Membrane microdomains are crucial for Mycobacterium marinum EsxA-dependent membrane damage, escape to the cytosol and infection

  1. Cristina Bosmani
  2. Angélique Perret
  3. Florence Leuba
  4. Aurélie Guého
  5. Nabil Hanna
  6. Thierry Soldati

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Infection by pathogenic mycobacteria such as Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-Containing Vacuole (MCV). The key effector EsxA, secreted via the ESX-1 type-VII system, is pivotal in this process, yet its membranolytic activity is not fully elucidated. Infecting the amoeba Dictyostelium discoideum with Mycobacterium marinum , we demonstrate that the composition of the MCV membrane, notably its sterol-rich microdomains, significantly influences damage and rupture. Disruption of these microdomains through the knockout of organizing proteins, termed vacuolins, or through sterol depletion, markedly diminishes M. marinum -induced membrane damage and cytosolic escape, thereby increasing cellular resistance to infection. Furthermore, we establish that vacuolins and sterols are essential for the in vitro partitioning of EsxA within membranes. Extending our findings to mammalian cells, we show that the role of microdomain organizers and sterols is evolutionarily conserved; specifically, flotillin knockdown and sterol depletion enhance the resistance of murine microglial cells to M. marinum infection. Our results underscore the critical role of host membrane microdomains in facilitating mycobacterial membranolytic activity and subsequent cytosolic access, pivotal for a successful infection.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC-2024-02655

    Corresponding author(s): Thierry SOLDATI

    1. General Statements [optional]

    The emergence of powerful model organisms for infection studies accelerates discoveries in innate immunity and conserved cell-autonomous defence mechanisms. Using the genetically tractable Dictyostelium discoideum/Mycobacterium marinum infection platform, we explored the critical interplay between pathogen-induced membrane damage and host repair pathways.

    Recent findings highlight evolutionarily conserved membrane repair pathways as crucial for cellular integrity against both sterile and pathogenic insults. We previously demonstrated the involvement of ESCRT and autophagy machineries in repairing membrane damage and containing pathogenic mycobacteria within vacuoles. Crucially, we uncovered that TrafE, an evolutionarily conserved TRAF-like E3 ubiquitin ligase, coordinates these machineries to repair membrane damage, preventing cell death.

    Here, we reveal that pathogenic mycobacteria manipulate host membrane microdomain scaffolding proteins and sterols to enhance toxin activity and facilitate bacterial escape. Genetic knockout of these microdomain organizers and sterol depletion significantly reduce membrane damage and bacterial escape, effectively containing mycobacteria and increasing host resistance. The conserved roles of flotillin and sterols are confirmed in murine microglial cells, underscoring evolutionary conservation.

    These discoveries significantly advance understanding of intracellular host-pathogen interactions, offering broad implications for cellular microbiology and immunology and have already attracted wide interest at major international scientific meetings.

    Thanks to the constructive criticisms and suggestions of the referees, we were able to significantly enhance the manuscript by integrating novel experimental strategies and improving presentation and discussion of previous results that together further strengthen our evidence.

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The proposed study aims to elucidate the role of membrane microdomains and associated proteins-Vacuolin A, B, and C-during the infection of Dictyostelium discoideum (Dd) amoebae by Mycobacterium marinum (Mm). The results demonstrate that Vacuolins are required for Mm virulence, and that the presence of membrane microdomains is essential for phagosome membrane damage and bacillary escape into the cytosol-key steps in establishing a successful infection and subsequent bacterial proliferation. The study is well-designed, employing methodologies with which the authors have demonstrated expertise. Overall, it is methodologically sound, and most conclusions are well-supported by the presented data. However, some points require clarification.

    We thank the referee for their positive evaluation of the scope and strengths of our manuscript. The constructive criticisms of the referees were important to guide our revisions. We are convinced that the new data now integrated further strengthen our evidence.

    Major Points:

    The study aims to link the function of Dd Vacuolins to their potnetial facilitating role in phagosome escape and overall infection by Mm. To phenocopy the effect of Vac-KO, the authors used MβCD. Strikingly, this compound had a more significant impact on phagosome escape compared to Vac-KO, which either did not affect or only mildly affected this process. This likely reflects a difference in the underlying mechanisms being studied. Vac-KO cells may lack well-organized membrane domains but could retain a similar overall membrane composition. In contrast, MβCD disrupts these domains by chelating cholesterol, thus altering both the membrane composition and the domains themselves. This may explain why EsxA partitioning is more affected by MβCD than by triple KO. Consequently, this suggests that cholesterol, rather than the mere presence of membrane domains, plays a critical role in EsxA partitioning and activity in the phagosome. And even if LLOMe activity was lower in Vac-KO cells, this might be explained by the compartment targeted, the lysosomes which membrane composition may differ from the MCV. These points should be further discussed in the discussion section.

