Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript entitled “CPP-PNA Conjugate-Mediated Inhibition of pdxA Gene Impairs Vitamin B6 Biosynthesis and Growth in Acinetobacter baumannii”, the authors developed a PNA that targets pdxA, suppressing the growth of A. baumannii. They further show that the CPP-conjugated PNA does not reduce the pdxA mRNA, however, it decreases vitamin B6 levels as shown by Western blot and ELISA.

Overall, I find that the authors have carried out sufficient work to characterize the pdxA-targeting CPP-PNA that they developed. Upon addressing the following comments, I recommend the manuscript is accepted for publication.

 

1. Either in the introduction or in the discussion, discuss further the choice of PNA. Did you consider a PMO? Would you expect that the PMO version of the 10-mer oligo you identified eventually would be equally effective or perhaps more effective compared to the PNA?
2. Major comment: For the results summarized in Tables 2 and 3, you should show the plotted data that result in the generation of MIC i.e. the graphs with the sigmoidal curves for each CPP-PNA tested. The tables with the summarized data are helpful, but the readers will appreciate seeing where the MICs came from.
3. For the data showing sequence-specificity of the PNA, it would be beneficial to show (perhaps in the SI) where the 10mer CPP-PNA targets (non-specifically) in the other bacteria species.

         Also, in line 128, you should avoid saying “No antimicrobial activity was observed in these       bacteria species…” as there are reported MICs for E. coli and P. aeruginosa albeit higher than in A. baumannii.

4. For Figure 1, mention the incubation time in the Results section as well. Moreover, do you have cytotoxicity data for the PNA alone or the CPP alone, either at all concentrations or at the higher concentrations i.e. 12.5uM or 25uM? This will be important to determine whether the cytotoxicity is due to the CPP or the PNA.
5. In Figure S7 and in the original Western Blot image, the anti-PdxA blot shows more than one bands. What do the other bands represent? How did you determine the PdxA band? The two set of bands at the top of the blot and the lowest set of bands also show dose-dependent protein reduction. Can you comment on that?

Author Response

1. Either in the introduction or in the discussion, discuss further the choice of PNA. Did you consider a PMO? Would you expect that the PMO version of the 10-mer oligo you identified eventually would be equally effective or perhaps more effective compared to the PNA?

• L187–194: We have added a discussion regarding PMO.

 

2. Major comment: For the results summarized in Tables 2 and 3, you should show the plotted data that result in the generation of MIC i.e. the graphs with the sigmoidal curves for each CPP-PNA tested. The tables with the summarized data are helpful, but the readers will appreciate seeing where the MICs came from.

• While presenting data as sigmoidal curves is useful, using MIC values allows for a simpler comparison of multiple PNAs at once. To generate sigmoidal curves, all PNAs would need to be resynthesized and re-tested. Since selecting the most effective PNA would lead to the same conclusion, we believe the MIC-based representation is also appropriate. We kindly ask for your reconsideration.

 

3. For the data showing sequence-specificity of the PNA, it would be beneficial to show (perhaps in the SI) where the 10mer CPP-PNA targets (non-specifically) in the other bacteria species. Also, in line 128, you should avoid saying “No antimicrobial activity was observed in these bacteria species…” as there are reported MICs for E. coli and P. aeruginosa albeit higher than in A. baumannii.

• Line 134–137: The expression has been revised. We have taken care not to state “No antimicrobial activity was observed in these bacterial species” as MICs for coli and P. aeruginosa have been reported, albeit higher than for A. baumannii.

 

4. For Figure 1, mention the incubation time in the Results section as well. Moreover, do you have cytotoxicity data for the PNA alone or the CPP alone, either at all concentrations or at the higher concentrations i.e. 12.5uM or 25uM? This will be important to determine whether the cytotoxicity is due to the CPP or the PNA.

 

- Line 152: Incubation time has been specified.

-  Low cytotoxicity of PNA alone has been reported in multiple studies (e.g., Antibacterial Peptide Nucleic Acids—Facts and Perspectives).

- Although not included in our previous publication (Targeting carA Using Optimized Antisense Peptide Nucleic Acid–Cell-Penetrating Peptide Conjugates in Acinetobacter baumannii: A Novel Antibacterial Approach), we observed in related experiments that GFP conjugated to different CPPs showed higher cytotoxicity for some CPPs.

- Additionally, in prior studies using different PNA sequences, cytotoxicity was observed at 12.5–25 µM.

 

5. In Figure S7 and in the original Western Blot image, the anti-PdxA blot shows more than one bands. What do the other bands represent? How did you determine the PdxA band? The two set of bands at the top of the blot and the lowest set of bands also show dose-dependent protein reduction. Can you comment on that?

- The band discussed in the manuscript corresponds to the predicted size of PdxA.

-  As is well-known, polyclonal antibodies can produce non-specific bands. Genes involved in vitamin biosynthesis, such as pdxA, may also influence other related genes. Related studies on PdxA–PdxJ in vitamin B6 biosynthesis support this observation (Vitamin B6 biosynthesis: formation of pyridoxine 5'-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein).

