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PREreview of “Simple Sequence Repeats Mediate Phase Variation of the Mucoid Phenotype in Hypervirulent Klebsiella pneumoniae” by Augustin DUFOUR, Margaux LAIRESSE, Bahria NDOYE, Morganne PIFFERO, Julien RAMUSCELLO, Peter REDDER.

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

It took us 3 sessions to finalise 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 Peer-review pertains to the BioRxiv version of the manuscript published on 12/09/2025.

The manuscript entitled “Simple Sequence Repeats Mediate Phase Variation of the Mucoid Phenotype in Hypervirulent Klebsiella pneumoniae” by Oliwia Stawarska et al. describes a poly-G repeat which they discover can mutate with high frequency to turn a regulator gene on or off, thus generating a reversible phase variation of the Klebsiella pneumoniae mucoid phenotype. They moreover show that the phase variation mechanism is a conserved feature of all hypervirulent Klebsiella pneumoniae, and the OFF state facilitates epithelial cell infection while the ON state enhances resistance to the bactericidal activity of human serum and systemic spread in a mouse model.

Major comments:

Figure 1C and E, it is unclear how the statistical test was done. It is also unclear what is statistically significant compared to what. In addition, the authors mention Line 125 a “significant loss of mucoidy”, however figure 1c shows “NS” symbols which seem contradictory.

Figure 1G has no legend.

All figures: The signs “>” and “<” are inversed in most of the indications of p-value (see for example the legend to figure 1).

Line 199: The wording used might be too strong since it appears on figure 3C that not all bacteria in the top fraction are “entirely resistant” to sedimentation.

Lines 302-345: The paragraphs are very dense and difficult to follow. Please consider rewriting them to help the reader.

Line 310-312: Based on the Alphafold models, the authors conclude that changes in repeat length does not affect RmpA protein function. However, Alphafold predicts protein structure but does not tell anything about the dynamics, stability, or in vivo activity, so the functional consequences of repeat length variation can’t be fully excluded based on these structural predictions. The sentence should be reformulated to tone down the conclusion.

Minor comments:

Figure 1A: It is difficult to see the bacterial cells within (or adhered to) the human cells. It would be ideal to change the colour scheme to something with more contrast.

Figure 2:

Title of the figure is a bit misleading because it implies that both rmpa truncation and plasmid loss are responsible for mucoidy loss but rmpa truncation plays a bigger part : Fig 2C + “with very few rmpA-LOST colonies recovered in these assays” (line 167) + “these data indicate that HVKp host cell infection is associated with inactivating mutations in an intragenic poly-G tract within rmpA, and occasional loss of the large virulence plasmid”. Maybe the title could be reformulated to be “Loss of mucoidy is primarily driven by rmpA truncation and occasionally by loss of the large virulence plasmid.” instead?

Fig 2A : The drawing makes it looks like the two routes can coexits (at least to us) when they can’t. It could be improved by adding an “or” between the two routes.

Line 147: “200kb were missing” Where are these 200 kb located? Perhaps give a start and end position of the deletion? (or first and last genes that are missing?) This information would be ideal to have in the supplementary data.

Figure 4:

1. The method used to assess the mucoidy for 4b is not described in the materials and methods.

2. Are the reversions stable? Or can an OFF-strain go back to “ON”? And with what frequency?

Line 218: Please provide a reference for “given that repetitive DNA is known to…”

Line 817: typo “dat are representative” instead of data

Figure 7: Links between text and figure panels could be clearer. For example, Figure 7b (Alphafold) could explicitly indicate what we as readers should observe.

Figure 7A: This panel could be moved to supplementary materials, to lighten the figure.

Figure 1B and D, a statistical test for invasion between cells A549 and THP-1 could highlight a preferential invasion for one cell type.

On figure 3A it is difficult to understand why the title is mentioning “RmpA allele 2” as it is mentioned nowhere in the text.

On figure 3A the black protein domain is difficult to see, an arrow might help the reader seeing it.

On figure 3A the pink and yellow titles could be inverted to facilitate the understanding of the figure.

On figure 3C the use of the color blue to indicate both “statistically significant resistance to sedimentation” and “rmpA-ON” is somewhat misleading and complexifies reading. It would be better for the reader if two different colours were used.

Figure 5A and B, the shades of blue used in the caption are not used in the graphs.

Statistical tests between the WT strain and the revertant are present in some figures (5C) but are absent on other (3 D and E, 5 A and B). Statistics should be presented in all figures.

The caption of figure 5C is mentioning colored “bars” whereas the graph is showing dots.

Line 248 and 250 figure 5C is referred to as “Fig 5c” which is not coherent with the rest of the text. The same typos are found line 161, 199, 209, 212, 225, 236, 375, 376, 377 and 536. It is easier for the reader if the naming is consistent, choosing either “Fig.” or “Figure”

Line 208 the mention “(prmpA)” could be added after “copy of rmpA on a plasmid” to help the understanding of figure 3.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.