    The referee is right on target, these are all excellent points, and we fully agree with the argumentation. If we compare EsxA to a cholesterol-dependent PFT such as SLO, the presence of sterol is an absolute requirement for pore formation, but the local concentration of sterols achieved via clustering and the organisation of lipids/sterols in microdomains "only" increases efficiency (see for example PMID: 39835825). Therefore, the respective impacts of vac-KO and CD treatment differ in "intensity", and are additive in most assays, but are not resulting from "different underlying mechanisms". The simplest and most plausible interpretation of the combined results is that EsxA requires a threshold of local concentration/clustering of sterols to act and vacuolins/flotillins is one of the means to achieve it. In other words, membrane composition inhomogeneities exist in physiological membranes, particularly sterol and sphingolipid clustering in rafts, and microdomain organisers probably regulate their size and dynamics. Without vacuolin/flotillin, these inhomogeneities persist. Only when sterol is depleted and/or redistributed, do they disappear. In brief, the local sterol concentration is the trigger for EsxA preferential partitioning and activity, and many factors besides microdomain organisers influence it.

    The second interesting point is that LLOMe is a lysosomotropic membrane damaging agent, whereas EsxA targets the MCV membrane. We have already documented that the MCV has some endo-lysosomal properties and potentially resembles most the "post-lysosomal" compartment, characterized by a mildly acidic pH (pH ~6), the presence of Rab7 and zinc, ammonium and cupper transporters, for example. Our experiments also show that LLOMe is active in the whole endo-lysosomal pathway, including these post-lysosomes (PMID: 30596802, PMID: 37070811). The exact lipid composition of the MCV and post-lysosomes is not known, but both accumulate sterols in a similar manner. Both compartments are also akin to multivesicular bodies. These data are no direct proof but are compatible with our conclusions that both LLOMe and EsxA require similar threshold of local sterol concentration and that vacuolins are a mean to achieve this.

    The presentation of these conclusions has been revised and enhanced in the discussion (for example lines 396-400 and 437-439).

    Despite these similarities between LLOMe and EsxA activities, note that the mature MCV can be distinguished from all other endo-lysosomal compartments by the use of a Flipper probe that is sensitive to lipid composition and packing (Fig. 7C, and see below). In addition, RNAseq analyses of the impact of vac-KO and sterol depletion on infected and non-infected cells also highlight the interdependence between sterol concentration and vacuolin expression (Fig. 3G, 4G and H, Fig. EV5 and 6, and see below).

    Based on this observation, in figure 2, does the D4H/filipin signal or association increase over time as the Vac signal "solidifies"? In Vac-KO cells, does the mScarlet-D4H signal change in intensity or pattern (building on fig. S1)? These insights could provide valuable information on cholesterol levels at the MCV in KO versus wild-type cells. If possible, the authors should quantify fluorescence or the frequency of signal association.

    Qualitatively, sterols, as visualised by filipin and D4H, are present at all stages of the endo-lysosomal pathway and of MCV biogenesis. Now, there are many technical difficulties linked to a quantitative assessment, and therefore, please, let me present the framework. First, despite their wide use, the exact mechanism of binding of both reporters and which pool of sterol they visualise is still a mystery. This is often expressed as "they detect the accessible pool" of sterol, whatever it is. In addition, filipin detects sterols in both leaflets (and in intra-lumenal vesicles and other lipidic structures), while D4H detects sterols only in the cytosolic leaflet, and it is not known whether both leaflets have the same concentration of sterols. It is also known that filipin signal is only indirectly proportional to the sterol quantity in a cell, as measured by other quantitative methods. One of the best examples comes from studying the cellular phenotype of Niemann-Pick Type C disease, because many publications report a strong increase of filliping staining, whereas lipidomic analyses show at best a two-fold increase in cholesterol in NPC deficient cells. Moreover, technically speaking, D4H is a live probe, and fixation leads to some loss of localisation, probably because sterols are not fixable. On the other hand, filipin is mainly used after chemical fixation, but again sterols are not fixable, and the signal is very likely restricted to the membrane of origin, but not necessarily to the microdomains.

    All this to admit that, despite numerous and rigorous tentatives, we have not been able to reliably obtain quantitative measurements of neither filipin nor D4H signals. Also, these features likely also explain why we were not able to document changes in "patterns" of signals during MCV maturation. We ask for the referee's indulgence about this. Vacuolins remain the best microdomain morphology reporters.

    We nevertheless present additional qualitative D4H and VacC colocalization images in Fig. EV1C.

    Additionally, since Vacuolins do not have a significant impact on phagosome damage or escape, the difference in intracellular growth may be indirect, as suggested in the team's previous study on Vacuolins (DOI: 10.1242/jcs.242974). The authors measured MCV pH in figure S6-could they repeat this experiment to test whether Vacuolins affect MCV maturation? This was investigated in a previous version of the pre-print (DOI: 10.1101/2021.11.16.468763), and if the results still hold, it would strengthen the hypothesis that Vacuolins promote escape by modulating membrane organization, rather than influencing phagosome maturation.

