PREreview of Enhanced virulence and stress tolerance are signatures of epidemiologically successfulShigella sonnei

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: 

Enhanced virulence and stress tolerance are signatures of epidemiologically successful Shigella sonnei

Sydney L. Miles, Dilys Santillo, Vincenzo Torraca, Ana Teresa López Jiménez, Claire Jenkins, Stephen Baker, Kate S. Baker, Vanessa Sancho-Shimizu, Kathryn E. Holt, Serge Mostowy

bioRxiv 2025.02.05.636615; doi: https://doi.org/10.1101/2025.02.05.636615

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: Shigella sonnei is a major cause of gastrointestinal disease globally, with Lineage 3 being the most virulent strain among five lineages. To date, our understanding of S. sonnei Lineage 3 virulence is largely extrapolated from infection models of lab-adapted strains that may not properly reflect the enhanced virulence of the Lineage 3 strain. Miles SL, et al., previously generated assembled genomes of S. sonnei isolates, revealing loss of genes encoding putative immunogenic proteins. Motivated by this finding, they investigated factors that could contribute to Lineage 3 infection outcomes using a zebrafish model, followed by confirmatory studies in primary human neutrophils. From the infection model conducted in zebrafish larvae, Lineage 3 exhibited similar pathogenesis tactics to other lineages (i.e., Lineage 2.8), such as the presence of a large virulence plasmid (pINV) encoding a type three secretion system (T3SS) that aids in host cell invasion. Additionally, like other lineages, the virulence in the S. sonnei Lineage 3 strain was also associated with the induction of leukocyte cell death, and macrophage and neutrophil recruitment. Conversely, the heightened virulence of Lineage 3 could be due to its increased tolerance to complement-mediated killing, efficient growth in acidic conditions, and early recruitment of neutrophils relative to other lineages. Finally, the authors investigated the role of group 4 capsules (G4C) synthesis genes in the enhanced tolerance of Lineage 3. Overall, this study provides useful new information about the molecular underpinnings of S. sonnei lineage 3.

OVERALL ASSESSMENT: We commend the authors for addressing significant knowledge gaps about an important human pathogen. The authors leveraged a zebrafish infection model to understand the virulence and tolerance of S. sonnei Lineage 3. However, we have identified some areas where the study methodology, analyses, and descriptions could be improved to strengthen the manuscript. We also recommend minor modifications of some figures, along with more exposition in the figure legends, to improve readability.

STRENGTHS: The study provides a better understanding of the heightened virulence and tolerance S. sonnei Lineage 3, which remains poorly understood. The study showcases how genomics can be powerful in generating testable hypotheses. This is because the authors first analyzed assembled genomes from different strains of S. sonnei, subsequently finding that Lineage 3 has lost most of the immunogenic-linked genes which are present in other lineages.

WEAKNESSES: We identified some discordance between certain data and conclusions drawn from them, with notable inconsistencies between the zebrafish larvae and human cell infection models. Additionally, although the study improves the understanding of S. sonnei Lineage 3 virulence, it is not clear why control treatments are lacking in some experiments. The in vitro Congo Red-induced secretion assay was interesting, but its relevance to the zebrafish infection model was unclear.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments  

·     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: While the figure strongly supported the conceptualization of the initial hypothesis that formed the basis of this study, we have the following suggestions:

o   Add more exposition/detail in the figure legend to aid reader comprehension.

o   Flip the figure horizontally for a better view

o   Simplify the figure by removing unnecessary details. For example, the authors can focus on genes that were unique to the S. sonnei clade 3 isolates, or perhaps focus only on fimbriae genes. This will focus the reader’s attention on points of difference.

o   The figure could be re-structured to represent the data as strain by strain and number of virulence genes, rather than focusing on presence or absence of individual genes in a particular strain.

o   In lines 121-122, the authors mention that the genes that were found to be absent in Lineage 3 included ‘fimBHGF’. However, from Figure 1, we can clearly see that fim ‘B’ is present in S. sonnei Lineage 3.

·       Fig. 2:

Fig 2 A-B: Since there are only two data points, the survival curve does not properly represent the trend. A box plot may be more appropriate. Additionally, a control experiment was lacking in this figure and therefore using Lineage 1.5 as reference point sparked a question of whether it was avirulent. The authors should also comment about the effect of raising temperature on the viability/health of the zebrafish larvae, which is a limitation of the model.

2C-D: The use of parametric tests such as ANOVA without informing the reader whether the distribution of the data points shown in the figure was normally distributed. In case the data was not normally distributed, then non-parametric test such as the Kruskal-Wallis rank sum test with Dumm post hoc test (correcting for multiple comparisons) would be more appropriate.

Fig 2E-F: In the text (lines 151-152) the authors refer to this figure and say that the increased dissemination indicates that the immune response is subverted. However, the data in this figure does not agree with that statement. Additionally, we would like the authors to comment on why there was no difference in CFU across the lineages when they continue to state that Lineage 3 is evading immunity. Our thought was that the increased dissemination could be also due to something else like increased capacity to cause tissue damage (b/c of things like increased neutrophil recruitment, etc...).

