Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 The manuscript  “Biosynthesis of the inner core of Bordetella pertussis LPS: Effect 2 of mutations on LPS structure, cell division, and TLR4 activation” focuses on structural modifications of the inner core of B. pertussis and their effect on endotoxicity.

I believe that there should be no abbreviations in the title of the article, please explain LPS and TLR. Further, I don’t see an explanation of TLR and Kdo in the abstract.

I think that in the abstract it is good to describe in one sentence the pathogenicity of Bordetella pertussis, which also argues the applied purpose of your research.

I think that in the abstract it is good to describe in one sentence the pathogenicity of Bordetella pertussis, which also argues the applied purpose of your research.

I also suggest that the second paragraph of L78 be moved to the beginning of the Introduction so that you can introduce the readers to the essence of the problem.

L99 The explanation of Kdo has already been introduced.

In general, the introduction presents enough information to introduce us to the research work, but my advice is that it be rewritten and properly organized.

L135 Place the figure immediately after its first citation.

L203 It's not entirely clear to me how you came to the conclusion that the electrophoretic mobility has been changed in all the strains producing KdtAEc? In the original gels, I do not see a molecular marker, nor is the whole gel represented, but again only a part of it.

All original gels do not present a picture of the entire gel, but only of a section of it, missing molecular markers and labeling of the molecular markers where they are present. Also, the photos are black and white and it is difficult for me to find out which is SDS PAGE and which is WB. Please, indicate your original SDS PAGE gels and Western blot which is which?  

L602 After electrophoresis, proteins or LPS were stained with Bradford reagent [58] or with silver [59].

This explanation is not correct, can you explain how you use Bradford reagent for protein staining?

 

Western blot protocol is not clearly explained, please include Abs concentration!

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper demonstrates the effects of inner-core structure genetically modified by genes from Francisella or E. coli to the lipid A ability to be recognized by TLR4. The results are interesting, and profitable to the development of B. pertussis vaccine. However, the volume of data is very large, and not easy to understand in detail for general readers. To simplify the manuscript, some modifications should be considered.

1. Introduction can be shortened, because this manuscript is for the Special issue of LPS, and basic knowledge of LPS would be described elsewhere. 

2. The names of enzymes and genes for them for lipid A and core synthesis can be incorporated to Figure 1 for the visual image for the readers.

3. Some data for phenotypic characters of constructed strains are presented in the supplementary file, so data in Figure 6 can also be moved to supplementary data.

Minor points for consideration:

1. Most of the TLR4 recognition assays are performed using whole cells. If authors have assay data using LPS, those should be included, especially in Figure S1.

2. In Figure 4, one peak is present at around m/z 3600 in both panels A and B. Is this a peak of some contaminant or LPS?

3. Page 4, Line 119: "be added en bloc to the core" Is this typing error?

4. Page 9, Line 245 and Figure S3: Growth temperature should be described here because it may affect the phenotypes of strains or structure of LPS.

5. Page 17, Line 449: "PPEA" should be "PEA".

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf