A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli
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Evaluation Summary:
This manuscript describes how a small plasmid containing a quinolone resistance determinant changes the cellular response to sub-inhibitory concentrations of Tobramycin. Authors have found that Escherichia coli cells carrying this plasmid undergo nitrosative stress mediated by two previously uncharacterized genes, which leads to induction of the SOS response, a well described stress response in bacteria. These findings are relevant for readers across the microbiology and genetics fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli , sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD -plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD . Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide ( FAD )-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase ( FNR )-type transcription factor, related to an O 2 -responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli . Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.
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Evaluation Summary:
This manuscript describes how a small plasmid containing a quinolone resistance determinant changes the cellular response to sub-inhibitory concentrations of Tobramycin. Authors have found that Escherichia coli cells carrying this plasmid undergo nitrosative stress mediated by two previously uncharacterized genes, which leads to induction of the SOS response, a well described stress response in bacteria. These findings are relevant for readers across the microbiology and genetics fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript aims at reporting a novel role of a small fluoroquinolone resistance qnrD plasmid and elucidating relevant mechanisms that allow extreme low sub-inhibitory levels of aminoglycoside antibiotics to induce SOS response in E. coli.
The authors identified two qnrD plasmid-encoded products, FAD-dependent oxidoreductase and FNR-type transcriptional repressor, which contribute, respectively, to elevated nitric oxide production and reduced nitric oxide detoxification, subsequently resulting in the SOS response. The contribution from the oxidative stress was ruled out. These novel findings were achieved in vitro by using a range of clearly-defined genetic backgrounds of isogenic strains (include gene deletion mutants and in-trans complementation) and by measuring the expression of SOS response …
Reviewer #1 (Public Review):
This manuscript aims at reporting a novel role of a small fluoroquinolone resistance qnrD plasmid and elucidating relevant mechanisms that allow extreme low sub-inhibitory levels of aminoglycoside antibiotics to induce SOS response in E. coli.
The authors identified two qnrD plasmid-encoded products, FAD-dependent oxidoreductase and FNR-type transcriptional repressor, which contribute, respectively, to elevated nitric oxide production and reduced nitric oxide detoxification, subsequently resulting in the SOS response. The contribution from the oxidative stress was ruled out. These novel findings were achieved in vitro by using a range of clearly-defined genetic backgrounds of isogenic strains (include gene deletion mutants and in-trans complementation) and by measuring the expression of SOS response indicator genes, which is the strength of the study. The experiments were overall conducted both logically and carefully for understanding the mechanisms responsible for observed qnrD plasmid-dependent induction of SOS response by aminoglycosides, followed by proposing a justifiable model that summarizes the relevant nitrosative stress pathways leading to the SOS response.
However, the target(s) (molecules) with which aminoglycoside interact as inducers remain unknown, although the aminolgycoside effect is dependent on the GO repair system and indirectly results in DNA damage. Moreover, the magnitude of gene expression changes affected by aminoglycosides in the presence of a qnrD plasmid was relatively small, mostly within about 2- to 3-fold alterations at the transcriptional level, raising an open question on the interpretation of the significance of aminoglycoside-induced SOS response in the contribution to DNA mutations and resistance emergence. The latter warrant future additional in vitro and in vivo studies to shed light on the importance of aminoglycoside-triggered SOS response.