    First, we respectfully disagree that vacuolins have no impact on membrane damage, we explained above why this impact is limited, but nevertheless additive with sterol depletion in most assays, during infection and sterile damage.

    We thank the referee for their excellent knowledge of the literature. Indeed, we previously went to extreme experimental sophistication to interrogate the impact of vac-KO on endo-phagosomal maturation. We were able to demonstrate that the major impact is on the recycling of phagocytic receptors and therefore on the cytoskeleton- and motor-induced deformation of the membrane in a cup that is essential for efficient phagocytosis (but not macropinocytosis). We also demonstrated a minimal effect on maturation, on the kinetics of pH change and delivery/recycling of hydrolases, but these cell biological differences did not translate in an impact on bacteria killing and digestion. As mentioned above, the MCV shares characteristics with post-lysosomes but minimal alterations of endo-lysosomal maturation in vac-KO cells should not be responsible for the strong effect on Mm infection. In other words, we are convinced that these minimal (mainly loss-of-function) perturbations that do not impact killing of food bacteria do not lead to an increased phagosomal "ferocity" and restriction of tough mycobacteria.

    Consequently, we decided not to repeat experiments to measure the pH around wt Mm in vac-KO cells, as it is anyway only slightly and transiently acidified in wt host cells, and previous work did not reveal major differences in endolysosomal compartment pH control (PMID: 32482795). But we agree with the referee that some of the MCV maturation data presented in the previous bioRxiv version are interesting for specialists, despite the indications of extremely small alterations between wt and vac-KO host cells. These data document that in absence of vacuolins, MCV characteristics are slightly altered, but we found no indication that they are more bactericidal in vac-KO cells (Fig. EV8F-H).

    Finally, as a substantial part of this manuscript relies on microscopy and image analysis, the methods section should detail how these analyses were performed. Specifically, for figure 1f, it is unclear how the cells were segmented and fluorescence quantified-was total fluorescence per cell measured, or was an average value used? Figures 5c and 5h could be moved to the supplementary material, and the segmentation method should be explained in the methods section. Additionally, statistical analysis should be more clearly described, justifying the use of one-way or two-way ANOVA, and specifying the post-hoc tests used for group comparisons.

    We fully agree with the referee and have therefore improved the detailed description of image analyses. For example, details for cell segmentation in images originating from infection and LLOMe experiments are succinctly described in the Materials & Methods section (lines 585-588, 594-597, and 639-640), but we now also refer to a methods chapter in press that describe in detail the whole segmentation pipeline (Perret et al. 2025).

    Concerning specifically Fig. 1F, we distinguished infected or bystander cells by the presence of bacteria and quantitated the maximal fluorescence intensity for each cell. Then, we decided on an arbitrary threshold of intensity of 5,000, that corresponds to the maximal signal observed for cells in mock conditions. Then, we quantified the percentage of bystander and infected cells with a higher-than-threshold (>5,000) vacuolin signal intensity. This clarification is now added to the legend of Fig. 1F.

    The statistical analyses applied are described in more detail in each figure legend.

    Reviewer #1 (Significance (Required)):

    This study provides the first direct evidence of the importance of membrane composition and organization in the virulence of Mycobacterium marinum, particularly in facilitating phagosome damage and bacillary escape. Using the well-established model of Dictyostelium discoideum infected with M. marinum, which has frequently been predictive of Mycobacterium tuberculosis behavior within phagosomes, the authors contribute critical insights into the mechanisms of mycobacterial phagosome escape-a key step in cellular invasion and dissemination. These findings have the potential to inform strategies aimed at blocking this escape mechanism, which, as demonstrated in this study, could prevent intracellular bacterial growth.

    This work is significant for advancing our understanding of mycobacterial pathogenesis, particularly by linking membrane microdomain composition to bacterial virulence. It will be highly relevant to researchers studying mycobacteria, intracellular pathogens, and host-pathogen interactions. While the study's use of M. marinum provides valuable insights, a limitation is that these results may not fully translate to M. tuberculosis, and further testing with the latter pathogen will be essential.

    We sincerely thank the referee for their very strong appraisal of our contributions, past and present, much appreciated. We agree that the translation of our findings to Mtb and macrophages is not guaranteed ... but has turned to be surprisingly and satisfyingly consistent in the past. To our delight, a recent article in Nature Communications reports about "Paired analysis of host and pathogen genomes identifies determinants of human tuberculosis" and clearly identified flotillin-1 as a susceptibility factor for tuberculosis (PMID: 39613754). We have introduced a sentence in the discussion that reads "Importantly and consistently with our findings, recent work has revealed flotillins as a major determinant of the fate of Mtb infection in patients, because overexpression of flotillin-1, resulting from particular allele variants, is a host susceptibility factor for Mtb infection (PMID: 39613754)." (Lines 477-480)

