From the journal RSC Chemical Biology Peer review history

04-Aug-2020

Dear Dr Blaskovich:

Manuscript ID: CB-ART-07-2020-000118
TITLE: Fluorescent Macrolide Probes – Synthesis and Use in Evaluation of Bacterial Resistance

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Reviewer 1

Summary: The authors describe a series of experiments that are collectively directed at probing the utility of fluorescently-derivatized macrolide antibiotics for evaluation of macrolide uptake/efflux in representative Gram-negative and Gram-positive organisms. Efflux is one of the potential pathways that bacteria can use to achieve a measure of resistance against this important class of antibiotics. As a result, the work described in this manuscript has the potential to lead to novel insights that may be used to combat macrolide resistance.

The family of probes utilized in these experiments are fluorescently-modified roxithromycin derivatives wherein the fluorescent subunit was installed via a copper-activated azide-alkyne cycloaddition reaction. This portion of the work described in this manuscript is not particularly innovative, but I do not think that this is an issue at all since chemical innovation is not the major thrust of the work described. After establishing their antibiotic activity profiles, these derivatives were examined in a series of assays directed at determining their uptake/efflux.

It is worth pointing out that experiments utilizing fluorescently-modified macrolide antibiotics to quantify uptake/efflux can provide results that are difficult to interpret. This is especially true in this case. One can rightly note that peripheral changes to an antibiotic can affect both its cellular uptake as well as its ability to function as a substrate for a given transporter. Does a diminished intracellular fluorescence signal reveal substantial efflux or does it reveal lack of uptake? This is just one of several very difficult hurdles that must be addressed in work of this nature.

From my reading of the manuscript one of the significant issues that is not addressed is fluorescence that may arise from non-specific binding. For example, the images in Figures 2A,E and 3A,E appear to show a significant amount of non-specific binding of the fluorescently-modified antibiotic. Is this due to binding to the cell membrane or is it due to random intracellular distribution within the cytoplasm? If it is the former, this can have a substantial impact on uptake measurements. This may also explain the diminished activity of the fluorescently-modified macrolides relative to that observed for the parent compounds. In fact, the very nicely designed experiments aimed at quantification of intracellular labeling appear to bear this out. That said, it would appear that this level of rigor is required to gain useful information from this approach; macroscopic evaluation is largely precluded by non-specific binding.

There was also an important observation that would benefit from some additional discussion. It was noted that reduced uptake was not observed in efflux upregulated Gram-positive bacteria. This was in contrast to the observations in Gram-negative bacteria. The manuscript could be strengthened with a rationale for this or through inclusion of additional experiments directed at unraveling this surprising observation. The experiments utilizing the efflux pump inhibitor (CCCP) should also be included.

The manuscript is well-organized, is supported by a strong supporting information section, and is generally well-written. There are some very minor typographical errors that can be addressed in the next round of review. I will be happy to provide a copy of the pdf illustrating these, if it will help in the revision process.

Overall, I think this manuscript will be embraced by the community that studies bacterial resistance to macrolides as well as the chemical biology community, in general. The corresponding author is an expert in the design and use of fluorescently-modified antibiotics to unravel important biological questions. I would support its publication upon submission of a revised manuscript addressing the concerns noted above.

Reviewer 2

Stone, Blaskovich and coworkers in their work entitled “Fluorescent Macrolide Probes – Synthesis and Use in Evaluation of Bacterial Resistance” describe the potential to use two erythromycin probes against the problem of antibiotic resistance. The authors perform semi-synthesis to access the probes and a series of biological experiments to evaluate them. The authors would like to claim that fluorescent antibiotics haven’t been used in the past nor properly evaluated. Here they present some fluorescent microscopy to show that fluorescent macrolides could be useful in the design of new antibiotics. I have found discrepancies in the work and would like to see the authors address them. I am extremely sensitive to the conditions caused by COVID and if the authors cannot address them experimentally – I would appreciate more detailed information to increase the rigor of this manuscript.


1. Page 5, first full paragraph, fourth line: “Although
fluorescent analogues of antibiotics were used in the second half of the 20th century to investigate the mode of action of antibiotics,5 they have been applied sparingly to help address the modern crisis of resistance.”

