Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane

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 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 outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. 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 cytoplasmic membrane 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.

Data availability

Source data for all figures has been deposited at Dryad: https://doi.org/10.5061/dryad.98sf7m0hh

The following data sets were generated

Article and author information

Author details

  1. Akshay Sabnis

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Katheryn L H Hagart

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Anna Klöckner

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Michele Becce

    Department of Materials, Department of Bioengineering, Institute of Biomedical Engineering, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Lindsay E Evans

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. R Christopher D Furniss

    Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5806-5099
  7. Despoina A I Mavridou

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Ronan Murphy

    National Heart and Lung Institute, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Molly M Stevens

    Department of Materials, Department of Bioengineering, Institute of Biomedical Engineering, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7335-266X
  10. Jane C Davies

    National Heart and Lung Institute, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Gérald J Larrouy-Maumus

    MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Thomas B Clarke

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. Andrew M Edwards

    MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
    For correspondence
    a.edwards@imperial.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7173-7355

Funding

Medical Research Council (PhD Studentship)

  • Akshay Sabnis

Wellcome Trust

  • Andrew M Edwards

NIHR Imperial Biomedical Research Centre

  • Andrew M Edwards

DFG

  • Anna Klöckner

Horizon 2020

  • Anna Klöckner

Rosetrees Trust

  • Molly M Stevens

Cystic Fibrosis Trust

  • Jane C Davies

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: The use of mice was performed under the authority of the UK Home Office outlined in the Animals (Scientific Procedures) Act 1986 after ethical review by Imperial College London Animal Welfare and Ethical Review Body (PPL 70/7969).

Reviewing Editor

  1. Philip A Cole, Harvard Medical School, United States

Publication history

  1. Received: December 16, 2020
  2. Accepted: March 31, 2021
  3. Accepted Manuscript published: April 6, 2021 (version 1)
  4. Version of Record published: May 4, 2021 (version 2)

Copyright

© 2021, Sabnis et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Akshay Sabnis
  2. Katheryn L H Hagart
  3. Anna Klöckner
  4. Michele Becce
  5. Lindsay E Evans
  6. R Christopher D Furniss
  7. Despoina A I Mavridou
  8. Ronan Murphy
  9. Molly M Stevens
  10. Jane C Davies
  11. Gérald J Larrouy-Maumus
  12. Thomas B Clarke
  13. Andrew M Edwards
(2021)
Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane
eLife 10:e65836.
https://doi.org/10.7554/eLife.65836
  1. Further reading

Further reading

    1. Medicine
    2. Microbiology and Infectious Disease
    Yan Wang, Xiaohui Liang ... Wenkui Yu
    Research Article

    Background:

    Severe pneumonia is one of the common acute diseases caused by pathogenic bacteria infection, especially by pathogenic bacteria, leading to sepsis with a high morbidity and mortality rate. However, the existing bacteria cultivation method cannot satisfy current clinical needs requiring rapid identification of bacteria strain for antibiotic selection. Therefore, developing a sensitive liquid biopsy system demonstrates the enormous value of detecting pathogenic bacterium species in pneumonia patients.

    Methods:

    In this study, we developed a tool named Species-Specific Bacterial Detector (SSBD, pronounce as "speed") for detecting selected bacterium. Newly designed diagnostic tools combining specific DNA-tag screened by our algorithm and CRISPR/Cas12a, which were first tested in the lab to confirm the accuracy, followed by validating its specificity and sensitivity via applying on bronchoalveolar lavage fluid (BALF) from pneumonia patients. In the validation I stage, we compared the SSBD results with traditional cultivation results. In the validation II stage, a randomized and controlled clinical trial was completed at the ICU of Nanjing Drum Tower Hospital to evaluate the benefit SSBD brought to the treatment.

    Results:

    In the validation stage I, 77 BALF samples were tested, and SSBD could identify designated organisms in 4 hours with almost 100% sensitivity and over 87% specific rate. In validation stage II, the SSBD results were obtained in 4 hours, leading to better APACHE II scores (p=0.0035, ANOVA test). Based on the results acquired by SSBD, cultivation results could deviate from the real pathogenic situation with polymicrobial infections. In addition, nosocomial infections were found widely in ICU, which should deserve more attention.

    Funding:

    National Natural Science Foundation of China. The National Key Scientific Instrument and Equipment Development Project. Project number: 81927808.

    Clinical trial:

    This study was registered at ClinicalTrilas.gov (NCT04178382).

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Takeshi Imai, Ryuta Tobe ... Hisaaki Mihara
    Research Article Updated

    Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.