    I am an expert in the infection of macrophages by Mycobacterium tuberculosis, the phagosome escape mechanism, and its associated effectors. I also have expertise in microscopy and image analysis. However, I do not specialize in Dictyostelium discoideum biology.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    the authors of this manuscript reported that EsxA, a secreted virulent factor of Mtb or Mm, causes membrane lysis in sterol-rich micro domain. They used the Mm-infected amoeba as an infection model, and characterized the effects of microdomain in Mycobacterium-containing Vacuole (MCV) on EsxA-mediated membrane disruption. They found that disruption of the micro domain through knockout of vacuolins or sterol depletion diminished Mm-induced membrane damage and cytosolic escape. They also found that vacuolins and sterol are essential for EsxA inserting into the membranes in vitro, and flotillin knockdown and sterol depletion conferred the resistance of murine microglial cells to Mm infection. The experiments were well designed and controlled, and the data were convincing.

    We thank the referee for this snappy summary of our main findings and for the positive comment on study design.

    My major comment is that the authors need to justify the use of BV-2 cells that are murine microglial cells, instead of macrophage cell lines, which are more relevant to Mtb/Mm infection.

    We understand the referee's concerns about the host used for Mm infection. First, we would like to argue that it is very true that the detailed biological processes accompanying the infection by Mtb, Mm or in fact any other pathogen depend on the origin and status of the host cell. In the TB field, a plethora of host macrophages, from murine and human origins, primary or immortalised, alveolar or interstitial, M1 or M2 have been used through the decades. Beside a robust agreement on many processes (phagosome maturation arrest, MCV membrane damage, role of xenophagy etc...), some of the crucial outcomes, for example the susceptibility or resistance to Mtb infection and the type of host cell death, have been hotly debated and depend on the host phagocyte identity and status.

    Now, it is true that microglial cells have only rarely been used for Mtb (or Mm) research, but it does not mean that this is not relevant. First, we would like to remind the referee that TB is not only a pulmonary disease, and that among the most disastrous extra-pulmonary sites of infection is the brain, resulting in the devastating tuberculous meningitis. In fact, tuberculous meningitis is the most severe form of tuberculosis with a fatality rate of 20-50% in treated individuals (doi: https://doi.org/10.1101/2025.03.04.641394). A quick literature survey on the topic reveals over 9,000 publications, including very significant contributions, using both Mtb and Mm in animal and human models (PMID: 38745656, PMID: 38264653, PMID: 36862557, PMID: 32057291, PMID: 30645042, PMID: 29352446, PMID: 27935825, PMID: 26041993).

    We have introduced a brief mention of these arguments in the discussion (Lines 456-459).

    In addition, we have already shown that this BV-2 cell line is reliable, they are adherent, motile and constitutively phagocytic and thus do not need to be differentiated with mega-doses of PMA, or any other stimulus. They beautifully recapitulate our findings in the Dd-Mm model (PMID: 38270456, PMID: 25772333), including when used as a host phagocyte to validate anti-infective compounds that were primarily identified using the Dd-Mm platform (PMID: 29500372).

    We have introduced a brief mention of these arguments in the results section (Lines 329-334).

    We also introduced two novel experimental evidence to strengthen the link between the Dd and BV-2 model systems. First, we show using qRT-PCR that, like vacuolins, flotillin-1 is upregulated in BV-2 at 32hpi (Fig. EV9B). Excitingly, as mentioned as response to referee #1, a recent article in Nature Communications reports about "Paired analysis of host and pathogen genomes identifies determinants of human tuberculosis" and clearly identified flotillin-1 as a susceptibility factor for tuberculosis (PMID: 39613754). We have introduced a sentence in the discussion that reads "Importantly and consistently with our findings, recent work has revealed flotillins as a major determinant of the fate of Mtb infection in patients, because overexpression of flotillin-1, resulting from particular allele variants, is a host susceptibility factor for Mtb infection (PMID: 39613754)." (Lines 477-480)

    Second, we used for the first time the LysoFlipper probe to monitor MCV lipid composition and packing during infection (Fig. 7C). These results indicate that in BV-2 cells, as in Dd, the membrane characteristics of the MCV are profoundly different from the standard endo-lysosomal compartments.

    Reviewer #2 (Significance (Required)):

    It is well known that EsxA is membrane-lytic protein playing a role in Mtb/Mm-mediated phagosomal escape. There are other studies that have indicated lipid raft or micro domains in the membrane may play a role in EsxA-mediated membrane damage. This study further confirmed that the sterol-rich micro domain on the membrane has significant influence on the EsxA-mediated membrane disruption both in vitro and in vivo. While this finding is expected, but confirmation with solid experimental evidence is welcomed. This study also identified the genes or proteins required for micro domain organization, vacuolins and flotillin, which could be a target of host-directed therapy. Overall, this study is performed well and the results are convincing.

