Journal article Open Access
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:
Souvik Bhattacharyya, David M. Walker, Rasika M. Harshey. Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms. BioRxiv 2020.02.26.966986; doi: https://doi.org/10.1101/2020.02.26.966986
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: In this manuscript, Bhattacharyya, et al. investigated the mechanism of how the death of a sub-population of swarming bacterial cells increased survival of the remaining cells. They called this phenomenon STRIVE (swarming with temporary resistance in various environments). Using E. coli and several other bacteria, the authors identified a candidate STRIVE factor called AcrA that was released by dead cells. They also demonstrated that the outer membrane protein TolC on the live cells is involved in the STRIVE response, possibly interacting with AcrA released from dead cells. Microscopy studies of mutant bacteria suggested that AcrA binds to TolC on the exterior of live cells and triggers STRIVE. Finally, the authors investigated the gene expression profile of the swarming cells and proposed that increased AcrA-dependent efflux and ROS catabolism, and reduced cell permeability, are potential mechanisms of STRIVE. In summary, this work reveals that AcrA is an enhancing factor of STRIVE that interacts with TolC to stimulate the reprogramming of live cells.
· The work provides a new insight of how the dead subpopulation contributes to swarming cell resistance. The findings are novel and impactful and merit further exploration.
· The authors thoroughly investigated the mechanisms of necrosignaling/STRIVE using a suite of complementary approaches.
· The reader needs more thorough Introduction and Discussion sections to put these new discoveries (necrosignaling/STRIVE) in proper context in the field. These sections are quite underdeveloped.
· Modifications in a few figures and more detailed methodology will help the reader interpret the data.
· Statistical analyses are not well described for most data.
DETAILED U.P. ASSESSMENT ("1" represents the highest quality)
OBJECTIVE CRITERIA (QUALITY):
1. Quality: Experiments (1-3 scale) SCORE = 1.5
Figure by Figure, do experiments, as performed, have the proper controls?
· Fig 1: This figure is properly controlled, but the readability of this section of the Results is poor, because the reader must flip back-and-forth between the Supplemental figures and Figure 1 to properly understand the experimental approach and the results. The authors should consider including parts of Figs S1 and S3, as well as a smaller version of the cartoon in Figure S2, to help the reader understand method and important findings. The current compressed format that relegates important data to the supplement, does not serve the reader well.
· Fig 2: The findings displayed in Fig. 2C would be stronger if a wild-type control was included.
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?
Is there the appropriate technical expertise in the collection and analysis of data presented?
· Fig S1: We think that the relative susceptibility of swarm cells at early times and low doses of antibiotic is quite subtle, and unless the reader consults the supplemental data, they might misunderstand this subtle finding. The description of this finding in the Results text should acknowledge the modest difference in susceptibility. Moreover, we wonder whether this small difference could be an artefact resulting from the collection method rather than true antibiotic susceptibility. However, this experiment was incompletely described in the methods, which makes it difficult to interpret. Appropriate statistical tests should be performed on the data in Fig. S1 to determine whether these modest differences are significant or not.
Do analyses use the best possible (most unambiguous) available methods, quantified via appropriate statistical comparisons?
· Fig 3c & 3d: The slopes of these curves are shown, but statistical analyses would be helpful in determining whether true significant differences are achieved.
· When error bars and p values are presented, the type of error (SD or SEM) and statistical method used should be described.
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 experiments are consistent with previous findings that swarming cells develop adaptive antibiotic resistance.
2. Quality: Completeness (1-3 scale) SCORE = 1.5
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.
· Most of the major conclusions are supported by the experimental results. The only egregious unsupported statement is on line 38: "...our findings may also be relevant to chemotherapy-resistant cancers". The link to cancer chemotherapy resistance is not made clear in the manuscript and is not directly addressed by any of the data. It should be removed.
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.
· We think that further description of the RNA-seq analysis is needed to clarify whether functional categories were significantly different overall or if other non-related categories were also significantly different. It is especially important that the authors reveal how they identified the gene families shown: were all gene families of porins/ROS metabolism/efflux pumps shown, or were the families shown selected from a group?