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Reviewer #2 (Public Review):
Strengths and Weaknesses:
The manuscript from Babosan and coworkers is nicely written and presented and describes a well-conducted experimental work using appropriate methodology. Overall, this is an interesting manuscript describing an important phenomenon, the induction of the SOS response by aminoglycosides (AGs) in cells carrying a plasmid conferring resistance to another drug class. The main points are clearly addressed and demonstrated: SOS induction by AGs in cells carrying the qnrD-containing plasmid; the involvement of nitrosative stress; identification of ORFs 3 and 4 as the responsible for the nitrosative stress. The major weakness of the work is that it does not assess whether the SOS induction by AGs leads to increased mutagenesis as speculated. Also, the qnrD plasmid containing ORFs 3 and 4 is …
Reviewer #2 (Public Review):
Strengths and Weaknesses:
The manuscript from Babosan and coworkers is nicely written and presented and describes a well-conducted experimental work using appropriate methodology. Overall, this is an interesting manuscript describing an important phenomenon, the induction of the SOS response by aminoglycosides (AGs) in cells carrying a plasmid conferring resistance to another drug class. The main points are clearly addressed and demonstrated: SOS induction by AGs in cells carrying the qnrD-containing plasmid; the involvement of nitrosative stress; identification of ORFs 3 and 4 as the responsible for the nitrosative stress. The major weakness of the work is that it does not assess whether the SOS induction by AGs leads to increased mutagenesis as speculated. Also, the qnrD plasmid containing ORFs 3 and 4 is not commonly found in E. coli (Table S3 and Figure S1). It is adequate that most of the work has been performed in this genetically tractable organism. Nevertheless, it would be important to assess whether the same AG-mediated SOS induction occurs in bacteria that usually carry this type of plasmid (P. mirabilis or other Morganellaceae). This is a simple qRT PCR experiment which could add a lot to the significance of the paper. Perhaps the nitrosative stress (which is obviously a deleterious effect) is particular to E. coli and that could be one of the reasons why this particular plasmid architecture occur rarely (or never?) in this species.
Other important points for consideration by the authors:
- How the experiments in figure S2 addressed plasmid stability during growth by measuring OD? Plasmid stability in the experimental conditions is an important issue.
- Comparing Figures 3B and 1E, it can be observed that deletion of both recF and recB have significant effects on SOS induction by AG in cells carrying the qnrD plasmid. These results are very important for their model and a qRT measurement of sulA in recF and recB mutants would be an important confirmation for the results obtained with plasmid based assays, which could be influenced by the reporter plasmid copy number and stability in the different genotypes analyzed.
- The complementation of ORF 3 and 4 mutants (Figures 4A and 4C) is incomplete, the same SOS induction level as observed in the wt is never reached. Could this be due to the deletion of one ORF influencing on the expression of the other? Additionally, a more proper comparison with cells carrying the empty expression vector may help to solve this issue. -
Reviewer #3 (Public Review):
The authors use the term "aminoglycosides" in multiple sections of the manuscript, including the title, the abstract and the discussion. However, only one aminoglycoside (tobramycin) has been used throughout the study. Gentamicin was used at the beginning of the study but disappears at some point. Please clarify.
In contrast with the detailed analysis described in the initial sections, the information provided on the products of ORF3 and ORF4 is scanty. This is unfortunate as these products and their activities are the main findings of the story. Hence, more detailed characterization of these putative proteins seems advisable to support the authors's model. Bioinformatic analysis should be shown in more detail, the existence of the predicted proteins should be proven and the putative DNA binding activity of …
Reviewer #3 (Public Review):
The authors use the term "aminoglycosides" in multiple sections of the manuscript, including the title, the abstract and the discussion. However, only one aminoglycoside (tobramycin) has been used throughout the study. Gentamicin was used at the beginning of the study but disappears at some point. Please clarify.
In contrast with the detailed analysis described in the initial sections, the information provided on the products of ORF3 and ORF4 is scanty. This is unfortunate as these products and their activities are the main findings of the story. Hence, more detailed characterization of these putative proteins seems advisable to support the authors's model. Bioinformatic analysis should be shown in more detail, the existence of the predicted proteins should be proven and the putative DNA binding activity of the ORF4 product should be shown. Otherwise, the possibility of indirect effect will remain open.
If 8-oxoG is the problem indeed, assays in a strain lacking formamidopyrimidine-DNA glycosylase (fpg) might provide support to the authors' hypothesis about NO production.
The magnitude of SOS induction reported in the manuscript is small. Hence, the authors' hypothesis that induction of the SOS response will increase the mutation rate can be questioned as error-prone repair is unlikely to operate under such circumstances. To sustain the authors' hypothesis, mutation rates should be tested.
Absolute numbers of beta-gal activities should be provided when lac fusions are used. This would help the reader to assess the magnitude of the phenomena described in the manuscript.
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