Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules

Round 1

Reviewer 1 Report

In their manuscript, Stein et al studied the extraction of membrane components from Neisseria gonorrhoeae using catanionic surfactant vesicles to study the bacterial surface molecules. Over-all, the study is interesting, though I feel that there are few revisions are warrants.

1. Please provide the transmission electron microscopy images of surfactant vesicles.
2. Please discuss the proliferation of THP-1 cells by purified bare vesicles in higher dilution.
3. Section 3 should be written as results and discussion as contains both results and discussion (too little)
4. Discuss all results, Discussion must be elaborated further.
5. Section 4 should be written as Conclusion 

 

Author Response

In their manuscript, Stein et al studied the extraction of membrane components from Neisseria gonorrhoeae using catanionic surfactant vesicles to study the bacterial surface molecules. Over-all, the study is interesting, though I feel that there are few revisions are warrants.

1. Please provide the transmission electron microscopy images of surfactant vesicles. We have previously published these Ems.  We  have added a reference to this manuscript in the discussion “J. AM. CHEM. SOC. VOL. 131:5471 (2009)”

2. Please discuss the proliferation of THP-1 cells by purified bare vesicles in higher dilution. (There really is no significant increase in proliferation. While at first glance, it might appear that the rate increases, the error bars for this later point are quite large and thew differences are not statistically significant)

1. Section 3 should be written as results and discussion as contains both results and discussion (too little) (This has been modified)
2. Discuss all results, Discussion must be elaborated further (A discussion section has been added as section 4, and the existing section renamed conclusion and modified)
3. Section 4 should be written as Conclusion (Changes were made to make this conclusions)

Reviewer 2 Report

This article was well constructed describing the utilization of surfactant vesicles fro the study of bacterial membrane surface features. The work was carefully done, and is well described. Some of the gels shown are a little sloppy where it appears that one was broken before the photo was taken (Panel B figure 1), the gel may have dried too much before the photo was taken (Figure 3), or there was a poor transfer for the western (panel B figure 4). However, overall the somewhat sloppy presentation does not change the results. Two of the authors are associated with superscript 3, but there is no superscript 3 in the affiliations. In figure 5 and 6, there should be a note stating that the error bars represent one standard of deviation (I am assuming that is what is shown). In my version of the manuscript it appears that the figure legend in Figure 7 has been squished. Overall, the work appears to be sound, and warrants publication.

Author Response

This article was well constructed describing the utilization of surfactant vesicles fro the study of bacterial membrane surface features. The work was carefully done, and is well described. Some of the gels shown are a little sloppy where it appears that one was broken before the photo was taken (Panel B figure 1), the gel may have dried too much before the photo was taken (Figure 3), or there was a poor transfer for the western (panel B figure 4). However, overall the somewhat sloppy presentation does not change the results.

I can appreciate the reviewers comments and would have preferred better images.  Unfortunately, due to the times, the funding has run out and the personnel associated with this work have left the University.

Two of the authors are associated with superscript 3, but there is no superscript 3 in the affiliations.(This has been added) In figure 5 and 6, there should be a note stating that the error bars represent one standard of deviation (I am assuming that is what is shown).  (This has been added) In my version of the manuscript it appears that the figure legend in Figure 7 has been squished (This has been fixed).  Overall, the work appears to be sound, and warrants publication.

Reviewer 3 Report

Toxicity is shown with the surfactants CTAT and SDBS in your figure 5.  Now when they are incorporated/formulated into vesicles and unincorporated components are removed, the toxicity of CTAT and SDBS is eliminated.  It would be good to explain why this occurs.  Have any hypotheses been developed to explain this.

Has there been any work to determine the shelf life of these vesicles?  Is it possible that deterioration of these vesicles could affect their use as vaccines?

Very good paper.  Thank you.

Author Response

Toxicity is shown with the surfactants CTAT and SDBS in your figure 5. Now when they are incorporated/formulated into vesicles and unincorporated components are removed, the toxicity of CTAT and SDBS is eliminated. It would be good to explain why this occurs. Have any hypotheses been developed to explain this. We had addred this to the discussion

Has there been any work to determine the shelf life of these vesicles? Is it possible that deterioration of these vesicles could affect their use as vaccines?  We know that the vaccine formuations are stable for at least a month, and are currently addressing this question

Very good paper. Thank you.

Round 2

Reviewer 1 Report

The authors made the requested revisions.