprisingly, residues important for ubiquitin's specific degradation interactions are not sensitive to substitutions in the EccD3 Ubl domain." Does this mean that proper folding of Ubl is less critical for ESX_3 function? Please elaborate on this further.

    We apologize for any confusion. Our data shows that residues which side chains extend into the hydrophobic core of the Ubl domain are intolerant to swaps to charge residues. We hypothesize these missense mutations disrupt this hydrophobic core, and lead to destabilization of this domain. These intolerant missense mutations each have negative Enrich2 scores, implying a loss of ESX-3 function, and that proper folding of the Ubl is critical for ESX-3 function. We have updated our text to clarify this point:

    Unsurprisingly, residues important for ubiquitin function's specific interactions are not sensitive to substitutions in the EccD3 Ubl domain. There is no simple discernable preference within the Ubl domain to any side that maintains protein-protein interactions, implying that the scores are dominated by stability effects and that the Ubl domain must maintain a stable β-grasp fold for ESX-3 function.

    Figure 4, panel C: the surface does not provide residue-specific information, hence this panel is not very informative.

    We agree with the reviewer that Figure 4 panel C was not very informative, and so we have removed it from Figure 4 for the sake of brevity.

    Results text "T148 extends out from transmembrane helix 1 into a hydrophobic pocket between transmembrane helices 1, 2, and 3." Could this please be illustrated in one of the structural presentations?

    We have updated figure 5 to include a snapshot of this residue and the hydrophobic pocket it extends into, as panel E.

    Results text, last paragraph, Figure 5C-D: interpretation of the experimental ESX-3 data based on ESX-5 models is problematic, without showing proof of conservation of relevant sequence/structural features. As mentioned above, I would encourage the authors to establish a hexameric ESX-3 model and interpret the data starting from there. Extrapolation of the interpretation of data to other ESX systems, including ESX-5, would expand the scope by generalization, which however would open another chapter. The ESX-5 structure does not explain e.g. why W227 when mutated is less sensitive to iron depletion as opposed to iron being present.

    We do not believe we can use AI to predict a hexameric ESX-3 model. We will update our supplement to include a figure showing proof of conservation between the EccD3 and EccD5 sequences. We can superpose the ESX-3 dimer structure onto the ESX-5 hexamer structure, and see that this dimeric complex overlays quite well on top of an ESX-5 subcomplex. We can imagine this hexamer as a trimer of dimers, where three copies of this dimeric complex interact to form the hexamer. The superposition is not perfect and there are slight rearrangements to different helices to allow for hexamer formation, but these do not imply we cannot compare these two homologous structures.

    We have included a new structure snapshot in Figure 5, where panel D is the ESX-3 dimer (PDB: 6umm) shown as a side and top-down view. This allows for a comparison with panel C, the snapshot of the ESX-5 complex (PDB: 7np7) where in two protomers the EccB, EccC, and EccD proteins are colored the same way as ESX-3, and the other ESX-5 protomers are colored white. Note that in this hexamer, EccE is missing. We see the EccD membrane vestibule is conserved in both structures.

    Significance

    Strength and Limitations: already assessed under "Evidence, reproducibility and clarity".

    There is scope for further interpretation using experimental structural and modeling data. There is also scope for applying complementary assays for selected mutants, most likely within a lower throughput format.

    Advance: The contribution demonstrates well the power of DMS for systematic screening, in the context of Type VII secretion. The main advance is in the raw data generated and deposited.

    Audience: microbiology with a specific interest in secretion, structural biology

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Trinidad et al. provides a deep mutational scanning (DMS) analysis to investigate the functional roles of residues from the EccD3 subunit of the Type VII ESX-3 secretion apparatus from M. smegmatis. A previously published structure of ESX-3 from M. smegmatis by the Rosenberg group (Oren Rosenberg is also an author of this paper) is used as basis for structural interpretation of the DMS data presented in this contribution. A shortcoming of the previous structure, despite being very rich in terms of structural details, was in the lack of hexameric pore formation, which has been established more recently by structures of the related ESX-5 system.

