From the journal RSC Chemical Biology Peer review history

24-Jul-2023

Dear Dr Walvoort:

Manuscript ID: CB-COM-06-2023-000110
TITLE: Evaluation of Kdo-8-N₃ Incorporation into Lipopolysaccharides of Various <i>Escherichia coli </i>strains

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Reviewer 1

Zeynep et al. synthesize 8-azido-Kdo and assess the incorporation into the LPS of various E. coli strains via SDS-PAGE and fluorescence microscopy. The authors test various strains, different bioorthogonal fluorophores and growth media to this end.

The paper is very well-written and the data largely support the conclusions. The major novelty is the comparison of a host of strains, including clinical ones, as well as the discovery that incorporation patterns are different between the strains. The data are interesting and in my view of interest to the readers of RSC Chem. Biol.

An issue is the characterization of compounds - the isolates appear to be mixtures of several compounds which makes it more difficult to assess whether the observed labeling is actually due to glycosylation by Kdo itself. It would seem necessary to repeat one or two key experiments with 8-azido-Kdo that is purified to homogeneity, or by just using commercial 8-azido-Kdo if that is more pure. Furthermore, can the authors please show 1H NMR spectra from 0 ppm to 8.5 ppm?

A limitation to the work presented here is that it is not proven that 8-azido-Kdo is actually introduced into LPS by glycosylation, rather than non-enzymatic attachment such as glycation which could, in theory, happen. However, I believe that Nilsson and colleagues have already shown incorporation by mass spectrometry, whcih the authors could just cite (10.1074/jbc.M117.814962). To this end, it is curious that certain E. coli strains exhibit a shortening of chains upon incorporation of 8-azido-Kdo. I wonder if this is associated with the azide being a chain-terminating structure that prevents further elaboration.

As a note on the copper-mediated click reaction, I believe that the toxicity seen because is due to the high concentration of Cu(II) used as well as the use of THPTA as a ligand. Newer ligands such as BTTAA (10.1002/anie.201101817) are much better in reducing the toxicity as they remain extracellular and allow Cu(II) salts to be used at much lower concentration than the ligand itself. It would be good if the authors could mention this in their manuscript.

Reviewer 2

Evaluation of Kdo-8-N3 Incorporation into Lipopolysaccharides of Various Escherichia coli strains.
The communication by Ziylan et al. describes the synthesis of 8-Azido-3,8-dideoxy-α/β-ᴅ-manno-oct-2-ulosonic acid (Kdo-8-N3) and its metabolic incorporation into membrane-bound lipopolysaccharides (LPS) of different non-pathogenic and pathogenic E. coli strains. The incorporation into the LPS was confirmed by covalent attachment of different fluorophores using either copper-catalyzed or strain-promoted (DBCO) click chemistry and subsequent visualization of labelled LPS in SDS-PAGE or intact E. coli with fluorescence microscopy. The authors tested different conditions for optimal metabolic labelling (LB, M9 media) and compared labelling efficiency in different E. coli strains.
The manuscript is overall well written and the results are clearly presented and discussed in the figures and the manuscript text. While metabolic incorporation of Kdo-8-N3 has been previously established and reported, this work contains some novel insights into labelling efficiency and labelling pattern in different E. coli strains, which could be potentially useful with regard to investigating, in particular, pathogenic strains that are clinically relevant.
Below there are two minor comments that the authors may want to consider in their revision to further improve this excellent manuscript.
The error bars in Figure 3A (and some in 3B) are not visible, presumably because they are very small. The authors may want to consider adding a table in the SI that states the values of the three individual measurements and the respective standard deviation to support this figure.
The authors state that the copper-catalyzed click chemistry gave only faint bands of labelled LPS and speculated that incubation with copper resulted in cell lyses and loss of LPS. Was this hypothesis supported by visual confirmation of E. coli disruption in presence of copper? The conditions for the copper-catalyzed click-chemistry might have not been optimal. Incising the copper:Ligand ratio to 1:4 or 1:6 can reduce toxicity of CuAAC in live cells. In addition, using BTTP or BTTPS instead of THPTA can significantly accelerate the click reaction and thus shorter incubation times (5-7 min instead of 20 min) can be used, which can also reduce the risk of cell lysis. For E. coli BTTP may work slightly better than the negatively charges BTTPS or BTTES, which can have repulsive interaction with the negatively charged bacteria. However, since the strain-promoted click chemistry gave the desired results, this comment may just be informative for the authors, in case CuAAC in live cells becomes relevant again in future studies.

13 September 2023

Evaluation of Kdo-8-N3 Incorporation into Lipopolysaccharides of Various Escherichia coli strains
Manuscript ID: CB-COM-06-2023-000110

We thank the reviewers for their assessment of our manuscript and the several suggestions that they offered to improve it.

