Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii

Round 1

Reviewer 1 Report

The manuscript ‘Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii’ builds on the authors previous publication, examining the impact of ctp disruption in A. baumannii strain ATCC17978. In the current manuscript the authors examine the subcellular localisation of Ctp, and the impact of its loss on phenotypes including biofilm formation, OMV production and OMV cytotoxicity.

 

Unfortunately, the authors have failed to include suitable controls for some assays, present conflicting data for others and throughout the manuscript fail to address the fact that a ctp mutant has significantly reduced (almost a 50% reduction) viability in stationary phase compared to the parental or complement strains - This observation from their previous publication questions the significance of their current observations, given the vast majority of the experiments are conducted using cultures grown for 24hrs. The manuscript would benefit from significant reductions in the presentation of repetitive information, i.e. numerous figures could be moved to supplementary, biofilm related assays could be condensed to two (max. 3) multiple panel figures, with supporting microscopy included where provides clear evidence to support the conclusions being drawn and surplus figures removed. Some experiments require the inclusion of specific controls and some conclusions are currently not consistent with the data presented or not supported by the experimental evidence provided (see my comments below). My specific concerns are detailed below.

 

Methods/Results

1. Section 2.4 – Subcellular localization of Ctp
   1. The method of cell fractionation is not an acceptable method for accurately separating inner and outer membranes of gram negative bacteria– this should be achieved using density gradient separation (ie sucrose or percol)
   2. Fractionated cells should be probed using additional control antibodies specific to known inner membrane and outer membrane proteins to confirm the purity of these fractions.
2. Section 2.5 Capsular Polysaccharide Visualization and Quantification –
   1. The methodology applied as described in references #38 and 39 to the best of my knowledge has not previously been applied to Acinetobacter. Can the authors provide a citation confirming that this methodology is specific for isolating only capsule (and does not result in the isolation of other proteinaceous contaminates) in this species.
   2. During capsule isolation, was the supernatant filtered prior to the addition of TCA? Otherwise can the authors confirm that the sample does not contain residual cells?
3. Section 2.6 – Biofilm Assay
   1. “The supernatant was then transferred to fresh 96 well plates and OD600 was measured to determine the planktonic growth defects” – Please explain the rationale for this – Given the previous publication highlights that a ctp mutant has a planktonic growth defect this does not add new information and is merely repetition of the already published work.
   2. “...after which it was gram stained and visualized under a light microscope at 100× magnification” can the authors please clarify why they have elected to use Gram stain? And secondly, please provide additional information concerning the microscope used to obtain these images.
4. Section 2.7 - Quantification of the Biofilm Components
   1. The authors use Congo Red binding as a quantitative measure of biofilm polysaccharide – Given the viability defects observed with the mutant, can the authors provide a suitable citation to confirm that Congo Red will only bind polysaccharide and not other cell debris (present as a result of cell lysis).
   2. “We also quantified the viable cells within the biofilm matrix by making a serial dilution....” Cells grown in a biofilm are held together tightly and require sonication and vortexing to return them to a homogenous solution – were these samples treated in some way prior to dilution and enumeration? If so please provide details.
5. Section 2.9 – TEM and CLSM Analysis of Biofilm Cells
   1. Live/Dead staining - Can the authors please provide a clear rationale for conducting this experiment. Similar observations were reported in the previous publication, this assay looks to be repetitive of the previous findings and does not provide new information beyond the cells being grown in a biofilm rather than planktonic growth.
6. Section 2.11 OMV isolation, visualisation and Quantification
   1. “DNA-bound SYTO9 to detect eDNA associated with OMVs” – Please rephrase this statement, as this does not distinguish between extracellular DNA associated with the OMVs and DNA contained within the vesicle.
7. Section 2.12 Nanoparticle Tracking Analysis of OMVs
   1. “In brief, 1mg/mL OMV..” please clarify is this in reference to protein concentration or sample weight

