Write a comment

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: 

A connection between Vibrio cholerae motility and inter-animal transmission

Ian W. Campbell, Ruchika Dehinwal, Alexander A. Morano, Katherine G. Dailey, Franz G. Zingl, Matthew K. Waldor

bioRxiv 2025.02.12.637895; doi: https://doi.org/10.1101/2025.02.12.637895

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: Campbell and colleagues studied Vibrio cholerae in a neonatal murine orogastric infection model. Their objective was to use Tn-seq to identify gene mutations that alter colonization of the neonates. However, a critical limitation of Tn-seq in animal infection models is a colonization bottleneck resulting in many mutants being unable to colonize irrespective of their mutation, making results challenging to interpret. Recent work by another group demonstrated a way to overcome the colonization bottleneck using high density libraries of barcoded transposon mutants. Here, Campbell, et al., used this modified Tn-seq approach to pool and recover mutants from several infant mice to increase the number of unique mutants in their screen. From this, they identified mutants that are important for growth in the neonatal infection model but not in broth culture, which served as their control. An interesting result from this screen was the observation that motV mutants (bacteria unable to reverse direction or tumble) displayed increased colonization in the infant mice. This was confirmed by generating a motV deletion in a clinical V. cholerae strain and comparing the CFUs from small intestines infected with wild-type (WT) or the ΔmotV strain. The ΔmotV strain demonstrated increased colonization of both the proximal and distal small intestine compared to WT and in direct competition with WT. Furthermore, a competition experiment employing fluorescence microscopy and fluorescently labeled ΔmotV and WT strains showed that ΔmotV was better able to colonize small intestine crypts. This was expanded upon to implicate the ΔmotV strain in occupying the crypts earlier in infection compared to WT. The authors suggested that the enhanced ability of ΔmotV to enter crypts could be due to its motility phenotype of constitutive straight swimming. Next, they demonstrated the ΔmotV strain can overcome the initial host bottleneck better than WT and establish a greater founding population. Additionally, increased virulence of ΔmotV during infection was seen by increased diarrheal discharge, small intestine fluid accumulation, and weight loss, as well as reduced survival of ΔmotV infected infant mice. Finally, the authors investigated transmission from infected (‘seed’) to uninfected (‘contact’) littermates. There was greater transmission of the ΔmotV strain than WT strain and greater intestinal colonization of ΔmotV in contact mice contributing to the authors’ classification of the ΔmotV mutant as being ‘hyper-transmissible’. The authors also implicated cholera toxin as being important for the transmission of ΔmotV mutant bacteria but documented residual transmission in the absence of the toxin, suggesting mechanisms of toxin-independent transmission.

OVERALL ASSESSMENT: We found the paper to be well written with a logical flow. The experiments were well designed and demonstrated a breadth of knowledge in different experimental techniques while leveraging a strong animal model. The ΔmotV mutant data about colonization and transmissibility was thorough and compelling; however, their reasoning for inconsistent use of other mutants as comparators is unclear. While the importance of the mutant phenotype in humans was demonstrated, it would be interesting to consider the mutant in the greater context of the full Vibrio lifecycle.

STRENGTHS: The paper begins with a strong introduction and continues with a logical experimental design of starting with a sequencing based ‘hit’ and mechanistic studies that employed a variety of methodologies including a robust infection model. Appropriate methodologies were pursued throughout to directly address research questions.

WEAKNESSES: An important feature of Vibrio species is their dual host life cycle. It would have been nice for the authors to include a consideration of how the ΔmotV mutant might survive in the natural environment. Additionally, the authors rightly consider similarities to the well-described ΔcheYmutant and should therefore consider further exploration of the parallels (and differences) between ΔmotV and ΔcheY mutants.

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]

·       Throughout the Results, there was some inconsistency between the mutants that were studied in addition to ΔmotV (i.e. sometimes using ΔcheY and other times using ΔctxAB). Additional justification for these decisions in different experiments would be helpful.

·       Fig. 5: Based upon the interesting findings presented in Fig. 6 regarding how the double mutant of ΔmotV and ΔctxAB rescued some of the transmissibility phenotype from the single deletion of ΔctxAB it would be interesting to include the ΔmotVΔctxAB and ΔctxAB strains in these experiments as well. It appears that some of these experiments were done and presented in Fig. S6. We recommend incorporating this data into Fig. 5 or providing more exposition about Fig. S6 in the Results text to better highlight this important data.  

·       Fig. 6: Building upon the previous comment, it is interesting that there is no difference between the single ΔctxAB and double ΔctxABΔmotV mutants in Fig. S6, but differences are evident in Fig. 6. The reader would benefit from some discussion about these apparently discordant findings with respect to transmissibility vs. fluid accumulation and weight loss.

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. 

The experiments and analyses were generally excellent throughout and provide support for the title- and abstract-level conclusions. However, we thought that the title was too general; could it be revised to be more descriptive and name the mutant gene that is the major focus of the study? Authors may consider removing “motility” from the title as the authors only conducted a single motility experiment; instead, the title could highlight what was more thoroughly investigated such as the colonization and inter-animal transmission phenotypes. The Discussion would benefit from a little more speculation into the variability of results from the single ΔctxAB vs double ΔctxABmotV mutant. Authors may consider discussing the implication of the relationship between quorum sensing and production of the cholera toxin (low quorum and high toxin production, and vice versa when bacterial density increases) and how that may contribute to the results.

·     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. 

Throughout the paper the authors consistently link their findings to the constitutive straight-swimming phenotype, however they only conducted a single experiment to confirm the phenotype. We understand that the phenotype has previously been described and suggest a stronger emphasis upon what the previous paper demonstrated and connect that to the authors’ results when initially describing motility (the end of the first section in the Results). This will help the reader appreciate the known linkages to the motility phenotype as they read the remainder of the Results.

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? 

We noticed some inconsistencies between the data in Fig. 5A and Fig. S6; the findings for ΔmotV strain differ, but it appears to be a similar experiment). As we assume it is just a different set of replicates of the same experiment; perhaps describing these differences more clearly would be helpful. Fig. S6A lacks quantification of diarrheal discharge, which could be provided to better match the presentation of Fig. 5A.

·     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? 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? 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.

If possible, consider improving the image quality of the model figure (Fig. 7).

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)

While the mutant phenotype has been well described, the paper could benefit from greater discussion of the significance of the ΔmotV mutant. To help the reader appreciate the potential impact of the study, the authors may consider discussing the likelihood and potential outcome of human infection with this mutant bacterium, and/or the potential fitness cost that this mutant could face in the environment and how this could contribute to human infections. Additionally, readers could better appreciate impact if the authors could measure the kinetics of how the ΔmotV bacteria are shed in their infection model and how this contributes to transmissibility phenotype; are the bacteria shed at the same rate despite their localization deeper in the crypts and more proximal to the SI?. Including additional speculation about the mutant in the broader context of V. cholerae’s dual host-aquatic lifecycle could more directly address the overall impact of the author’s findings.

Competing interests

The authors declare that they have no competing interests.