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Comment by Patrice Polard
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Dear Alizée Malnoë, Tahreem Zaheer, Josy Joseph, Carter Collins, Camryn Guenther, and one anonymous author of IU Bloomington Biology
Thank you for reviewing our manuscript and providing your comments in a peer-review format, following the editorial procedure commonly used by scientific journals. Your analysis is a very helpful complement to improve the presentation and clarity of our work, and it also provides an initial assessment of our study with the same public visibility as that offered by bioRxiv.
We are particularly grateful for your very positive feedback on our fundamental and integrative study that we conducted in S. pneumoniae to unveil the unprecedented modes of action of the bacteriocin RumC1 and of its cognate immunity protein RumIc1. In addition, we were delighted to read your assessment of the importance of our study in the frame of the global antibiotic resistance crisis, and the emergency to develop innovative tools to fight against multi-drug pathogenic bacteria.
Below, we respond to your comments in detail for the two major points and more generally for the minor points you raised.
Major comments
Point #1: … it would be helpful to include fluorescence quantification, as represented in Figure 6. Similarly, for Figures 2C and D, consider incorporating the quantification for cell width….
You are absolutely right to raise these points, which are fully feasible. We will perform these fluorescent and cell-shape quantifications and include them in the final revised version of the manuscript.
Point #2: To strengthen the conclusion that variants of RumIc1 do not confer immunity, consider including evidence that the protein variants are stable by showing an immunoblot…
Addressing this point rigorously would require either (i) generating antibodies against RumIc1, or (ii) engineering functional immuno-tagged fusions of the WT and point-mutant RumIc1 proteins, followed by Western blot analysis of total cell extracts to demonstrate that the mutations do not destabilize the protein, but specifically impair its proteolytic activity. Importantly, both approaches are not fully guaranteed to succeed (e.g., the antibodies might lack sensitivity, and the tagged WT fusion might not be functional). For these reasons, we instead chose to provide direct biochemical evidence that RumIc1 has proteolytic activity, which is the primary objective of its functional characterization. As shown in Fig. 6C, RumIc1 clearly displays proteolytic activity. In addition, the RumIc1 point mutant that we purified to demonstrate processing of the pentapeptide in this experiment was produced at levels comparable to the WT protein and showed similar solubility when expressed in E. coli, which generally indicates that the mutation does not affect the overall fold of the protein and is unlikely to compromise its stability.
Minor comments
Thank you very much for this well-organized list of minor points, which will help us improve the final version of the manuscript. As mentioned above, we will not provide here a point-by-point reply (as typically done in response to referees’ comments during journal peer review process); instead, when appropriate, most of your suggestions will be incorporated into the revised and finalized version after acceptance.
In particular, we appreciate your identification of errors in the text and figures, which will be corrected. We also noted your suggestion to explain more clearly what is known about WalRK, which we will address in the revised manuscript. Finally, we understand the proposal to include a capsulated strain of S. pneumoniae in the analysis; however, it is known and published that capsulated strains of S. pneumoniae are sensitive to RumC1. Thus, including a capsulated strain (more difficult to manipulate as you acknowledge) would have substantially expanded the manuscript focused on the functional analysis of RumC1 and RumIc1 to characterize their mode(s) of action. To this end we selected our laboratory strain of S. pneumoniae as powerful ‘genetical chassis’ and an advanced model for single-cell microscopy analysis for such analysis. Nonetheless, now that we know that RumC1 interacts and interferes with PG homeostasis, an important perspective is to pursue its analysis in other sensitive pneumococcal strains with different cell envelopes.
In conclusion, we warmly thank you for taking the time to select and peer-review our manuscript and, again, for your very positive feedback on our work. Your comments support our study describing the novel antibacterial mode of action displayed by the RumC1 bacteriocin, together with the unique self-protection provided by the immunity protein RumIc1, both targeting the synthesis and maturation of the peptidoglycan – two discoveries that define the take-home message of our manuscript.
Patrice Polard
Competing interests
The author of this comment declares that they have no competing interests.