- I think that this statement fails to capture two major recent advances in the field. Both fluorescent glycopeptides and b-lactams have been used widely in the field to address human interactions as well as resistance mechanisms. The discussion in this paragraph should be expanded to capture the following:

a. Suanne Walker’s work with glycopeptides: c Natl Acad Sci U S A
2006 Jul 18;103(29):11033-8.
doi: 10.1073/pnas.0600829103.Epub 2006 Jul 10.

b. Erin Carlson’s catalog of work with fluorescent b-lactams:

most recent manuscript and references therein:

Chem Biol 2020 May 15;15(5):1242-1251. doi: 10.1021/acschembio.9b00977. Epub 2020 Mar 20.

-- Same topic, Page 6, line 8: “there are relatively few reports …. “ -- please clarify that you mean macrolide. As other classes of antibiotics have been used.

2. For the synthesis of the azido linked erythromycin – compound 5 in the experimental. Please provide IR for this compound. Also, for the modification at the ketone, could the authors comment on the ability for this group to make contact with the ribosome. The crystal structure with erythromycin and the ribosome has been solved. Finally, for the use of the NBD probe, the authors should reference back to Matijasic and co-workers, as they have successful used this modification on the macrolide core.


3. The paragraph regarding activity of the antibiotics should be re-worded to highlight that the two modified compounds retain activity. If the DMACA probe does not retain activity, should it be used as a probe moving forward (see my comments in the next section). The data presented in this paragraph go against this statement on page 10 of the manuscript “The reason for this difference from NBD probe 9 is unclear but given the similarity in results from the quantitative assay (see Tables 1-2 and below), the results obtained from 10
should be representative of both probes, and the parent antibiotic”. And this statement in the conclusion: “The resultant probes retained the same pattern of antibiotic activity as the parent drugs, and labeled both Gram-positive
and -negative bacteria as visualized by confocal microscopy. Could the authors please explain this discrepancy?

4. Figure 2: It is unclear what the authors are trying to show here. I understand that they are showing that the probe is the cytoplasm. But are they trying to argue that the probe localizes with nucleotide (as it should – should bind to RNA). But that is not clear from their data nor the text in which they describe these results. As a negative control, the dye alone (uncoupled to the antibiotic) must be shown. I would image the dye would show a similar phenotype as Figure 1 and would be removed upon over expression of an efflux pump.

5. Figure 4: Single cell experiments. Again these experiments should be repeated with dye alone. They could easily be a report of the dye behavior and not the macrolide. I understand that these controls are used in the lysis experiments – but they need to be used in the microscopy experiments as well. The (assumed) negative background will make these images and subsequent findings more convincing. It is a necessary control.

6. For the lysis and isolation experiments, would the authors expect to find the macrolides in the ribosomal fraction? Did they attempt to isolate the ribosomes?

7. The conclusion should be reworked to highlight what the probes can really tell us in addition the crystal structures that already exist.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

9 October 2020
Prof Andrea Rentmeister Associate Editor
RSC Chemical Biology
Dear Prof Rentmeister,
Please find attached our revised manuscript, "Fluorescent Macrolide Probes – Synthesis and Use in Evaluation of Bacterial Resistance”, a research article describing the synthesis, characterisation and microbiological evaluation of fluorescent probes derived from the macrolide class of antibiotics. We appreciate the feedback that the reviewers have provided, and believe that we have substantively addressed all of the reviewer concerns, as indicated in the separate response to reviewers. We have included versions of the main manuscript and supplementary material with changes highlighted as additional documents.
We trust that the revised manuscript is now suitable for acceptance, and look forward to your response.
Yours sincerely,
Mark Blaskovich
IMB Fellow
Director, Centre for Superbug Solutions Institute for Molecular Bioscience
The University of Queensland

RESPONSE TO REVIEWER REPORTS:
Referee: 1
Comments to the Author Summary:
The authors describe a series of experiments that are collectively directed at probing the utility of fluorescently-derivatized macrolide antibiotics for evaluation of macrolide uptake/efflux in representative Gram-negative and Gram-positive organisms. Efflux is one of the potential pathways that bacteria can use to achieve a measure of resistance against this important class of antibiotics. As a result, the work described in this manuscript has the potential to lead to novel insights that may be used to combat macrolide resistance.