    We thank the referee for their expert views and comments on the function of EsxA and the lipidic environment in which it is supposed to act. We agree that EsxA has been the centre of attention for decades, but we respectfully disagree that its precise mode of action is known, neither in vitro nor in vivo. First, historically, it took the best of a decade for the field to accept that Mtb was not a strictly vacuolar pathogen. And even when the escape to the cytosol became a fact, the implication of EsxA remained extremely debated. For example, a "petition" was signed and published, arguing against its direct membrane damaging activity (PMID: 28119503). We agree that cumulated evidence now converges against a canonical "pore-forming" activity, but in favour of a "membrane-disrupting" activity. On the other hand, it is true that researchers have reached a form of consensus on the role of low pH to dissociate the EsxA-B dimer, and on the importance of the "physiological" composition of the acceptor membrane (PMID: 31430698, PMID: 35271388, PMID: 17557817). We are convinced that our evidence is not merely expected and confirmatory, but represents a novel, complete, solid, biochemical in vitro, molecular and genetics in vivo demonstration of the role of sterols clustering and microdomain organisers as susceptibility factors for Mm infection in evolutionary distant phagocytes.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Bosmani, Perret et al examines the role of Dictyodistelium discoideum vacuolin proteins in the integrity of the Mycobacterium marinum vacuole membrane. The data demonstrates that loss of vacuolins, similar to sterol depletion, reduced vacuole membrane damage meaning less cytosolic escape of the pathogen and subsequently reduced bacterial replication. The authors demonstrate functional analogy in a mammalian model of infection - where flotillin plays a similar role to the vacuolins - and this is an important demonstration of the utility of the D. discoideum model. The data is well presented and clear.

    We thank the referee for this positive summary of our main findings and of the clarity of results, interpretations and working model.

    Major Comments:

    There is no evidence presented in the manuscript of "microdomains" - while I believe this is likely a true description of what is happening on the vacuole membrane there is no visualisation of this. Both the GFP-Vac vacuole staining and the filipin staining show complete coverage of the vacuole. Perhaps at the 1 hour time points this is more convincing but I think it is worth looking at more of these earlier time points and quantifying these "microdomains" - i.e. proportion of vacuole membrane that is positive for the Vacs. Is it possible to look at the GFP-Vac signal and filipin staining at the same time? And other vacuole markers too?

    We agree with the referee that microdomains are the central characters of our study. Now, we would like to argue with the referee that one has to distinguish between structural, morphological evidence for the existence of microdomains and the biochemical and genetic evidence of their functional implication.

    On the one hand, microdomains are in fact nanometer-scale and are thus under the resolution limit of most optical microscopies. We and others already documented that during phagosome maturation, vacuolin distribution is patchy, reflecting the clustering of nanometer-scale inhomogeneities, and that the coating becomes more continuous with progressing maturation. The transition we observed here for vacuolins, as microdomain organisers, from a patchy to continuous coating reflects indirectly their macroscopic coalescence. As discussed above in response to the first referee, visualisation of the underlying lipidic clusters and microdomains is for technical reasons almost undoable. One cannot fix sterols. As replied to the first referee, we have not been able to improve much on the spatial resolution of lipidic microdomains, and, despite numerous and rigorous tentatives, we have not been able to reliably obtain quantitative measurements of neither filipin nor D4H signals, nor to document changes in "patterns" of signals during MCV maturation. We nevertheless present additional qualitative D4H and VacC colocalization images Fig. EV1C.

    On the other hand, we respectfully disagree that our manuscript lacks in strong and direct evidence for the functionality of sterol-rich microdomains as susceptibility factors required for a full mycobacteria infection in evolutionary distant phagocytes.

    In addition to the evidence presented previously, we have now added a large set of RNAseq analyses of the impact of vac-KO and sterol depletion on infected and non-infected cells, which also highlight the interdependence between sterol concentration and vacuolin expression (Fig. 3G, 4G and H, Fig. EV5 and 6). Moreover, we have now used a Flipper probe sensitive to lipid composition and packing to distinguish the mature MCV from all other endo-lysosomal compartments in microglial cells (Fig. 7C). Altogether, the simplest and most plausible interpretation of our cumulated evidence is that sterol-rich microdomains are necessary for EsxA-mediated MCV damage and escape to the cytosol.

    I really like the data presented in Figure 1 that demonstrates the specific upregulation of Vacuolin C during M. marinum infection. This is an intriguing result that brings up a lot of new questions e.g. how is this regulated? In response to membrane damage? Sensed by what? Does this upregulation also hold true for flotillin in the mammalian model? (and more!) however none of these ideas are pursued in the manuscript and by the end I was wondering why this data was included in the manuscript because all of the phenotypic data uses either a VacBC or ABC mutant. The link between figure 1 and the rest of the manuscript would be aided by characterisation of a specific VacC mutant.

    We share the referee's fascination with these data showing that VacC is a specific reporter of virulent mycobacteria infection. First, VacC expression at the transcriptional level, but also at the protein accumulation level both point toward a correlation with an infection with damage-causing mycobacteria. Specifically, one can distinguish two stages, one transient upregulation of all three isoforms that becomes sustained only for VacC and only when wt Mm causes damage (as opposed to the DRD1 mutant or M. smegmatis). This is clearly presented in multiple places in the manuscript (for example lines 377-380).

