We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

Spreading of a virulence lipid into host membranes promotes mycobacterial pathogenesis

CJ Cambier, Steven Banik, Joseph A. Buonomo, Carolyn Bertozzi

bioRxiv 845081; doi: https://doi.org/10.1101/845081

We adhered to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. https://doi.org/10.1016/j.cell.2019.11.029

SUMMARY: Cambier CJ, et al. investigated the mechanism by which a virulence lipid on the outer mycomembrane (OM) of Mycobacterium marinum prevents host immune activation. They report on a system for extracting lipids from the OM, and also for reconstitution of OM lipids. Using this technique, the authors confirmed that the virulence lipid, phthiocerol dimycocerosate (PDIM), is required for avoiding host toll-like-receptor (TLR)-dependent immune responses to aid bacterial infection. The authors describe a novel method for labelling specific lipids on the OM. Using this approach, the authors demonstrated that PDIM spreads away from the M. marinum OM and into the membranes of nearby zebrafish host cells. By fixing PDIM to the OM to prevent spreading, they demonstrate that this spreading phenotype is required to avoid the host TLR-dependent immune response.

Overall, we thought this was an excellent paper. Although we were confused about several findings, we believe the authors could likely address our main concerns by including clarifying statements in the text.

OVERALL ASSESSMENT: 

STRENGTHS: Overall, we thought that a clear rationale was provided for each experiment. The manuscript introduced interesting new techniques like lipid reconstitution and labelling. New insights into PDIM spreading into host membranes could be valuable for better understanding Mycobacterium pathogenesis in humans and warrants further research. 

WEAKNESSES: Several results were difficult to interpret and could be misleading without further clarification. In particular, distinguishing between levels of TLR-independent and TLR-dependent macrophage activation was confusing. In at least one instance, a control experiment that would greatly aid interpretation is missing.

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY): 

1.     Quality: Experiments (1-3 scale) SCORE = 1.5

Figure by Figure, do experiments, as performed, have the proper controls? 

·       In general, we thought the authors did an excellent job of including appropriate controls. For instance, we were convinced by their hydroxylamine control experiments in Figure 4E that the fluorescent modifications themselves were unrelated to the in vivo spreading of PDIM that they report.

·       However, in Figure 2C the bacterial volumes for both M. marinum strains should also be reported in MyD88-deficient zebrafish. This would allow us to make clearer comparisons to the findings in Figure 2D.

Are specific analyses performed using methods that are consistent with answering the specific question? Is there the appropriate technical expertise in the collection and analysis of data presented?

·       Overall the authors used the appropriate methods for answering their specific questions (although we lack some relevant expertise, as noted below). Our issues are not with these methods, but instead with interpretation of results. However, we did have issues with several analyses, which may be resolved by adding clarification in the main text.

·       We were confused about the results reported in Figure 2D regarding macrophage recruitment by bacteria. If WT lipids are required to circumvent the TLR response, we believe that the expected result would be low macrophage recruitment for the WT lipid treatment regardless of whether the zebrafish are MyD88-deficient. Instead, the recruitment levels seem similar to the result with mmpL7 knockout lipids when MyD88 is present. At the very least, this finding requires more explanation in the text.

·       Most of the in vivo spreading experiments are focused on spreading specifically to macrophages. However, azido-DIM spreading to epithelial cells occurs in the absence of macrophages (Figure 4F and G). Although the authors do point this out, it remains unclear whether there is a difference in affinity between macrophages and epithelial cells.

·       For Figure 6E, we were also confused why the WT Live samples had such high macrophage recruitment. Again, this result seems at odds with the conclusion that WT lipids enable the bacterium to down-regulate TLR-dependent activation of macrophages. The confusion here may arise due to the difficulty in distinguishing macrophages recruited from TLR-dependent and TLR-independent signaling. Again, at the very least this distinction should be clarified in the main text. In particular, the authors should emphasize that the data in Figure 6E (and Figure 2D) is consistent only with PDIM-deficient bacteria or bacteria unable to spread PDIM onto host membranes to require TLR signaling to recruit macrophages. Based on the macrophage recruitment findings alone it is not convincing that these cells are detrimental. Also, the authors should emphasize that macrophage recruitment is high irrespective of lipid composition.  

Is there the appropriate technical expertise in the collection and analysis of data presented? 

·       To the best of our knowledge the authors have the appropriate expertise. However, we should note that many of the techniques in this study, and particularly the chemistry work, is outside our own expertise.

Do analyses use the best possible (most unambiguous) available methods, quantified via appropriate statistical comparisons? 

·       We did find certain approaches ambiguous (see above points), which should be clarified.

·       Many of the comparisons from Figure 2 onwards did not include any statistical tests. We do not anticipate that the key findings of this paper will change after including statistical tests, but nonetheless these should be included to make the results more convincing. In addition, significance stars should be indicated on the figures and the authors should describe all statistical tests they perform.

·       Several times in the text the authors mention their result is representative of three separate experiments. Based on this wording, it is unclear whether all reported differences were reproducible across separate biological replicates. If there were no differences in interpretations across different experiments this should be clearly stated. Alternatively, if there was a lot of variation across experiments, then this additional data should be included in the manuscript (perhaps as supplementary figures depending on the extent of disagreement).

Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least 2 examples in the literature of such results. To address this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study. 

·       The key findings of this paper are consistent with other work in this area.