· Despite the attempt to identify critical residues for AcrA-TolC interaction, it is unclear how this extracellular interaction contributes to increased efflux and regulation of other signaling pathways, since AcrA usually acts as a periplasmic protein. As a major conclusion of this manuscript, the authors should at least discuss what they think is the most likely mechanism in the Discussion, or state that it is still unclear how this mechanism works.
· It would be nice to have a piece of data like an SDS-PAGE of the purified AcrA (could be placed in the Data Supplement) to show the quality of their purification, as any other residual E. coli proteins could influence the results.
· The authors should explain how they chose the antibiotics used in the study. In addition, it is unclear why Kan50 was used in Fig 1b and Kan70 in Fig 1d.
3. Quality: Reproducibility (1-3 scale) SCORE = 2
Figure by Figure, were experiments repeated per a standard of 3x repeats or 5 mice/cohort etc.?
· Except for the RNA-Seq analysis, most figures and methods do not indicate how many repeats were performed, especially for those that had error bars and p values like Fig 1a, 3b, & 3d.
Is there sufficient "raw data" presented to assess rigor of the analysis?
Are methods for experimentation and analysis adequately outlined to permit reproducibility?
· Methods for the Kanamycin survival curves and MIC determination (Fig S1) were not described.
· More details are needed on how the RNA-seq data was analyzed. We also suggest that the authors deposit the code that was used to create the simulations and do the RNA-seq analysis in public repository such as github.
4. Quality: Scholarship (1-3 scale), generally not the basis for acceptance/rejection: SCORE = 3
Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?
· Yes. In the Discussion the authors mention how their finding is different from previous studies that reported other functions of cell lysis.
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 Introduction and Discussion should be expanded to help readers appreciate previous findings in the field and to put the current work in proper context of larger themes like chemotaxis and quorum sensing.
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.
· The switching between "STRIVE" and "SR" is rather confusing. It would be clearer to use just one or the other consistently throughout the text.
· The resolution of pictures in the main figures should be increased if possible (this may be an issue with the BioRxiv uploader).
o Fig 2c: Mutated amino acids are not visible.
o Fig 2e: Panel FM 1-43 - deltaTolC/pTolC, looks like cellular debris; it is difficult to see any bacterial morphology, and we suggest that this image should be replaced by a clearer picture if possible. Many figures are missing figure keys.
· The heatmaps in each figure should all include figure keys so that the reader doesn't have to find this information in the figure legend. Fig 1f and S1 should also have a figure key.
· Fig 1a: It would be clearer to directly label "1" and "2" as "homogeneous" and "heterogeneous".
· Fig 2c: We suggest changing numbers I, II, III and IV (in diagram) to TolC mutants, WT AcrA, WT AcrA, AcrA mutants, respectively.
· Fig S12: The use of color (Blue, SR+; Maroon, SR-) is confusing as it appears different from the other figure legends.
· Although it is described in the Methods section, we suggest that the authors describe how they prepared the samples for the MS/MS analysis in the Results section (lines 111-113). This would help the readers understand the massive work performed to identify the final 5 common proteins.
· Some lines of text are grey in colour instead of black (lines 48, 49, 427).
· The short Introduction, short Discussion, and the extensive Supplementary figures make the manuscript very difficult to read.
· Grammatical error:
o Line 64: "The aim of present study" should be changed to "The aim of the present study".
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.
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.
· We think this work is very interesting in terms of the overall rationale and experimental design. That being said, more background and discussion should be provided to assess its novelty and impact on the field. Specifically, (i) how necrosignaling differs from previously known chemotaxis/quorum sensing mechanisms, and (ii) whether chemotaxis toward products from the lysis of other bacteria in the same species has been described.
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).
· More rationale is needed to determine extensibility. Specifically, the reader needs more discussion about how STRIVE-induced adaptive resistance differs from that induced by biofilms and how it relates to what we already know about quorum-sensing and chemotaxis.
· The necrosignaling phenomenon could be further investigated in other bacterial species such as Gram-positive bacteria like Listeria monocytogenes, Listeria innocua, Proteus vulgaris, Clostridium spp., etc., and the Discussion could describe potential extensibility of this aspect.