    Technically, DMS is state-of-the art and a powerful approach to systematically scan residues of potential functional interest. Therefore, the data presented here, provide a remarkable repository for further interpretation in this contribution and by other future investigations. The experimental data have been deposited in Github enabling access by others in the future.

    Overall, the paper would benefit from an improved overall organisation. I found in part hard to extract some of the main points from the way the data are presented. In essence, two separate screens were performed, the first one focusing on the EccD3 Ubl domain and adjacent linker regions and a second one on the EccD3 TM region. I think the paper could be better structured accordingly. Tables of residues with strong effects in iron-deficient and iron-sufficient media, together with their structural annotation, would facilitate extracting main messages from this manuscript. Without going too much in detail, there is also scope for improvement of most of the structural figures. More consistency in terms of color coding with the previous paper by Powileit et al. (2019) would also help navigation.

    A potential weakness of the paper is in the limited scope of interpretation of the data in the context of the dimeric ESX-3 assembly, which is actually acknowledged by the authors. Computational AI-based methods should allow generating a complete pore model of ESX-3, which would allow interpretation of some of the data in a more functional relevant context. This would enhance the validity of the current interpretations presented.

    The use of full names and acronyms needs to be more consistent. As an example, the terms "ubiquitin-like" and ubiquitin-like (Ubl) and UBl are used in parallel throughout the manuscript. The percentages given in various places of the paper could be reduced to integers, as they generally relate to relatively small data sets. Please express numbers with a precision, reasonable matching expected statistical significance.

    Some of the DMS experiments have been repeated three-fold, which should be a minimal number to allow extracting statistical significance, other experiments have only been repeated two-fold. Could this be clarified, please?

    Specific comments on text and figures:

    Figure 1: The EM densities shown considerably deviate from those that were shown in the original publication by Poweleit et al (2019). If there is an aim is to reinterpret the data this needs to be described in sufficient technical detail. There may be a case for this, in light of recent advances in computational AI-vased structural biology.

    Introduction, statement "We made comparisons to a prior DMS on ubiquitin to increase signal-to-noise in our interpretation of the Ubl domain mutagenesis data." Could this be further explained please? I could not find anything in addition in the Methods section and elsewhere.

    Description of "vestibule" as a core feature of the ESX-3 structure. As mentioned above, this is very much a result of the presented dimeric arrangement. In the context of a complete pore model, these features may change or even disappear.

    Figure 2, panel B: Isn't right that "positive" and "negative" need to exchanged? Perhaps, there is something I misunderstood.

    Figure 2, panel F: it is hard to extract the assignments from the overlaid curves.

    Figure 3, caption "from low (red) to white (tolerant)": for the sake of consistency, please either put the color in parentheses, or functional description. Does this statement relate to panel A or B? "All other residues are colored white". I can't see this.

    Results text "In contrast to ubiquitin, all hydrophobic core residues in the EccD3 Ubl domain are equally intolerant to charged residue swaps. Unsurprisingly, residues important for ubiquitin's specific degradation interactions are not sensitive to substitutions in the EccD3 Ubl domain." Does this mean that proper folding of Ubl is less critical for ESX_3 function? Please elaborate on this further.

    Figure 4, panel C: the surface does not provide residue-specific information, hence this panel is not very informative.

    Results text "T148 extends out from transmembrane helix 1 into a hydrophobic pocket between transmembrane helices 1, 2, and 3." Could this please be illustrated in one of the structural presentations?

    Results text, last paragraph, Figure 5C-D: interpretation of the experimental ESX-3 data based on ESX-5 models is problematic, without showing proof of conservation of relevant sequence/structural features. As mentioned above, I would encourage the authors to establish a hexameric ESX-3 model and interpret the data starting from there. Extrapolation of the interpretation of data to other ESX systems, including ESX-5, would expand the scope by generalization, which however would open another chapter. The ESX-5 structure does not explain e.g. why W227 when mutated is less sensitive to iron depletion as opposed to iron being present.