Response to Referee 1:
1. An issue is the characterization of compounds - the isolates appear to be mixtures of several compounds which makes it more difficult to assess whether the observed labeling is actually due to glycosylation by Kdo itself. It would seem necessary to repeat one or two key experiments with 8-azido-Kdo that is purified to homogeneity, or by just using commercial 8-azido-Kdo if that is more pure.

To confirm the labeling was indeed due to incorporation of Kdo-8-N3, we purchased commercial 8-azido-Kdo (Kdo-Azide) with >90% purity from Click Chemistry Tools (CAS 1380099-68-2, catalog #1241-10). The non-pathogenic E. coli BW25113 strain was incubated either in the presence of our synthetic Kdo-8-N3 or the commercial Kdo-8-N3, and labeled structures were separated by SDS-PAGE. The new gel image shows efficient labeling of LPS with comparable labeling efficiency between our chemically synthesized Kdo-8-N3 and the commercial Kdo-8-N3. The new gel image is added to Figure 5 and discussed in the main text, lines 181-185 and lines 193-195. The legend of Figure 5 was adjusted according, lines 214-216.

2. Furthermore, can the authors please show 1H NMR spectra from 0 ppm to 8.5 ppm?

We adjusted the 1H NMR spectra of all compounds in the supplementary information document to show the ppm range from 0 to 8.5.

3. A limitation to the work presented here is that it is not proven that 8-azido-Kdo is actually introduced into LPS by glycosylation, rather than non-enzymatic attachment such as glycation which could, in theory, happen. However, I believe that Nilsson and colleagues have already shown incorporation by mass spectrometry, which the authors could just cite (10.1074/jbc.M117.814962).

We have included the suggested citation in our manuscript to support our observations, in the main text lines 204-205, to support the incorporation of 8-azido-Kdo into LPS by glycosylation.

4. As a note on the copper-mediated click reaction, I believe that the toxicity seen because is due to the high concentration of Cu(II) used as well as the use of THPTA as a ligand. Newer ligands such as BTTAA (10.1002/anie.201101817) are much better in reducing the toxicity as they remain extracellular and allow Cu(II) salts to be used at much lower concentration than the ligand itself. It would be good if the authors could mention this in their manuscript.

We thank the referee for this suggestion. We have added a more detailed discussion of our observations and the suggested citation into our manuscript, main text lines 228-232.

Response to Referee 2:

1. The error bars in Figure 3A (and some in 3B) are not visible, presumably because they are very small. The authors may want to consider adding a table in the SI that states the values of the three individual measurements and the respective standard deviation to support this figure.

We have created an additional supplementary document (Microsoft Excel) consisting of a table showing the three individual OD values measured in each indicated growth condition present in Figure 3A and 3B, and their respective standard deviation values.

2. The authors state that the copper-catalyzed click chemistry gave only faint bands of labelled LPS and speculated that incubation with copper resulted in cell lyses and loss of LPS. Was this hypothesis supported by visual confirmation of E. coli disruption in presence of copper?

This hypothesis was based on the observations from an experiment where we investigated the effect of copper presence on the level of LPS present in the samples on SDS-PAGE gel images. However, we did not perform additional experiments to confirm cell lysis under the microscopy. On the silver stained gel image, we observed that when we added copper and THPTA into the click mixture the intensity of the LPS bands were less compared to the LPS bands in the samples incubated without the presence of copper and THPTA. We have included a more detailed explanation of our hypothesis supported by citations including the paper mentioned by referee 1 on the toxicity of copper on live cells. This is discussed in the main text, lines 228-235 and 241.

3. The conditions for the copper-catalyzed click-chemistry might have not been optimal. Incising the copper:Ligand ratio to 1:4 or 1:6 can reduce toxicity of CuAAC in live cells. In addition, using BTTP or BTTPS instead of THPTA can significantly accelerate the click reaction and thus shorter incubation times (5-7 min instead of 20 min) can be used, which can also reduce the risk of cell lysis. For E. coli BTTP may work slightly better than the negatively charges BTTPS or BTTES, which can have repulsive interaction with the negatively charged bacteria. However, since the strain-promoted click chemistry gave the desired results, this comment may just be informative for the authors, in case CuAAC in live cells becomes relevant again in future studies.

We thank the referee for the kind information and suggestion for future studies. We have incorporated this information in the main text, lines 229-235 and added some citations as referee 1 suggested as well.

25-Sep-2023

Dear Dr Walvoort:

Manuscript ID: CB-COM-06-2023-000110.R1
TITLE: Evaluation of Kdo-8-N₃ Incorporation into Lipopolysaccharides of Various <i>Escherichia coli </i>strains

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Reviewer 1

The authors have addressed all queries.