A Kayvirus Distant Homolog of Staphylococcal Virulence Determinants and VISA Biomarker Is a Phage Lytic Enzyme
, Jakub Boreczek 1 and Małgorzata Łobocka 1,*Round 1
Reviewer 1 Report
This study describes the characteristics of Tgl in association with the lytic activity. It should need further revision. Overall results were not properly stated and the discussion was not clearly interpreted. The specific comments are attached.
Comments for author File: 
Comments.pdf
Author Response
We thank the reviewer for helpful comments to our manuscript. Our responses to the comments are below. They are numbered according to the page and comment numbers in the reviewed manuscript.
Comments to Page 1
Number 1: Corrected (L. 31-32)
Number 2: Corrected (L. 32)
Number 3: Corrected (L. 32)
Number 4: Corrected (L. 33)
Number 5: Corrected (L. 35)
Number 6: Corrected (L. 35-36)
Page 2
Number 1: The subsection "Bacterial Strains, Bacteriophages and Growth Conditions of Bacteria" was divided into two subsections, as requested (L. 87-100).
Number 2: The goal of our study was to determine the function of kayviruses Tgl protein and to verify, whether it can decrease the sensitivity to vancomycin of S. aureus cells that produce it, similarly to SceD which is a staphylococcal distant homolog of Tgl and whose overproduction is associated with VISA phenotype. The overproduction of SceD has not been observed in MRSA strains that did not acquired the VISA phenotype, and in VRSA strains, whose mechanism of resistance to vancomycin is entirely different than that in VISA strains. Thus, we did not include MRSA and VRSA strains in our study.
Page 3
Number 1: The manuscript was verified to correct the writing of all genus and species names and gene names.
Number 2: The description of plasmids was simplified and shortened, as requested (L. 102-139).
Page 5
Number 1: Overproduction of the SceD protein (a distant homolog of Tgl) is tightly associated with the VISA phenotype - vancomycin intermediate sensitivity. It has not been detected in MRSA and VRSA strains. Thus, to verify suspicions that the Tgl protein production in S. aureus cells infected with a Kayvirus may become more tolerant to vancomycin, we tested only the sensitivity of S. aureus cells producing Tgl to vancomycin.
Page 6
Number 1: We shortened, reorganized and put together former paragraphs 2 and 4 of the subsection 3.1 of the Results section. We also removed the redundant part of the text, as requested (L. 279-283).
Number 2: The unclear terms were re-stated (L. 274-275)
Number 3: The unclear terms were re-stated (L. 279)
Page 8
Number 1: The sentence with the indicated error was removed from the manuscript (to avoid redundancy suggested in previous comments).
The changes in the manuscript are highlighted in yellow. The English of the manuscript was corrected by the MDPI editing service.
Author Response File: Author Response.pdf
Reviewer 2 Report
In this manuscript the authors characterised the role of a protein, Tgl, encoded by some Staphylococcus aureus bacteriophages belonging to the Kayvirus genus. Overall, this is a very nice piece of work. The experiments were well designed, and many different experimental approaches were used to characterise the function of this protein. My only criticism about this paper is the title and the section related with the tolerance to the vancomycin, which I found irrelevant in this study. Since the Tgl protein is produced by lytic phages, the infected cells will die at the end of the infective process, making irrelevant is they are sensitive or resistant to vancomycin. Other than that, I really enjoyed reading this paper.
Author Response
We thank the reviewer for helpful comments. As requested the title of our manuscript has been changed. However, we did not remove the section related to the tolerance to vancomycin. Our preliminary results indicate that kayviruses can, under certain conditions, coexist with their bacterial hosts, forming a relation that resembles pseudolysogeny. Thus, we cannot exclude that under these conditions the tgl gene is expressed by infected cells in which phage development is temporarily inhibited. To indicate that such possibility may exist and to provide the reasoning for our experiment with vancomycin, we added the relevant explanation in the discussion section (see L. 588-596). The changes in the manuscript are highlighted in yellow. The English of the manuscript is under corrections by MDPI editing service.