2F: The figure caption should tell the reader what lineage was used to infect the zebrafish larvae.

·       Fig. 3: The protein secretion assay in Fig 3F sparked a discussion in our review session about the appropriateness of the in vitro assay considering that it is performed on bacterial cultures, and not in the infection model. Beyond this point, we thought the assay would benefit from a loading control like a whole cell lysate, which is a standard approach in the field. The SDS-PAGE gel should be run longer to improve resolution of protein bands. From the figure, it appears that CR- S. flexneri was secreting SepA as expected, in a T3SS-independent manner, but there is no corresponding band for the S. sonnei sample; does S. sonnei lack SepA? More detail should be provided about the temperature conditions (28˚C or 32.5˚C) for the experiment.

·       Fig. 4: It’s unclear why PBS was introduced at this stage rather than earlier. In Fig. 4F, if the higher points are excluded as outliers, it may be justified. If not, the text (lines 200-201) should be revised to reflect the data as upregulated but not significant.

·       Fig. 5: The G4C gene visualization in Fig 5E does not give the reader a clear picture of the results of the study. Therefore, it can go to supplemental, or it could be expanded on with more data from the supplementals. Instead of emphasizing on findings related to acid or stress resistance, the authors should shift the focus to results concerning complement resistance and capsule results.

·       Fig. 6: The data reported here suggests that there should be an increase in CFU compared to Figure 2. It would have been interesting to investigate timepoints beyond 1 hpi to determine whether increases in S. sonnei lineage 3 CFU plateau and decline. This would better cohere with the zebrafish data (i.e., some kind of early transient advantage). Additionally, one hour is quite an early timepoint to see a strong cytokine response.

·       Table 1: Acronym OUCRU = Oxford University Clinical Research Unit is referenced in the caption but not featured anywhere in the table. The author may replace this by defining the acronym GFP which appears as labels to some strain ID’s.

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

·     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  

·     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. 

·   Yes

·     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. 

·   Yes

3. Quality: Reproducibility  

·     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? 

·   The authors need to provide additional experimental details concerning the protein assay and gel experiment in Figure 3F. Additionally, the temperature conditions employed for experiments in Figure 3 should be clearly stated.

·     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  

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

·       Figure 3C-E compares T3SS expression of the tested S. sonnei lineages. Is this representative of what’s happening in the infected zebrafish, since they are grown at very different temperatures? (32.5^oC vs 37^oC).

·       For the secretion assay, could a simple proteomics experiment be more valuable compared to less informative stained SDS-PAGE gel?

·       The narrow range of time points used in this study may be problematic. Perhaps if authors were to extend these experiments, they would see a plateau in growth (refer to Fig 2) where all lineages would even out, which would support the zebrafish survival data. Especially since zebrafish (to the best of our knowledge) have a functional complement system.

·     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? 

In line 135, we suggest the authors to consider citing the following study to justify choice of temperatures for conducting experiments:

Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO. Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J. 1998 Dec 1;17(23):7033-43. doi: 10.1093/emboj/17.23.7033. PMID: 9843508; PMCID: PMC1171051.

·     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 arrangement of the data in multi-panel figures could be improved and harmonized. For example, there is little justification for combining complement killing figure panel with a capsid figure panel.  

·       Lines 150-151 states that “significantly increased dissemination was recorded for Clades 3.6 and 3.7 relative to other Lineages (Fig 2E-F)” but the authors only looked at one other lineage in 2E-F (lineage 2.8), so this statement should be revised. This is also immediately followed up with “F), consistent with an increased propensity to subvert the immune response”, a strong statement insufficiently supported by the primary data. Revise for clarity and accuracy.

·       Fig 6F is the only graph in Fig 6 where the authors plot “relative CFU” relative to lineage 2.8 rather than relative to inoculum. We suggest this to be changed to relative to inoculum for consistency with other graphs in the figure.

MORE SUBJECTIVE CRITERIA (IMPACT): 

Impact: Novelty/Fundamental and Broad Interest  

How big of an advance would you consider the findings to be if fully supported but not extended? 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)? 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)

·       The authors excelled in i) developing new zebrafish infection model for the difficult-to-model S. sonnei infection and ii) strongly situating the context/importance of their work in terms of better understanding clinically relevant S. sonnei lineage 3 which remain poorly characterized to date.

References:

·       Falconi, M., Colonna, B., Prosseda, G., Micheli, G., & Gualerzi, C. O. (1998). Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature‐dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H‐NS. The EMBO journal.

·       Shad, A. A., & Shad, W. A. (2021). Shigella sonnei: virulence and antibiotic resistance. Archives of Microbiology, 203(1), 45-58.

Competing interests

The authors declare that they have no competing interests.