    Now, how is MCV damage sensed is extremely interesting and is the focus of numerous past and on-going studies in our laboratory but is out of the scope of this article. Just to mention a few lines of research as food for thoughts, membrane damage (by EsxA and by LLOMe) triggers the recruitment of the E3 ubiquitin ligase TrafE (PMID: 37070811), and subsequently of the ESCRT and autophagy machineries (PMID: 37070811, PMID: 30596802). Upstream of TrafE, we know that decrease of membrane tension is one parameter, because transient hyperosmolar shock also recruits TrafE to endo-lysosomal compartments (PMID: 37070811). On-going experiments demonstrate that calcium leakage from endo-lysosomes and MCV is another major triggering factor.

    As mentioned above, and in more direct response to the referee's questioning, we have now included RNAseq experiments that unequivocally indicate the link between vac-KO and sterol depletion and the direct effect on reducing membrane damage, because the two conditions lead to a down-regulation of the damage-dependent transcriptomic signatures of the ESCRT and autophagy related genes (Fig. 4G-H and Fig. EV5). Moreover, it clearly establishes that sterol depletion, which decreases sterile and EsxA-mediated damage, decreases vacuolin expression in infected cells (Fig 3G). Finaly, qRT-PCR on infected BV-2 microglial cells indeed documents an up-regulation of flotillin-1, reminiscent of vacC regulation in Dd (Fig. EV9B).

    All in all, we would like to respectfully ask the editor and referee to acknowledge that the signalling pathway between damage sensing and the vacuolin responses will be the focus of future studies.

    We understand that investigating the phenotypic consequences of only a single vacC-KO might be interesting, but we would like to argue that it is superfluous. First, for intricate biological reasons, KO of single and combinations of vacuolin genes result in very qualitatively and quantitatively similar phenotypes associated to motility, phagocytosis, endosome maturation etc... (PMID: 32482795). The present study extends this remarkable phenomenon by interrogating multiple parameters, reporters and phenotypes linked to infection, some shown and some unpublished (for example Fig. EV3B and Fig. 4D-E).

    Are the MMVs positive for all three vacuolins? It would be great if you could quantify which are present together or whether there are more distinct populations that are positive for just one or all three for example.

    The referee points to an interesting mechanistic aspect. We have therefore directly assessed the colocalization of pairs of vacuolin isoforms (Fig. EV1B), which clearly indicate that every MCV is coated with two vacuolins, which therefore arithmetically implies that all three isoforms are present together and that there is no isoform-specific MCV (Fig 2B). This is potentially also corroborated by earlier studies that showed vacuolin hetero-oligomerization (PMID: 16750281), a characteristic shared by flotillins (PMID: 38985763).

    Minor Comments:

    Fig 1F - this graph is quite striking but I think the individual data points should be presented as it is unclear whether this intensity threshold is an arbitrary value or genuinely represents two different populations. Perhaps better represented as a scatter plot?

    We fuly agree with the referee and have accordingly replotted all the graphs where this improved the visualisation and contributed to the interpretation of the data. We did not change the representation in Fig. 7E and G, Fig. EV3C, because the error bar already represents the deviation of the Area Under the Curve (AUC) that was calculated for the average curves resulting from a biological triplicate of experiments.

    The bar graphs early in the manuscript should shoe the individual data points from replicates. While the presentation is clear and differences are striking I think this article explains why showing the replicate data is important: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128

    We fully agree with the referee and have accordingly replotted all the graphs where this improved the visualisation and contributed to the interpretation of the data.

    In Figure 2: F and G should include quantification, in G the arrow on the24 hpi filipin panel is not in the right location

    As mentioned in response to referee #1 and #2, qualitatively, sterols, as visualised by filipin and D4H, are present at all stages of the endo-lysosomal pathway and of MCV biogenesis. Now, there are many technical difficulties linked to a quantitative assessment, and therefore, please, let me present the framework. First, despite their wide use, the exact mechanism of binding of both reporters and which pool of sterol they visualise is still a mystery. This is often expressed as "they detect the accessible pool" of sterol, whatever it is. In addition, filipin detects sterols in both leaflets (and in intra-lumenal vesicles and other lipidic structures), while D4H detects sterols only in the cytosolic leaflet, and it is not known whether both leaflets have the same concentration of sterols. It is also known that filipin signal is only indirectly proportional to the sterol quantity in a cell, as measured by other quantitative methods. One of the best examples comes from studying the cellular phenotype of Niemann-Pick Type C disease, because many publications report a strong increase of filliping staining, whereas lipidomic analyses show at best a two-fold increase in cholesterol in NPC deficient cells. Moreover, technically speaking, D4H is a live probe, and fixation leads to some loss of localisation, probably because sterols are not fixable. On the other hand, filipin is mainly used after chemical fixation, but again sterols are not fixable, and the signal is very likely restricted to the membrane of origin, but not necessarily to the microdomains.