We appreciate the positive comments.

The family of probes utilized in these experiments are fluorescently-modified roxithromycin derivatives wherein the fluorescent subunit was installed via a copper-activated azide-alkyne cycloaddition reaction. This portion of the work described in this manuscript is not particularly innovative, but I do not think that this is an issue at all since chemical innovation is not the major thrust of the work described. After establishing their antibiotic activity profiles, these derivatives were examined in a series of assays directed at determining their uptake/efflux.

It is worth pointing out that experiments utilizing fluorescently-modified macrolide antibiotics to quantify uptake/efflux can provide results that are difficult to interpret. This is especially true in this case. One can rightly note that peripheral changes to an antibiotic can affect both its cellular uptake as well as its ability to function as a substrate for a given transporter. Does a diminished intracellular fluorescence signal reveal substantial efflux or does it reveal lack of uptake? This is just one of several very difficult hurdles that must be addressed in work of this nature. From my reading of the manuscript one of the significant issues that is not addressed is fluorescence that may arise from non-specific binding. For example, the images in Figures 2A,E and 3A,E appear to show a significant amount of non-specific binding of the fluorescently-modified antibiotic. Is this due to binding to the cell membrane or is it due to random intracellular distribution within the cytoplasm? If it is the former, this can have a substantial impact on uptake measurements. This may also explain the diminished activity of the fluorescently-modified macrolides relative to that observed for the parent compounds. In fact, the very nicely designed experiments aimed at quantification of intracellular labeling appear to bear this out. That said, it would appear that this level of rigor is required to gain useful information from this approach; macroscopic evaluation is largely precluded by nonspecific binding.

We believe that the quantification experiments performed support that the probes are localised in the intracellular fluid: we think it is unlikely that there is significant binding to the cell membrane, as this should be captured in the pelleted fraction e.g. Figure 7. It is difficult to say if the probes are "randomly" distributed in the cytoplasm or bound to the presumed target ribosome, as there is not clear literature precedent that the ribosome is found at a particular location in the cytoplasm. The confocal microscopy employed in Figures 2 and 3, clearly show no blue cellular outline which would be evident with membrane binding, with the red FM4-64X dye surrounding the intracellular blue probe. Figure 6 provides further evidence of minimal non-specific (outer) membrane binding, as no labelling of cells is observed when efflux pumps prevent intracellular accumulation.

There was also an important observation that would benefit from some additional discussion. It was noted that reduced uptake was not observed in efflux upregulated Gram-positive bacteria. This was in contrast to the observations in Gram-negative bacteria. The manuscript could be strengthened with a rationale for this or through inclusion of additional experiments directed at unraveling this surprising observation. The experiments utilizing the efflux pump inhibitor (CCCP) should also be included.

We agree that a rationale for the surprising result observed in Gram-positive bacteria would be useful, but at the moment we have insufficient information to confidently propose a hypothesis for this observation, other than suggesting that structural requirements for efflux differ substantially between the Gram-positive and Gram-negative efflux pumps, such that the probe is not an effective substrate in Gram-positive bacteria. A sentence suggesting this possibility has been added on p15, with some modifications to the previous sentence to clarify the uptake results: “The results suggest that the macrolide probe is not actually a substrate for the S. pneumoniae efflux pumps, with the higher MIC due to an alternative resistance mechanism” The confocal microscopy results in Figure 3 support the quantified accumulation results in Table 4, with similar uptake in susceptible and resistant S. pneumoniae. We agree that testing CCCP in the Gram-positive accumulation assays would be valuable, but CCCP addition experiments have been performed during the confocal microscopy experiments (see text below Fig 2) and did not show any visual change in accumulation. Due to COVID restrictions on laboratory work, additional experiments using CCCP in the quantitative measurement assays in Table 4 have not been able to be conducted.

The manuscript is well-organized, is supported by a strong supporting information section, and is generally well-written. There are some very minor typographical errors that can be addressed in the next round of review. I will be happy to provide a copy of the pdf illustrating these, if it will help in the revision process.

We have identified a number of additional errors, and trust that these are the ones the reviewer has identified.

