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Université Toulouse Paul Sabatier, Master Microbiologie Moléculaire

LMGM-CBI, Toulouse

This Pre Peer Review is an exercise performed by Master 2 students from University Toulouse Paul Sabatier (Master Microbiologie Moléculaire). Writing a pre-review from a bioRxiv article is part of their module “Scientific analysis”.

It tooks us 3 sessions to finalize the peer-review:

Session 1: presentation of the scientific edition landscape + article reading

Session 2: critical reading of each figure

Session 3: pooling students peer-reviews

The final text was slightly edited for grammar and syntax but the peer-review reflects the work of the students.

This manuscript entitled "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids" from Boyd and co-workers describes how the TcaP protein from the PLE satellite phage can replace the scaffold protein of the ICP1 host phage to form smaller capsids that are appropriate for the PLE genome, and by doing so prevents further ICP1 infections. The authors use an elegant and powerful genetic strategy to obtain escape ICP1 phages that are immune to TcaP, and they use these escapers to further characterize the role of TcaP. They present cryo-microscopy reconstruction structures of the PLE capsids with apparent external scaffold where they are then able to dock alphafold predictions of the capsid and TcaP proteins. The evolution of TcaP proteins from PLE satellites is then examined using old and new environmental isolates of PLE5.

The materials and methods are in general well explained in great and sufficient detail.

Legends including gene name, accession and Molecular Weight were greatly appreciated.

Major Issues :

• (1) There are no statistical tests for figures 1E, 2D, 5E and 5F.

• (2) Regarding Figure 2 : 

No quantification of ICP1 or PLE virions under the conditions shown in Figures 2A to 2C, fig 5. It would be preferable to have a small graph (potentially in supplementary data) with quantifications of each virion type. Currently the reader only has access to the few fields of the electron micrographs chosen by the authors to present in the manuscript.

Minor Issues :

• (1)  Regarding Figure 1:

(1.1) For non experts it is quite enigmatic what big size round shapes represent, or what seems to be double contracted tails. Is it possible to better annotate this panel? same for Figure 2

(1.2) Panels A and B: It seems there is a lot of cell debris in panel A and very little in panel B. Is that a general difference between the two types of virion preparations or is it specific to the fields chosen for this figure? Perhaps a comment on this from the authors will help the readers.

(1.3) Panel D: What are the particles of virion size but with black interiors?

(1.4) We found no details on the discovery of TcaP, even though PLE codes for no known homolog of a protein whose function is homologous to TcaP. Out of curiosity, how did the authors stumble upon TcaP as a candidate?

(1.5) No access to the source data used to create figure 1E and figure 2D, although it is stated so in the material and methods.

• (3)  Regarding Figure 3:

(3.1) There is a contrast between the figure 3 A-2 and 3 A-4,  is the difference in color caused by the presence (or not) of the "decoration" gene ? 

In contrast to the other fields, Figure 3 A-4 shows white capsids. We would like the authors opinion on this.

(3.2) In the figure 3B-4, we are not able to see ICP1's scaffold band with the coat on the SDS-PAGE. We would again ask the authors to comment on this.

(3.3) In figure  3B, a band is visible at the bottom of the SDS Page. Is it an artefact ? Something degraded by a protease in the figure 3-A ?

(3.4) It would be nice to have a quantification of ICP1 vs PLE sized procapsids for each conditions. Something like a red/blue/grey percentage scale (ICP1/PLE/spiral) next to every platforms to compare each of them, especially for A2-3-5.

(3.5) Regarding Figure 3 supplements: 

On Figure 3S1, the chosen axis scale on the second image prevents to see differences between non outlier values

Figure 3S3 isn't discussed or cited. 

Figure 3S4 labeling should be reshaped in accordance to the legend, and the figure in itself is quite difficult to read

• (4)  Regarding Figure 4:

(4.1) There is a small typography problem on Figure 4's legend line 2: "ICP1's cot",probably meaning  "ICP1's coat" (A few typos in the mat/met as well)

(4.2) It would be nice to have a representation of how ICP1 scaffold works to compare it with the TcaP's mechanism shown here.

(4.3) Adding the FSC treshold as a dotted line on the first supplementary figure 4 could be interesting. 

(4.5) Based on the AA proximity predictions, what about testing particle sizes for an inactive TcaP mutant (R133 for example) ?

• (5)  Regarding Figure 5:

(5.1) Numbers for AA and nt would help the readers to orientate themselves on the panel B

(5.2) For panel E, the lane of pEV isn't shown even though the comparison was done with it.

(5.3) For panels C, D E and F, which version of PLE5 is used (since the most recent is as functional as PLE1) ?

(5.4) There is no material and methods for the bioinformatic analyses.

• (6)  Regarding Figure 6:

(6.1) Since some escape phages were observed, maybe they could be added to the model, along with their frequency

(6.2) Questions and perspectives: Out of curiosity, one could ask about the actual length of packages in the different capsids, is it something that is currently under investigation?

Positive feedback

(1) Regarding Figure 1:

Honest about the correctness of the mutant construction: supplementary data with sequencing results

(2) Regarding Figure 2:

The presentation of the results is clear

(3) Regarding Figure 3:

Clear and comprehensible presentation overall, legends and figures are well though.

Investigation of the bands obtained in figure 3A-2 in a supp data (Figure 3S2A) to confirm it was protease cleavage is a nice addition. (except the suspicious one cf minor issues of figure 3B)

(4) Regarding Figure 4:

Material and methods are quite detailed

(5) Regarding Figure 5:

The genetic analysis looks complete.

Figures are easily readable. The choice of colors helps with the clarity.

(6) Regarding Figure 6:

Very clear model.

Competing interests

The author declares that they have no competing interests.

Competing interests

The author declares that they have no competing interests.