    We corrected the arrow localisation.

    Reviewer #3 (Significance (Required)):

    The key strength of this manuscript is the use of the Dictyostelium model to dissect host-pathogen interactions. This provides an interesting evolutionary lens to the research findings presented here and is further strengthened by the data demonstrating that these findings are relevant in a mammalian model as well. The weaknesses are articulated in my "major comments" section. The phenotypic data presented here is strong - it is clear that these vacuolin proteins are important for the intracellular success of M. marinum however the data demonstrating the mechanism for this is less clear.

    We thank the referee for this overall positive summary of our main findings and of the clarity of results, interpretations and working model. As detailed above, we respectfully disagree with the final conclusion and are pleased to note that the other two referees are more satisfied with the level of mechanistic evidence.

    I am an academic researcher who is interested in the molecular host-pathogen interactions mediated by intracellular microbial pathogens. Scientists in my research field will be a key audience for this research. Predominantly this is basic researchers but the interest will be broader than host-pathogen interactions as researchers in the membrane integrity and membrane dynamics field will be interested here.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Bosmani, Perret et al examines the role of Dictyodistelium discoideum vacuolin proteins in the integrity of the Mycobacterium marinum vacuole membrane. The data demonstrates that loss of vacuolins, similar to sterol depletion, reduced vacuole membrane damage meaning less cytosolic escape of the pathogen and subsequently reduced bacterial replication. The authors demonstrate functional analogy in a mammalian model of infection - where flotillin plays a similar role to the vacuolins - and this is an important demonstration of the utility of the D. discoideum model. The data is well presented and clear.

    Major Comments:

    1. There is no evidence presented in the manuscript of "microdomains" - while I believe this is likely a true description of what is happening on the vacuole membrane there is no visualisation of this. Both the GFP-Vac vacuole staining and the filipin staining show complete coverage of the vacuole. Perhaps at the 1 hour time points this is more convincing but I think it is worth looking at more of these earlier time points and quantifying these "microdomains" - i.e. proportion of vacuole membrane that is positive for the Vacs. Is it possible to look at the GFP-Vac signal and filipin staining at the same time? And other vacuole markers too?
    2. I really like the data presented in Figure 1 that demonstrates the specific upregulation of Vacuolin C during M. marinum infection. This is an intriguing result that brings up a lot of new questions e.g. how is this regulated? In response to membrane damage? Sensed by what? Does this upregulation also hold true for flotillin in the mammalian model? (and more!) however none of these ideas are pursued in the manuscript and by the end I was wondering why this data was included in the manuscript because all of the phenotypic data uses either a VacBC or ABC mutant. The link between figure 1 and the rest of the manuscript would be aided by characterisation of a specific VacC mutant.
    3. Are the MMVs positive for all three vacuolins? It would be great if you could quantify which are present together or whether there are more distinct populations that are positive for just one or all three for example.

    Minor Comments:

    1. Fig 1F - this graph is quite striking but I think the individual data points should be presented as it is unclear whether this intensity threshold is an arbitrary value or genuinely represents two different populations. Perhaps better represented as a scatter plot?
    2. The bar graphs early in the manuscript should shoe the individual data points from replicates. While the presentation is clear and differences are striking I think this article explains why showing the replicate data is important: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128
    3. In Figure 2: F and G should include quantification, in G the arrow on the24 hpi filipin panel is not in the right location

    Significance

    The key strength of this manuscript is the use of the Dictyostelium model to dissect host-pathogen interactions. This provides an interesting evolutionary lens to the research findings presented here and is further strengthened by the data demonstrating that these findings are relevant in a mammalian model as well. The weaknesses are articulated in my "major comments" section. The phenotypic data presented here is strong - it is clear that these vacuolin proteins are important for the intracellular success of M. marinum however the data demonstrating the mechanism for this is less clear.

    I am an academic researcher who is interested in the molecular host-pathogen interactions mediated by intracellular microbial pathogens. Scientists in my research field will be a key audience for this research. Predominantly this is basic researchers but the interest will be broader than host-pathogen interactions as researchers in the membrane integrity and membrane dynamics field will be interested here.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    the authors of this manuscript reported that EsxA, a secreted virulent factor of Mtb or Mm, causes membrane lysis in sterol-rich micro domain. They used the Mm-infected amoeba as an infection model, and characterized the effects of microdomain in Mycobacterium-containing Vacuole (MCV) on EsxA-mediated membrane disruption. They found that disruption of the micro domain through knockout of vacuolins or sterol depletion diminished Mm-induced membrane damage and cytosolic escape. They also found that vacuolins and sterol are essential for EsxA inserting into the membranes in vitro, and flotillin knockdown and sterol depletion conferred the resistance of murine microglial cells to Mm infection. The experiments were well designed and controlled, and the data were convincing.

    My major comment is that the authors need to justify the use of BV-2 cells that are murine microglial cells, instead of macrophage cell lines, which are more relevant to Mtb/Mm infection.

