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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: 

RNase-mediated reprogramming of Yersinia virulence

Ines Meyer, Marcel Volk, Ileana Salto, Theresa Moesser, Anne-Sophie Herbrüggen, Manfred Rohde, Michael Beckstette, Ann Kathrin Heroven, Petra Dersch

bioRxiv 2024.01.11.575149; doi: https://doi.org/10.1101/2024.01.11.575149

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: Bacterial pathogens tightly control the synthesis and deployment of virulence factors through a variety of mechanisms, including at the RNA level. In Yersinia pseudotuberculosis, the Type III Secretion System (T3SS) is upregulated in response to external stimuli, providing a conduit for Yersinia outer proteins (Yops) to be delivered into target host immune cells. Virulence-associated mRNAs are controlled at the post-transcriptional level by RNase-mediated degradation. In this preprint, Dr. Petra Derch’s team reports that endoribonuclease RNaseIII and exoribonuclease PNPase repress the synthesis of the thermally regulated, master virulence regulator LcrF. They constructed mutants with deletions of 11 putative RNAses in Y. pseudotuberculosis. Mutants lacking these RNases were found to have significantly higher levels of secreted proteins in secretion conditions (e.g. 37^oC, calcium depletion). The copy number of the virulence plasmid pYV where the T3SS is encoded is not influenced by RNase III or PNPase, suggesting the effects are primarily exerted through regulation of gene expression. Transcriptomic profiling revealed that induction of secretion coincided with a shift of gene expression from the chromosome to the pYV plasmid, a phenotype that was exacerbated in the RNase III deletion mutant Y. pseudotuberculosis. Overall, PNPase inhibits YopD expression, which subsequently inhibits LcrF expression by a negative-feedback loop. RNase III is an inhibitor of both PNPase and LcrF: loss of RNase III leads to increased availability of active RNA-binding protein carbon storage regulator (Csr) A by dimishing CsrB and CsrC expression, which stabilizes lcrF mRNA. 

OVERALL ASSESSMENT: The primary focus of this preprint was on roles for the endoribonuclease RNase III and exoribonuclease PNPase in regulation of Yersinia virulence. The authors engineered the necessary deletion and complemented mutants to demonstrate the effects of RNase III and PNPase on Yersinia virulence at the gene, gene product and phenotype levels. The evidence presented indicates that virulence gene expression is controlled by RNase-mediated regulation that exerts control over the master virulence regulator LcrF. It is convincingly demonstrated that RNase III and PNPase upregulation during infection conditions rapidly inhibits LcrF expression. This investigation into post-transcriptional regulation of virulence in Yersinia provides new insight into the importance of RNases in bacterial pathogenesis.

STRENGTHS: The authors are thorough and conduct a series of experiments that support the role of RNase III and PNPase in regulating Yersiniavirulence. The manuscript is well-written with only minor inconsistencies in text and data presentation. Techniques are consistent with best practices in the field. 

WEAKNESSES: The transcriptome data provides strong support for RNase-dependent mediation of virulence in Yersinia pseudotuberculosis, but “global reprogramming” of Yersisia gene expression does not accurately describe this regulatory mechanism. A revised title could instead focus on molecular mechanisms that govern the virulence phenotype.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

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

·     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].

·       Fig. 1 – 1A: Consider scaling the axes of graphs in panel A to be the same. Consider using statistics, e.g. repeated measures ANOVA or compare area under the curve, to more strongly support the findings presented in this graph. Using language in the text such as “more severe” and “much stronger” suggests striking differences but this argument would be better supported by a test that determines if the growth defects are statistically significant.

·       Fig. 3 – 3B: While RNaseIII and PNPase are the focus of the experiments, the YopE reporter experiment is only done with the RNase III mutant. The text should offer some rationale as to why a PNPase mutant was not also used for this assay. “Relative change” is a somewhat ambiguous term. While we can assume it means fold change relative to the wild type, this should be explicitly stated in the y-axis label, i.e. “Secreted YOP Proteins, fold change vs. control.” This can be applied to the other “relative change” axes in the figures.

·       Fig. 6 – 6B: Y-axis should be “fold change.”

·       Fig. 10 – 10B: Y-axis should be “fold change.”

·       Fig. 11 – 11B: Pie charts are harder to read than readily available alternatives. We suggest a stacked bar chart would suffice here. Here is some information regarding visualization and offering better alternatives to pie charts.

o    Siirtola, H. (2019, July). The cost of pie charts. In 2019 23rd International Conference Information Visualisation (IV) (pp. 151-156). IEEE.  

o    https://github.com/cxli233/FriendsDontLetFriends#10-friends-dont-let-friends-make-pie-chart          

·   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 = 2

·  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. 

·  The paper title is generally supported by the data, but it is quite vague. This paper only focuses on Yersinia pseudotuberculosis and while their RNA-seq data is compelling, the bulk of the data is really on the mechanistic interaction between RNAses and the YOP/T3SS. Revising the title to something more specific might could help readers, and including mention of the T3SS in the title could actually attract more readers. Suggested revised title: “RNAse-mediated regulation of the T3SS and virulence in Yersinia pseudotuberculosis” 

·  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.  No

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

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

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

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

·  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 (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

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

· The authors made effective use of the available literature to support their study and propose a well-supported model for RNaseIII and PNPase regulation of the Yersinia pseudotuberculosis T3SS.

· The Discussion was quite well written, drawing on interesting citations to support their Csr and translation factor data with regards to Yop-independent RNase III regulation of Lcrf levels. 

·     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? 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 be significant basis for decisions.

·  The Introduction was very helpful for a generalist audience, and the authors did a good job of describing their different growth conditions and what they represented (environment, T3SS inducing, etc). The first paragraph of the Results should be revised to be more concise and clearer, but from paragraph 2 onwards the Results section very well written. 

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 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? 

·       This impactful study significantly advanced our understanding of regulation of the Yersinia T3SS and virulence at the RNA level. 

·       Some outstanding questions remain regarding how RNAse III is reduced in wildtype Yersinia during infection. In this respect, we thought it might be helpful if outstanding questions and future directions were stated more explicitly at the end of the Discussion.

2.   Impact: Extensibility (1–4 or N/A 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, Box 2)

Competing interests

The authors declare that they have no competing interests.