2.     Quality: Completeness (1-3 scale) SCORE = 1

Does the collection of experiments and associated analysis of data support the proposed title/abstract-level conclusions? Typically, the major (title or abstract level) conclusions are expected to be supported by at least two experimental systems. 

·       The authors’ fluorescent labelling approach convincingly showed that PDIM spreads into host membranes. 

·       PDIM spreading was shown to be required for the TLR-dependent activation of macrophages (Figure 6E), although we are unclear why the numbers of macrophages would be so similar when recruited through a TLR-independent route (i.e. for the Live samples).

·       They also convincingly show that PDIM spreading into host membranes promotes pathogenesis, based on the observed increases in bacterial volume (especially in Figure 6F).

Are there experiments or analyses that have not been performed, but if "true" would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion/abstract that is clearly defensible with the experiments as presented, and one solution to ‘completeness’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. 

·       Again, we believe further clarification is needed to discuss the high levels of macrophage recruitment due to a TLR-independent process. If a clear explanation cannot be given, then there could be issues with how some of the results in this paper are interpreted. However, we think the main conclusions of the article are robust.

3.     Quality: Reproducibility (1-3 scale) SCORE = 1

Figure by Figure, were experiments repeated per a standard of 3x repeats or 5 mice/cohort etc.? 

·       The sample sizes of all experiments presented were sufficient.

Is there sufficient “raw data” presented to assess rigor of the analysis? 

·       Yes, the authors have done a great job of presenting a lot of data with sufficient controls to judge the data. 

Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

·       Yes, we have no concerns with how the methods are described.

If a ‘discovery’ dataset is used, has a ‘validation’ cohort been assessed and/or has the issue of false-discovery been addressed? 

·       N/A.

4.     Quality: Scholarship (1-3 scale), generally not the basis for acceptance/rejection: SCORE = 1

Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? 

·       No, the authors do not specifically discuss such relevant data. We do not have any specific recommendations for this point except that expanding the discussion would be useful and could aid interpretation of individual results (if that is permitted in the format).

Has the author cited and/or discussed the important works that are consistent with their conclusion and which a reader should be especially familiar when considering the work? 

·       The authors clearly describe similar work in the introduction that demonstrated that PDIM is required for Mycobacterium marinum to avoid the TLR signaling response in zebrafish. Their work clearly fits in a larger story that includes these studies as well.

·       The authors do discuss relevant work. In particular, they mention that the in vivo work complements a similar study that demonstrated that PDIM incorporates into host membranes in a macrophage in vitro model.

Specific (helpful) comments on grammar/diction, paper structure or data presentation (e.g. change a graph style or color scheme) go in this section, but scores in this area not to be significant bases for decisions. 

·       We noticed that a lot of introductory material is presented in the results section (e.g. the first paragraph of the results). We recommend that this information be moved to the introduction.

·       Write out (and indicate) acronyms in figure legends, for instance:

o    Indicate that dpi stands for days post infection in Figure 1 legend

o    Write out TLC, PDIM, and PNDIM in Figure 2 legend

·       We recommend that “wondered” be changed to “investigated” or re-worded to be “hypothesized” where appropriate (occurs several times - e.g. on page 3)

·       “Clickable” should be clearly defined upon first usage

·       Page 1: Change “A result” to “As a result”

·       Page 3: Change “do not repopulate the” to “do not repopulate in the”

·       We recommend that “where” be replaced with “in which” in certain places:

o    “we used the transgenic zebrafish line [...] where macrophages express [...]”

o    “PFA+GA-treated bacteria, where PDIM is no longer able to spread [...]” 

o    “our group developed metabolic labeling strategies where unnatural metabolic precursors [...] are fed [...]”).

·       At several points the authors discuss that results indicate that the presence of lipids at the onset of infection are required for virulence. We found this language a little confusing because it implies that the experiments included post-infection lipid modification. We recommend that this is clarified.

MORE SUBJECTIVE CRITERIA (IMPACT):

1.     Impact: Novelty/Fundamental and Broad Interest (1-4 scale) SCORE = 2

A score here should be accompanied by a statement delineating the most interesting/important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘1’ would be expected to be understood for the importance by a layperson but would also be of top interest (will have lasting impact) on the field. 

·       The key contributions are to confirm in an in vivo model that PDIM spreads to host membranes and is required for down-regulating the TLR-dependent activation of macrophages. 

·       We also think the methods that the authors have developed will be valuable for future work in this area.

·       We do not anticipate that this paper will necessarily be of interest to a lay audience, but it would be of interest to a broad group of microbiologists and immunologists.

How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Table 2). Citations (figure by figure) are essential here. 

These findings will be impactful without additional extension. The authors have performed numerous control experiments such as in Fig S1, Fig 1, and Fig 3 to convince us that their new methods are robust and reliable. In addition, the key results reported in Fig 6 will likely be of great interest to tuberculosis researchers. Of course, eventually extending this work into different model systems will be required to show that these results are important for understanding tuberculosis in humans.

2.     Impact: Extensibility (1-4 scale) SCORE = N/A

Has an initial result (e.g. of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g. in animals, or in a human cohort)? 

This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g. ‘repeat a mouse-based experiment in humans’) and difficulty that merely confirm, but do not extend. (see Bad Behaviors, Table 2).

·         Extending the work to explore the specific mechanism underlying the TLR response downregulation would be valuable.

·         In addition, this paper focused on zebrafish and it will be interesting to see how well the results hold up in mammals.

·         However, although we think extending this work will be useful, we do not believe any additional experiments are needed for this manuscript.