    Significance

    It is well known that EsxA is membrane-lytic protein playing a role in Mtb/Mm-mediated phagosomal escape. There are other studies that have indicated lipid raft or micro domains in the membrane may play a role in EsxA-mediated membrane damage. This study further confirmed that the sterol-rich micro domain on the membrane has significant influence on the EsxA-mediated membrane disruption both in vitro and in vivo. While this finding is expected, but confirmation with solid experimental evidence is welcomed. This study also identified the genes or proteins required for micro domain organization, vacuolins and flotillin, which could be a target of host-directed therapy. Overall, this study is performed well and the results are convincing.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The proposed study aims to elucidate the role of membrane microdomains and associated proteins-Vacuolin A, B, and C-during the infection of Dictyostelium discoideum (Dd) amoebae by Mycobacterium marinum (Mm). The results demonstrate that Vacuolins are required for Mm virulence, and that the presence of membrane microdomains is essential for phagosome membrane damage and bacillary escape into the cytosol-key steps in establishing a successful infection and subsequent bacterial proliferation. The study is well-designed, employing methodologies with which the authors have demonstrated expertise. Overall, it is methodologically sound, and most conclusions are well-supported by the presented data. However, some points require clarification. Major Points: The study aims to link the function of Dd Vacuolins to their potnetial facilitating role in phagosome escape and overall infection by Mm. To phenocopy the effect of Vac-KO, the authors used MβCD. Strikingly, this compound had a more significant impact on phagosome escape compared to Vac-KO, which either did not affect or only mildly affected this process. This likely reflects a difference in the underlying mechanisms being studied. Vac-KO cells may lack well-organized membrane domains but could retain a similar overall membrane composition. In contrast, MβCD disrupts these domains by chelating cholesterol, thus altering both the membrane composition and the domains themselves. This may explain why EsxA partitioning is more affected by MβCD than by triple KO. Consequently, this suggests that cholesterol, rather than the mere presence of membrane domains, plays a critical role in EsxA partitioning and activity in the phagosome. And even if LLOMe activity was lower in Vac-KO cells, this might be explained by the compartment targeted, the lysosomes which membrane composition may differ from the MCV. These points should be further discussed in the discussion section.

    Based on this observation, in figure 2, does the D4H/filipin signal or association increase over time as the Vac signal "solidifies"? In Vac-KO cells, does the mScarlet-D4H signal change in intensity or pattern (building on fig. S1)? These insights could provide valuable information on cholesterol levels at the MCV in KO versus wild-type cells. If possible, the authors should quantify fluorescence or the frequency of signal association. Additionally, since Vacuolins do not have a significant impact on phagosome damage or escape, the difference in intracellular growth may be indirect, as suggested in the team's previous study on Vacuolins (DOI: 10.1242/jcs.242974). The authors measured MCV pH in figure S6-could they repeat this experiment to test whether Vacuolins affect MCV maturation? This was investigated in a previous version of the pre-print (DOI: 10.1101/2021.11.16.468763), and if the results still hold, it would strengthen the hypothesis that Vacuolins promote escape by modulating membrane organization, rather than influencing phagosome maturation. Finally, as a substantial part of this manuscript relies on microscopy and image analysis, the methods section should detail how these analyses were performed. Specifically, for figure 1f, it is unclear how the cells were segmented and fluorescence quantified-was total fluorescence per cell measured, or was an average value used? Figures 5c and 5h could be moved to the supplementary material, and the segmentation method should be explained in the methods section. Additionally, statistical analysis should be more clearly described, justifying the use of one-way or two-way ANOVA, and specifying the post-hoc tests used for group comparisons.

    Significance

    This study provides the first direct evidence of the importance of membrane composition and organization in the virulence of Mycobacterium marinum, particularly in facilitating phagosome damage and bacillary escape. Using the well-established model of Dictyostelium discoideum infected with M. marinum, which has frequently been predictive of Mycobacterium tuberculosis behavior within phagosomes, the authors contribute critical insights into the mechanisms of mycobacterial phagosome escape-a key step in cellular invasion and dissemination. These findings have the potential to inform strategies aimed at blocking this escape mechanism, which, as demonstrated in this study, could prevent intracellular bacterial growth.

    This work is significant for advancing our understanding of mycobacterial pathogenesis, particularly by linking membrane microdomain composition to bacterial virulence. It will be highly relevant to researchers studying mycobacteria, intracellular pathogens, and host-pathogen interactions. While the study's use of M. marinum provides valuable insights, a limitation is that these results may not fully translate to M. tuberculosis, and further testing with the latter pathogen will be essential.

    I am an expert in the infection of macrophages by Mycobacterium tuberculosis, the phagosome escape mechanism, and its associated effectors. I also have expertise in microscopy and image analysis. However, I do not specialize in Dictyostelium discoideum biology.

  5. Version published to 10.1101/2024.08.19.608731 on bioRxiv