Results

8. Section 3.1 Endogenous Ctp is an Inner Membrane Protein
   3. “In this study, we first predicted the cellular location of Ctp by separately using the SACS MEMSAT, Gram-LocEN, iLoc-Gneg, PSORTb 3.0.2, and PSLpred servers. PSORTb 3.0.2 and PSLpred are reported to have an overall accuracy of 97.3% and 91.2%, respectively, for predicting subcellular localization of gram-negative bacterial proteins [48,49].” This is unnecessary detail for tools that have been previously published in peer reviewed journals
   4. Figure 1C requires additional controls to confirm the cell fractionation (see comment 1b) and the full (uncropped) membrane image should be provided in supplementary information
9. Section 3.2 ctp Mutation promotes CPS production and Cell Aggregation
   1. Figure 2A – Should be moved to supplementary
   2. Figure 2D –
      1. Can the authors confirm how they have determined that the material covering the cells is actually “polysaccharide-like structure’ and not just cell debris.
      2. Secondly, how is this different from images shown in figure 5 of the previous manuscript?
10. Section 3.3 More Biofilm Matrix Surrounded ctp Mutant Due to Cell Content Release by Autolysis
    1. 1. Figure 3 should be reduced to show only panel A. Panel B is repetition of previously published observations and panel C does not provide credible information without the addition of Live/Dead staining.
       2. The following comments specific to the corresponding text should be addressed
          1. “MR14 revealed aggregated cells, which are characteristic of increased biofilm-forming ability (Figure 3C).” – Given the significant reduction in the viability of a ctp mutant can the authors confirm that these aggregated cells are alive? And not merely an aggregation of dead cells? And if the later, is this really biofilm formation? Or just an aggregation of dead cells and cellular debris?
          2. “The lower planktonic growth in MR14 may be attributed to autolysis”– The authors identified this phenotype in their previous publication and I’m unsure how this differs and presents new information, please consider rephrasing to make this fact clear.
       3. Figure 3B - Graphical representations of bacterial growth must be presented on a log scale.
       4. “to verify the cell content released after autolysis in MR14” Can the authors please clarify what is meant by this statement, where else would the material go?
       5. Similar to above “The biofilm of MR14 also contained significantly more proteins and polysaccharides (Figure 4B,C)” – Can the authors provide more context regarding the significance of this, given the increased cell lysis. Is this really biofilm, or just cell elevated debris in the sample?
       6. “Greater planktonic growth in MR14 on the addition of DNase I at 0 h (Figure 5B) also proves the inability of MR14 to form a biofilm.” Please rephrase – this statement is not clear nor does it prove the strains inability to form biofilm, it merely reduces the presence of an extracellular agent that aids in cell aggregation
       7. Figure 5B – Bacterial growth should be presented on a log scale.

11. Section 3.4 Microscopic analysis of Biofilm cells of ATCC17978 and MR14
    1. Please define the term ‘ghost’ cells
    2. Please rephrase the statement “The results revealed greater aggregation of MR14 cells compared with the sparse distribution of ATCC 17978 cells (Figure 6B).” This appears to show an aggregation of dead cells, I’m not sure you can make conclusions regarding their dispersal.
    3. Figure 6C – Reports the same data set in two ways. Please report only the live or dead values.
    4. Figure 6D – Can the authors expand on how this figure is different from that presented in Figure 4D
    5. Figure 6E – Bacterial growth must be plotted on a log scale. I would also recommend removing this figure as I’m unclear as to the significance of this result – given the sample would started with fewer viable cells, therefore it’s expected that it will have reduced planktonic growth.
12. Section 3.5 ctp Mutant is sensitive to SDS and EDTA
    1. Figure 7A/C – Please be consistent with the choice of colour for each strain between figures.
    2. Can the authors confirm whether the starting cfu/mL values were determined for these cultures and found to be consistent between strains? Given the impact of ctp mutation on cell viability, and the contribution of OMVs and cell debris to optical density, adjusting overnight cultures to the same optical density may result in significantly different cell numbers in either sample.
13. Section 3.6 ctp Mutant causes Hypervesiculation phenotype
    1. Figure 8B – Please label the axis correctly – Protein concentration (g/ml)
    2. Can the authors expand on how they confirmed the identify of the protein band labelled 38kDa OmpA on the coomassie gel. As the intensity of this band is significantly different from that on shown on the western blot. Can the authors explain this discrepancy.
14. Section 3.7 OMVs from ctp Mutant are More Virulent than Parental Strain
    1. Can the authors please include the name of method by which mammalian cell cytotoxicity was determined
15. Section 4 Discussion
    1. “the higher cytotoxicity of MR14 OMVs is expected to be due to the presence of more OmpA than in ATCC 17978 OMVs” This statement is in direct conflict with the western blot data presented in figure 8B. Please either elaborate and explain the reasons for these conflicting observations or amend the statement accordingly.
    2. “OMVs were shown to be generated by MR14 cells undergoing autolysis.” Can the authors explain how they distinguish between OMVs and aggregated cell debris. Otherwise, I don’t feel the data entirely supports this statement and should be toned down.
    3. Figure 10 – Please remove.
       1. This figure reiterates what is an already well-established mechanism of OMV formation, it provides no additional information concerning the putative mechanism by which the loss of Ctp induces this phenotype.
       2. Secondly the figure implies that the hypervesiculation is what leads too cell lysis. This has not been addressed nor proven in the current study. Without the mechanism for how Ctp disruption impacts the cell this is speculation.
16. Section 5 Conclusions
    1. 1. Please rephrase the statement “Taken together, the study findings elucidate the relationship between ctp and OMV biogenesis and biofilm formation in baumannii.” The data does not show a relationship – its shows consequence of the ctp mutation. To show a relationship you need to overexpress Ctp and show this abolishes all OMV production.
       2. Please rephrase the statement “the production of vesicles may be highly regulated by Ctp,” – The data does not support this statement, it merely shows increased OMV production is a consequence of the loss of Ctp. This phenotype maybe an indirect effect caused by elevated cell envelope stress responses and the enhanced membrane defects caused by the loss of Ctp.
17. References
    1. 1. Citations are inconsistently formatted
       2. Please consider the citations selected, less than 50% are from within the last 5 years and this is a rapidly advancing field.
18. Supplementary Figures
    1. 1. Figure 1/2 – Can the authors confirm how they distinguish between OMVs and cell lysis resulting in the release of cell debris? As the images shown for MR14 show an absence of definition between the membranes, indicative of cell lysis.

