Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane
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Abstract
Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa , which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on February 15 2021, follows:
Summary:
The submitted manuscript presents an argument for a novel mechanism of action for the antibiotic colistin. The authors suggest that colistin kills bacteria through its action on lipopolysaccharides at the inner membrane. This is primarily supported by MCR-1 mediated colistin resistance conferring resistance only to cell lysis and not to outer membrane permeabilization. The authors extend this hypothesis to suggest that increasing the amount of LPS in the inner membrane should increase susceptibility to colistin. By inhibiting LPS transport with murepavadin, the accumulation of LPS in the cytoplasmic membrane increased. Combinations of colistin and murepavadin act synergistically to improve bacterial …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on February 15 2021, follows:
Summary:
The submitted manuscript presents an argument for a novel mechanism of action for the antibiotic colistin. The authors suggest that colistin kills bacteria through its action on lipopolysaccharides at the inner membrane. This is primarily supported by MCR-1 mediated colistin resistance conferring resistance only to cell lysis and not to outer membrane permeabilization. The authors extend this hypothesis to suggest that increasing the amount of LPS in the inner membrane should increase susceptibility to colistin. By inhibiting LPS transport with murepavadin, the accumulation of LPS in the cytoplasmic membrane increased. Combinations of colistin and murepavadin act synergistically to improve bacterial lysis and show efficacy in a murine lung infection model.
Essential Revisions:
- Observations demonstrating MCR-1 modification does not impact outer membrane perturbation and provides resistance to colistin induced lysis are supported by MacNair et al. They suggest that strengthened LPS packing provided by mcr-1 could play an important role in reducing the uptake and lytic activities of colistin. The author's should address that decreased colistin uptake could also result in reduced lysis. To support their hypothesis, the relationship between the amount of modified LPS in the inner membrane and resistance to cell lysis could be expanded on. https://www.nature.com/articles/s41467-018-02875-z
The authors use the lack of change in susceptibility of mcr-1 spheroplasts to daptomycin and nisin to support that there is no change to the biophysical properties of the phospholipid bilayer of the cytoplasmic membrane. However, whether the sensitivity of daptomycin and nisin to changes to membrane charge or fluidity remains unclear.
Murepavadin is used to increase LPS at the CM and as interpreted would support the hypothesis. However, it is also possible that in the whole cell assays, the OM disruption of colistin sensitizes the cells to the killing activity of murepavadin. Repeating the assays with a non-lethal OM permeabilizer like polymyxin B nonapeptide would eliminate this possibility and strengthen the authors conclusions.
The authors suggest that mcr-1 provides protection from colistin through the modification of LPS at the inner membrane and that outer membrane modification has no impact on colistin activity. In contrast, it has been demonstrated that mcr-1 decoration is capable of preventing outer membrane perturbation by polymyxin B nonapeptide (https://www.nature.com/articles/nmicrobiol201728). This suggests that modified LPS at both the inner and outer membrane may play a role in resistance.
Authors should discuss work in the synergy between novobiocin and colistin where novobiocin enhances colistin killing through the stimulation of LPS transport. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990483/
Hydrophobic NPN dye was used to explore the permeabilization of OM in this work. However, the uptake of NPN is not absolute proof that colistin is permeable. The authors should discuss this as a possible caveat of their mechanistic model.
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