Overall, I think this manuscript will be embraced by the community that studies bacterial resistance to macrolides as well as the chemical biology community, in general. The corresponding author is an expert in the design and use of fluorescently-modified antibiotics to unravel important biological questions. I would support its publication upon submission of a revised manuscript addressing the concerns noted above.

The authors appreciate the supportive comments.

Referee: 2
Comments to the Author
Stone, Blaskovich and coworkers in their work entitled “Fluorescent Macrolide Probes – Synthesis and Use in Evaluation of Bacterial Resistance” describe the potential to use two erythromycin probes against the problem of antibiotic resistance. The authors perform semisynthesis to access the probes and a series of biological experiments to evaluate them. The authors would like to claim that fluorescent antibiotics haven’t been used in the past nor properly evaluated. Here they present some fluorescent microscopy to show that fluorescent macrolides could be useful in the design of new antibiotics. I have found discrepancies in the work and would like to see the authors address them. I am extremely sensitive to the conditions caused by COVID and if the authors cannot address them experimentally – I would appreciate more detailed information to increase the rigor of this manuscript. 1. Page 5, first full paragraph, fourth line: “Although fluorescent analogues of antibiotics were used in the second half of the 20th century to investigate the mode of action of antibiotics,5 they have been applied sparingly to help address the modern crisis of resistance.” - I think that this statement fails to capture two major recent advances in the field. Both fluorescent glycopeptides and b-lactams have been used widely in the field to address human interactions as well as resistance mechanisms. The discussion in this paragraph should be expanded to capture the following: a. Suanne Walker’s work with glycopeptides: Proc Natl Acad Sci U S A 2006 Jul 18;103(29):11033-8. doi: 10.1073/pnas.0600829103.Epub 2006 Jul 10. b. Erin Carlson’s catalog of work with fluorescent b-lactams: most recent manuscript and references therein: Chem Biol 2020 May 15;15(5):1242-1251. doi: 10.1021/acschembio.9b00977. Epub 2020 Mar 20.

We appreciate the referee’s suggestions of additional relevant reference, and these have now been included. The following sentence has been added, which includes these references: “Examples of probes that have been successfully utilised include fluorescent glycopeptides6 and β-lactams.7-9”

-- Same topic, Page 6, line 8: “there are relatively few reports …. “ -- please clarify that you mean macrolide. As other classes of antibiotics have been used.

This sentence has been to modified to: “To date, no work has been reported utilising fluorescent macrolides for studying antibiotic resistance; indeed there are relatively few reports on the use of any macrolide-derived fluorescent probes.”

2. For the synthesis of the azido linked erythromycin – compound 5 in the experimental. Please provide IR for this compound. Also, for the modification at the ketone, could the authors comment on the ability for this group to make contact with the ribosome. The crystal structure with erythromycin and the ribosome has been solved. Finally, for the use of the NBD probe, the authors should reference back to Matijasic and co-workers, as they have successful used this modification on the macrolide core.

IR data for compound 5 has been added in the supplementary information. The following sentences have been added/modified to address the reviewer comments: “This ketone has been observed to not make significant interactions with the ribosomal active site in crystal structure studies.30” “Furthermore, conjugation of the NBD fluorophore was found to not impair biological activity for azithromycin,25 hence was considered a promising candidate for attachment by click chemistry.

3. The paragraph regarding activity of the antibiotics should be re-worded to highlight that the two modified compounds retain activity. If the DMACA probe does not retain activity, should it be used as a probe moving forward (see my comments in the next section). The data presented in this paragraph go against this statement on page 10 of the manuscript “The reason for this difference from NBD probe 9 is unclear but given the similarity in results from the quantitative assay (see Tables 1-2 and below), the results obtained from 10 should be representative of both probes, and the parent antibiotic”. And this statement in the conclusion: “The resultant probes retained the same pattern of antibiotic activity as the parent drugs, and labeled both Gram-positive and -negative bacteria as visualized by confocal microscopy. Could the authors please explain this discrepancy?