-  The additional bands observed at the top and bottom of the blot may represent non-specific binding or related proteins, and their dose-dependent reduction is consistent with effects on the vitamin biosynthesis pathway.

Reviewer 2 Report

Comments and Suggestions for Authors

Jeon and co-authors reported a CPP-PNA conjugate specifically killing A. baumannii by targeting the vitamin B6 biosynthesis. The rationales and outcomes of their experimental design have been clearly presented. Nonetheless, several major issues need further clarifications to increase the scientific depth of the work.

 

(1) there are several commonly used CPP, such as Tat and Penetratin. Why the (KFF)₃K was chosen? The rationale should be explained in detail. Please check the following reference 10.1007/978-3-319-66095-0_12

(2) from Table 3, the MICs have been detected for E. coli and S. aureus. Therefore, it did not seem to be reasonable to say “no antimicrobial activity was observed in these bacteria species…”

(3) the therapeutic index (IC₅₀/MIC) can be calculated and discussed in the section 2.4 to better explain the PNA-dyxA6 is the best candidate. 

(4) since the MIC determined for A. baumannii was 1.56 μM, why 50 μM was used to the experiments demonstrated in Figure 2 and Figure 3?

(5) the in vivo stability is an important criterion limiting the antibacterial applications of a molecule. Therefore, the enzyme or serum stability of PNA-dyxA6 were suggested to be examined and discussed: 10.1021/acs.jmedchem.4c02852; 

(6) as an important alternative of the clinically used antibiotics, the antibacterial peptides are extensively investigated rather the PNA. Some background information and comparison should be included in the Introduction section with the recent reviews and articles being cited and discussed.

 

several minor points:

(1) what are the numbers 1, 100, 256 in Table 1 represented for?

(2) all the tables and figures should be mentioned in the main text, including the supporting tables and figures.

(3) the spaces between 50 and μM were missing in Figure 2A and Figure 3

Author Response

(1) there are several commonly used CPP, such as Tat and Penetratin. Why the (KFF)₃K was chosen? The rationale should be explained in detail. Please check the following reference 10.1007/978-3-319-66095-0_12

 - Line 195–204: We have added a detailed rationale for choosing (KFF)₃K over commonly used CPPs such as Tat or Penetratin, and cited the suggested reference (10.1007/978-3-319-66095-0_12).

 

(2) from Table 3, the MICs have been detected for E. coli and S. aureus. Therefore, it did not seem to be reasonable to say “no antimicrobial activity was observed in these bacteria species…”

- Line 134–137: The sentence “no antimicrobial activity was observed in these bacterial species” has been revised.

 

(3) the therapeutic index (IC₅₀/MIC) can be calculated and discussed in the section 2.4 to better explain the PNA-dyxA6 is the best candidate. 

- Line 148: We thank the reviewer for the suggestion. We have added the therapeutic index (IC₅₀/MIC).

 

(4) since the MIC determined for A. baumannii was 1.56 μM, why 50 μM was used to the experiments demonstrated in Figure 2 and Figure 3?

- Although the MIC for A. baumannii is 1.56 μM, we used 50 μM in Figures 2 and 3 to ensure clear and reproducible effects.

- PNA does not directly degrade mRNA but interferes with ribosome binding, destabilizing the transcript and facilitating its degradation. Previous studies, including our prior submission (Targeting carA Using Optimized Antisense Peptide Nucleic Acid–Cell-Penetrating Peptide Conjugates in Acinetobacter baumannii: A Novel Antibacterial Approach), show that low concentrations do not always produce dose-dependent effects.

- Similarly, other studies, such as Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli, used higher concentrations to demonstrate clear antibacterial activity.

 

(5) the in vivo stability is an important criterion limiting the antibacterial applications of a molecule. Therefore, the enzyme or serum stability of PNA-pdxA6 were suggested to be examined and discussed: 10.1021/acs.jmedchem.4c02852; 

- Line 266–269: We revised the discussion to include PNA‑pdxA6 stability. Although in vivo/serum stability is an important factor limiting antibacterial applications (10.1021/acs.jmedchem.4c02852), this study focused on the validity of target selection. Future studies will consider CPP optimization and stability experiments.

 

(6) as an important alternative of the clinically used antibiotics, the antibacterial peptides are extensively investigated rather the PNA. Some background information and comparison should be included in the Introduction section with the recent reviews and articles being cited and discussed.

- Line 51–61: We have added a discussion in the Introduction highlighting the advantages of PNA over traditional antibacterial peptides, citing recent reviews and relevant articles.

 

several minor points:

(1) what are the numbers 1, 100, 256 in Table 1 represented for?

- We clarified that the numbers 1, 100, 256 represent CFUs.

 

(2) all the tables and figures should be mentioned in the main text, including the supporting tables and figures.

- All table (including the supplementary tables) and figures (including the supplementary figures) have now been referenced in the main text.

 

(3) the spaces between 50 and μM were missing in Figure 2A and Figure 3

- Spaces between values and units (50 μM) have been corrected in Figure 2A and Figure 3.