 

Author Response

I appreciate your precious time in carefully reviewing our paper and providing us valuable comments and constructive suggestions. Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen. Due to intrinsic and/or acquired resistance to multiple classes of antibiotics, it is involved in a number of severe infections and outbreaks that occur in hospital environments. Its ability to persist in these environments, as well as its virulence, is speculated to be a result of its ability to form biofilms. This work focused on an A. baumannii mutant, MR14, in order to investigate the role of mutation-induced autolysis in biofilm regulation in A. baumannii and to evaluate vesiculation caused by cell wall defects.

General comments

Acinetobacter baumannii has acquired inherent resistance to many antibiotics over time, so the treatment of infections caused by nosocomial strains of A. baumannii has become increasingly problematic. Consequently, the topic of the manuscript is interesting and current, although some changes are needed.

In detail

The Introduction section introduces the reader to the thematic question.

The Materials and Methods section is well structured and reports different experimental approaches.

The Results and Discussion section is written correctly and thanks to the methodical presentation of the results obtained from the experimentation, correct conclusions were drawn.

However, given the large number of abbreviations reported in the text, I suggest adding a table summarizing them to facilitate the reading of the article.

Author Response

I appreciate your precious time in reviewing our paper and providing us with suggestions to improve our manuscript. Thank you for your comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

Review of the article: “Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii”

Submission ID - microorganisms-1224280

The manuscript is interesting and well written. The experiments were well planned and performed. Below I have presented some issues which could be taken into account preparing the final version of the manuscript.

Abstract

Please explain abbreviation eDNA – I understand that it is obvious, but abstract should be clear also for the readers who are not specialist in this area can also (SDS, EDTA – also could be explained)

Introduction

In the firs sentence the word “infection” is repeated four times. Please try to rephrase this sentence.

The authors have presented a lot of information about Prc from different bacteria including E. coli, P. aeruginosa or Borrelia burgdorferi and Rhizobium leguminosarum. What about A. baummannii.

In this sentences: “In a previous study, we extracted a ctp mutant, MR14, and a complementation strain, MR14C, from ATCC 17978 [33]” It should be clearly written that it was A. baummannii.

Materials and methods

Please try to rephrase the sentence: “Vectors pET-30C (Novagen, Madison, WI, USA) and E. coli BL21 (DE3; TransGen Biotech, Beijing China) were used to induce and overexpress Ctp.”

Section 2.2. – what was the size of amplified DNA fragment,

Should be digested with the same enzymes not enzyme

Please provide information about moment of induction with IPTG

Why the protein was eluted with 8 M urea. Metal affinity chromatography was used for purification thus growing concentration of imidazole could be used for elution of the protein. Such a high concentration of urea probably caused protein denaturation.

Section 2.5 – bacteria were grown aerobically – do you mean with shaking?

Section2.10 – please explain why you decided to investigate different concentration of EDTA and the same analysis for SDS was not conducted.

Results

„In this study, we first predicted the cellular location of Ctp by separately using the SACS MEMSAT, Gram-LocEN, iLoc-Gneg, PSORTb 3.0.2, and PSLpred servers. PSORTb 3.0.2 and PSLpred are reported to have an overall accuracy of 97.3% and 91.2%, respectively, for predicting subcellular localization of gram-negative bacterial proteins” – it should be mentioned in MM section

„As shown in Figure 1C, Ctp was lacking in the crude lysate of MR14, and Ctp in A. baumannii was found to be localized in the cytoplasmic membrane, consistent with the results obtained using bioinformatics tools.” – it should be written more clearly which picture and which lane in figure 1 confirms this result.

„Moreover, MR14 was sensitive to increasing EDTA concentration (Figure 7B).” – in Figure 7B  the effect of EDTA on two other strains is also presented. A short comment would be necessary.

 

Discussion

The discussion is interesting and well presented. I do not have any critical remarks.

Conclusions

„… indicating that Ctp can be a potential target for the development of antibacterial drugs.” It is rather hypothesis not a conclusion from the research.

Final decision – minor revision.

Author Response

I appreciate your precious time in carefully reviewing our paper and providing us valuable comments and constructive suggestions to improve our manuscript. Thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have gone to significant effort to address my previous comments and the manuscript is vastly improved as a result, however the following minor corrections should be addressed.

1. While the authors have provided citations in their rebuttal, showing bacterial growth data which is also not presented on a log scale, this is merely propagating an inaccuracy regarding how this data should be presented and I therefore strongly recommend they edit figure 5B to present this data on a log scale.
2. Please increase the text size of the numbers next to the peaks in figures 8D and 8E.

Author Response

We greatly appreciate your valuable time and acknowledge your thoughtful comments. Thank you very much for your insightful suggestions. Your valuable comments have helped us improve our manuscript. 

Author Response File: Author Response.pdf