The existing wording in the activity paragraph did indicate both probes retain activity, but highlighted that the DMACA probe is less active than the NBD probe, even though it is still active – it has been reworded too more clearly indicate this intent. “Similarly, NBD probe 9 exhibited good to moderate antibacterial activity, with very similar activity as the azide 6 against Staphylococci and Streptococci (e.g. 4- to 8-fold less active than the parent roxithromycin). The DMACA probe 10 was also active at inhibiting bacterial growth, though was less potent than the other derivatives tested, with approximately 4- to 16- fold increased MIC compared to the NBD probe. However, both probes showed similar relative variations in potency across the different strains tested as the parent antibiotic.” The statement on p10 has been modified to “ The reason for this difference from NBD probe 9 is unclear but given the similarity in MIC (Tables 1-2) and uptake results from the quantitative assays (see Figures 5,7and Table 4 below), the results obtained from 10 should be representative of both probes, and the parent antibiotic.” The conclusion statement “The resultant probes retained the same pattern of antibiotic activity” refers to the fact that, while the DMACA probe was about four- to eight-fold less potent than the NBD probe, when comparing different bacteria, those that had a higher roxithromycin MIC also had elevated MICs for both the DMACA and NBD probes (e.g. Table 1, E. coli vs S. aureus), and those that were more sensitive to roxithromycin also showed greater susceptibility to both probes (e.g. Table 1, S. pyogenes vs S. aureus), with the same trends also seen in resistant strains (Table2).

4. Figure 2: It is unclear what the authors are trying to show here. I understand that they are showing that the probe is the cytoplasm. But are they trying to argue that the probe localizes with nucleotide (as it should – should bind to RNA). But that is not clear from their data nor the text in which they describe these results. As a negative control, the dye alone (uncoupled to the antibiotic) must be shown. I would image the dye would show a similar phenotype as Figure 1 and would be removed upon over expression of an efflux pump.

Images with the fluorophore alkynes has been added to the supplementary information (Figure S2) showing minimal uptake/labelling compared to the conjugated dye despite the much smaller size that should enable more efficient uptake, and the following sentence has been added to the main text: “This is in contrast to the fluorophore-alkynes, which did not significantly accumulate in the bacteria (Figure S2).”

5. Figure 4: Single cell experiments. Again these experiments should be repeated with dye alone. They could easily be a report of the dye behavior and not the macrolide. I understand that these controls are used in the lysis experiments – but they need to be used in the microscopy experiments as well. The (assumed) negative background will make these images and subsequent findings more convincing. It is a necessary control.

As mentioned above, control experiments have now been conducted for the confocal microscopy using alkyne-dyes alone, and showed minimal labelling/uptake, which matches the previously reported data that the alkyne fluorophore fluorescence was almost all contained in the wash fraction during quantitative uptake experiments (p13) so we anticipate we would see the same lack of labelling in the single cell experiments. We also see substantially different localisation and uptake behaviour when testing different antibiotics labelled with the same dyes in the microfluidics experiments (data not shown), further supporting that the experimental results are driven by the antibiotic, not the dye. We agree that testing dye alone in the microfluidic experiments would make a valuable control, but due to COVID restrictions on experiments are unable to perform the additional studies at this time.

6. For the lysis and isolation experiments, would the authors expect to find the macrolides in the ribosomal fraction? Did they attempt to isolate the ribosomes?

Yes, we would expect the macrolides to localise in the ribosomes, and isolation was performed. Preliminary experiments were carried out to try and measure interaction between ribosomes and probes, however fluorescence signals were found to be too low to quantify using spectrofluorometry.

7. The conclusion should be reworked to highlight what the probes can really tell us in addition the crystal structures that already exist.

The following sentences have been added to the conclusion. Fluorescent probes enable the researcher to visualise target interactions in a practical manner, and can be applied to experiments that range from studies of isolated targets through to investigations of mixed populations. Future implications include quantitatively distinguishing modes of macrolide resistance and uncovering heterogeneity in antibiotic response amongst bacterial populations.

28-Oct-2020

Dear Dr Blaskovich:

Manuscript ID: CB-ART-07-2020-000118.R1
TITLE: Fluorescent Macrolide Probes – Synthesis and Use in Evaluation of Bacterial Resistance

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Andrea Rentmeister

Reviewer 2

Thank you for addressing my comments/concerns. The manuscript is really nice and I look forward to future studies with your probes.