    Referee cross-commenting

    I especially second the comments of referee #1, major comments, point 3 (statistical significance of the data). Addressing this point is crucial for the paper. Referee #2, significance section "The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system." I was considering making the same point but did not at the end. Of course, ultimately, it would be great if all components of ESX-3 could be analyzed they way it was done for the EccD3 component. However, I am afraid such exercise could become quite open ended. Already by now, there is some compromise on the depth of mechanistic interpretation in light of a large data set generated.

    Significance

    Strength and Limitations: already assessed under "Evidence, reproducibility and clarity".

    There is scope for further interpretation using experimental structural and modeling data. There is also scope for applying complementary assays for selected mutants, most likely within a lower throughput format.

    Advance: The contribution demonstrates well the power of DMS for systematic screening, in the context of Type VII secretion. The main advance is in the raw data generated and deposited.

    Audience: microbiology with a specific interest in secretion, structural biology

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    Referee #2

    Evidence, reproducibility and clarity

    This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

    1. The authors engineered an M. smegmatis knockout strain with deletions of fxbA and eccD3. Deletion of fxbA renders the exocholin iron uptake system non-functional, forcing the bacteria to rely on siderophore-mediated iron uptake under iron-limiting conditions. This process, in turn, depends on ESX-3 secretion activity, as PPE4, a known ESX-3 substrate, has been previously implicated in iron utilization in M. tuberculosis (Tufariello et al., 2016). This experimental setup links EccD3 function to a growth phenotype under iron-limiting conditions, as mutations impairing ESX-3 secretion disrupt iron utilization and mycobacterial growth.
    2. By complementing the knockout strain with a library of EccD3 mutant variants, the authors systematically identify residues essential for protein-protein interactions within the ESX-3 core complex. Structural analysis corroborates the functional relevance of these residues, specifically those mediating interactions between EccD3 and other ESX-3 components, or those disrupting the hydrophobic core of the EccD3 ubiquitin-like (Ubl) domain.
    3. Structural comparisons with the MycP5-bound ESX-5 complex allow the authors to predict residues within EccD3 that may interact with MycP3 during ESX-3 core complex assembly. Furthermore, comparisons with the ESX-5 hexamer suggest residues that may stabilize or drive oligomerization of the ESX-3 dimer into its putative hexameric state. These insights are significant and provide testable hypotheses for future studies.
    4. The methodology is limited to ESX-3. The authors exploit the essentiality of ESX-3 for siderophore-dependent growth under iron-limiting conditions. However, this functional readout cannot be directly transferred to other ESX systems (ESX-1, ESX-2, ESX-4, ESX-5), which have distinct substrates, biological roles, and regulatory mechanisms.

    Significance

    This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

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    Referee #1

    Evidence, reproducibility and clarity

    This is a well-written manuscript that describes a thorough study of the functionality of individual residues of a central component of the ESX-3 type VII secretion system of Mycobacterium smegmatis, EccD3, in the essential role of this protein transport system in iron acquisition. Using the powerful and unbiased approach of deep mutational scanning (DMS), the authors assessed the impact of different mutations on a large number of residues of this component. This carefully executed research highlights the importance of hydrophobic residues at the center the ubiquitin-like domain, specific residues of the linker domain that connects this domain with the transmembrane domains and specific residues that connect EccD3 with the MycP3 component.

