PREreview of Attenuation of a DNA Cruciform by a Conserved Regulator Directs T3SS-1 mediated virulence in Vibrio parahaemolyticus

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:

Attenuation of a DNA Cruciform by a Conserved Regulator Directs T3SS-1 mediated virulence in Vibrio parahaemolyticus

Landon J. Getz1, Justin M. Brown1, Lauren Sobot1, Alexandra Chow1, Jastina Mahendrarajah1, Nikhil A. Thomas1,2 doi: https://doi.org/10.1101/2022.03.07.483294

We will adhere 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. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: Vibrio bacteria cause millions of life-threatening infections each year. These pathogens possess numerous disease-causing pathogenicity factors. Vibrio also encode DNA binding proteins that regulate virulence gene expression by altering DNA conformation. The HlyU family of winged helix-turn-helix (wHTH) DNA-binding proteins positively regulate a subset of virulence genes; however, the mechanism remains unknown. Several Vibrio strains have shown HlyU proteins outcompete binding of virulence genes by Histone-like Nucleotide-Structuring protein (H-NS) to prevent gene silencing. The HlyU and H-NS DNA binding sites do not often overlap, suggesting DNA topology or long-range protein-DNA interactions that could influence gene expression. The authors previously discovered that the exsBA intergenic region in Vibrio parahaemolyticus is an HlyU binding site that forms a cruciform DNA structure. DNA cruciform structures are present in all domains of life and have been implicated in repressing gene expression; however, functional DNA cruciforms in bacterial chromosomes are rare and no examples have been linked to virulence gene regulation. The authors aimed to investigate the mechanism of HlyU function in the context of exsA gene expression and subsequent T3SS-1 gene regulation. They hypothesized that a DNA cruciform structure is involved in regulation of exsA expression. The authors present data suggesting HlyU is responsible for attenuating a transcriptionally repressive DNA cruciform, leading to expression of exsA and resulting in V. parahaemolyticus T3SS-1 mediated virulence. They provide a stepwise model for the mechanism of DNA cruciform involvement in exsA activation and T3SS-1 activity. Overall, the authors present data suggesting a new role for HlyU in destabilizing a DNA cruciform structure that represses exsA expression, regulating virulence gene expression related to T3SS-1 activity. This reveals a previously overlooked de-repression mechanism of specialized DNA binding proteins to regulate gene expression.

OVERALL ASSESSMENT: This is a well-written manuscript that provides new insights into the regulation of virulence gene expression for an important human pathogen. Conclusions are well supported by the data. There are significant elements of novelty throughout, increasing mechanistic understanding of roles for HlyU and DNA cruciform structures in virulence gene regulation. We identified opportunities for further increasing clarity to reach a broader audience. The authors should take this opportunity to discuss the significance and potential applications of this work.

STRENGTHS: The writing is clear, and conclusions are well supported by the data. The authors provide new mechanistic insight into regulation of virulence gene expression. The novelty of this paper is a big strength as it suggests a previously unknown mechanism of gene regulation through a DNA cruciform structure and provides many new avenues for further research to understand virulence gene regulation in Vibrio spp. and other prokaryotes. 

WEAKNESSES: The only weakness we discussed was the supercoiling data, which if supported by further experiments could fit well within the dataset and provide stronger support for conclusions. In its current state, this data should be moved to the supplement, as it currently does not contribute to understanding of the model being established by the authors. Further explanation from the authors about the significance of the supercoiling data could help support its retention in the manuscript. 

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE =

● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

o Figures 1A and 1B were hard to interpret without detailed reading of the figure legend. It would be easier to interpret if the corresponding plasmids and gels were paired in one panel along with adding the enzymes used in each lane. This would make scanning of the gel for the correct bands in each lane easier. This change in labelling could be applied to Figure 5A as well.

o Figure 2 did not present results that strongly contributed to the storyline of the paper. By moving this figure to the supplementary data this would create a stronger, more linear story. Furthermore, supercoiling was not included in the model depicted in Figure 7, making it difficult for readers to understand the role it plays in this system. We suggest that sub-inhibitory levels of novobiocin may contribute to increased supercoiling; adding a reporter plasmid assay looking at linkage number changes would strengthen the supercoiling data. After the discussion in class and hearing from the authors about the role and significance of this figure to the story it was made clearer that this data is important. This should be clarified in the text so readers understand the full scope of this figure.

o The scales for Figure 2C are not the same and the labels and legend are small relative to the rest of the figure. This could be improved to make reading easier. 

o In Figure 3C, it seems that there was a reduction on unbound DNA and slow shifting bands in IR2, IV1 and IV2 HlyU positive group. It is unclear what those bands are and why they were not considered as DNA-HlyU binding complex. 

o There was insufficient explanation about the S1 and S2 shift in Figure 3C. It wouldn’t be much of an issue if both S1 and S2 disappeared in the mutant DNA group, but it may raise some questions in cases where only one band (S1 or S2) disappeared.

●     Are specific analyses performed using methods that are consistent with answering the specific question?

Yes

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

Yes

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

Yes

●     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 two examples in the literature of such results. Addressing 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 and discussion of discrepancies is an important aspect of scholarship.

Yes

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

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

o Generally good.

o Including more specific key words in the title would better represent the findings in the paper. There was also some discussion about the use of the word “attenuation” in the title and if it was the best word choice. 

●     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 and 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.

o No.

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

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

o Figure 6A and Figure 7C are missing statements in the figure legends that these experiments were repeated 3 times and a representative image is shown. This statement was appropriately included in all other figure legends.

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

o Yes

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

o Yes

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

o N/A

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE =

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

o Yes

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

o Yes

●     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 should not to be significant basis for decisions.

o Non-expert readers would benefit from more in-depth groundwork in the Introduction. In general, while reading the Into, we found it difficult to conceive the antagonistic activity of HlyU and H-NS, how this controlled expression of exsA, and how this then related to T3SS activity.

MORE SUBJECTIVE CRITERIA (IMPACT)

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

A score here should be accompanied by a statement delineating the most interesting and/or 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 (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 Box 2). Citations (figure by figure) are essential here.

o As the authors stated in their discussion, HlyU has shown de-repression of gene expression via competitive binding with H-NS. One of the novelties of this study is that it proposed another mode of action of HlyU: the binding of HlyU to its target DNA sequence has negative effect on the gene repression activity of DNA cruciform. This fundamental study provides a sufficiently important advance that does not require further extension. 

Impact: Extensibility (1–4 or N/A scale) SCORE = 

●     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)?

o The relevance of Vibrio species was well established in the introduction; however, this is not touched on again in the discussion. The authors conducted their experiments using Vibrio parahaemolyticus, further experiments to recapitulate their findings identifying cruciform structures in other Vibrio species were included only in the supplemental material. It would have been nice to carry another species forward for further experiments, especially to further investigate how V. anguillarum performed since it was identified to have a cruciform structure in silico but did not linearize with T7 endonuclease. It would also be nice to have some interpretation from the authors as to the significance and application of their findings to combating diseases caused by Vibrio spp. 

●     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, Box 2).