    Major comments

    Since the LOF effects in the iron-sufficient and iron-deficient condition differ less than expected, the differences of the DMS results between these two conditions should be better presented, explained and discussed:

    1. The authors discuss: "Of the 270 LOF mutations seen in the iron-deficient condition, 37 (13.7%) were tolerant in the iron sufficient condition, and 39 (14.44%) had strong LOF effects but weak LOF effects in the iron sufficient condition." Do the authors mean that 39 (14.44%) had strong LOF effects in the iron-deficient condition, but weak LOF effects in the iron-sufficient condition. In turn, does this mean that the remaining mutants (71.9%) had similar LOF effects in the two conditions?
    2. The diagonal shape of the scatter plot in Fig. 2C, which shows the correlation of the Enrich2 scores of all mutants in the two conditions, indicates that the growth of most mutants is affected similarly in these conditions, but in Fig. 2D lower graph, which shows only the Enrich2 scores of missense mutants, there are clear differences between the two conditions. How can this be explained?
    3. Regarding the authors' explanation for the observed LOF effects in the permissive condition, "This speaks to the sensitivity of next-generation sequencing compared to the strong differences observed between conditions in phenotypic growth curves." But this sensitivity does not explain the observed large LOF effects but no growth difference in the permissive condition, unless the analysis is less quantitative than expected? Could it be that there is local iron depletion in this mixed culture, causing selection pressure even in the iron-sufficient condition? Moreover, the severity of the growth defect at the time of sampling, i.e., after 24 hours of growth, is unclear. Indeed, the growth curve in Fig. 1 shows that the growth of the double mutant in iron-deficient conditions is significantly impaired at that timepoint. In the growth curve in Fig. 2B (and also slightly in Fig. 2F), however, the growth defect is less pronounced: the double mutant has a similar OD600 as the WT strain, although the error bar is larger. Is this variability between replicates also seen in the DMS analysis? In general, no statistics are shown for the DMS analysis and there is no information on the significance of the observed LOF effects. In addition, the legend should explain how many replicates the DMS data are based on.

    Minor comments

    1. Line and page numbering should be added to the manuscript to facilitate the reviewing process.
    2. "Knockout of the entire ESX-3 operon leads to inhibited M. smegmatis growth in a low-iron environment. When individual components of the ESX-3 system are deleted, growth is only available under impaired if the additional siderophore exochelin formyltransferase fxbA is also knocked out20." First, a reference should be added to the first sentence. Second, Siegrist et al. did not exactly show this. They showed that the fxbA/eccC3 double mutant grows slower that the fxbA single mutant. To my knowledge there is no publication showing that single esx-3 component mutants grow as WT in iron-deficient conditions. Do the authors have data demonstrating this? If true, it is surprising that mutating EccD3 has a milder phenotype compared the complete region deletion, as it is a crucial ESX-3 component.
    3. Reference to Table 1, should be a reference to Table S1.
    4. "Our heatmaps surprisingly reveal residues where substitutions are deleterious specifically in the iron-sufficient condition" Refer here to Fig. S2.
    5. "In the iron-deficient condition, 6/551 (1.08%) missense mutations have a weak LOF effect, and 0 have strong effects." More clearly explain this refers to the residues of the transmembrane region.
    6. "The MycP transmembrane helix has been hypothesized to be required for ESX complex specificity, targeting MycP to associate with the correct ESX homologue." I miss a reference here. And I thought that the transmembrane domain of MycP was required for complex stability not for specificity?
    7. "....role in ESX function relating to EccB3 and EccC3. In the transmembrane, ..... we" Insert "region" after "transmembrane"

    Significance

    The study provides insight into individual residues of a central component of the ESX-3 type VII secretion system for functionality, which is useful for those studying the functioning of mycobacterial type VII secretion systems. Moreover, because this system is essential for the growth of the important pathogen M. tuberculosis, this knowledge can be used to design new anti-tuberculosis compounds that block the ESX-3 system. Although the results mainly confirm previous observations (highlighting specific residues important for the stability of ubiquitin and residues of other parts of EccD important for protein-protein interactions within the ESX-3/ESX-5 membrane complex), to my knowledge this is the first time DMS has been applied to mycobacteria. This study is therefore of interest to mycobacteriologists.

  5. Version published to 10.1101/2024.08.23.609456 on bioRxiv