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Inhibiting fatty acid synthesis overcomes colistin resistance

Nature Microbiology volume 8, pages 1026–1038 (2023)Cite this article

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Abstract

Treating multidrug-resistant infections has increasingly relied on last-resort antibiotics, including polymyxins, for example colistin. As polymyxins are given routinely, the prevalence of their resistance is on the rise and increases mortality rates of sepsis patients. The global dissemination of plasmid-borne colistin resistance, driven by the emergence of mcr-1, threatens to diminish the therapeutic utility of polymyxins from an already shrinking antibiotic arsenal. Restoring sensitivity to polymyxins using combination therapy with sensitizing drugs is a promising approach to reviving its clinical utility. Here we describe the ability of the biotin biosynthesis inhibitor, MAC13772, to synergize with colistin exclusively against colistin-resistant bacteria. MAC13772 indirectly disrupts fatty acid synthesis (FAS) and restores sensitivity to the last-resort antibiotic, colistin. Accordingly, we found that combinations of colistin and other FAS inhibitors, cerulenin, triclosan and Debio1452-NH3, had broad potential against both chromosomal and plasmid-mediated colistin resistance in chequerboard and lysis assays. Furthermore, combination therapy with colistin and the clinically relevant FabI inhibitor, Debio1452-NH3, showed efficacy against mcr-1 positive Klebsiella pneumoniae and colistin-resistant Escherichia coli systemic infections in mice. Using chemical genomics, lipidomics and transcriptomics, we explored the mechanism of the interaction. We propose that inhibiting FAS restores colistin sensitivity by depleting lipid synthesis, leading to changes in phospholipid composition. In all, this work reveals a surprising link between FAS and colistin resistance.

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Fig. 1: Biotin and FAS inhibitors reverse colistin resistance against mcr-1-expressing Enterobacteriaceae.
Fig. 2: Established mechanisms do not explain the synergy between colistin and MAC13772.
Fig. 3: Inhibiting biotin or FAS induces cell envelope stress by modifying phospholipid composition.
Fig. 4: In vivo efficacy of Debio1452-NH3 and colistin in a murine infection model.
Fig. 5: MAC13772 modifies the lipidome to increase membrane fluidity, overcoming colistin resistance in mcr-1-expressing E. coli.

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Data availability

RNA sequencing data are available at the NCBI Sequence Read Archive under accession PRJNA824525. Lipidomics data have been deposited to the EMBL-EBI MetaboLights database with the identifier MTBLS7286. The complete dataset can be accessed at https://www.ebi.ac.uk/metabolights/MTBLS7286. The suppressor genome sequences and reference strain E. coli C0279 are deposited in NCBI Sequence Read Archive, SRA: PRJNA889379. E. coli K-12 (U00096) reference genome was used in this study. The source data underlying Figs. 1c, 3a,b,g and 4c are provided as Supplementary Tables 1, 8 and 9. The data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank G. Wright from McMaster University for bacterial strains from the Institute for Infectious Disease Research clinical collection, E. Parker and P. Hergenrother from the University of Illinois at Urbana-Champaign for providing ample Debio1452-NH3 for animal studies, M. Mulvey from the University of Manitoba for providing the environmental mcr-1-positive E. coli isolates, R. Melano at Public Health Ontario for bacterial strains GB687 and C0064, and D. Bikard from the Institut Pasteur for the plasmid, pFD152. The authors also thank the Centre for Microbial Chemical Biology at McMaster University, specifically N. Henriquez for LC–MS/MS and S. McCusker for providing bacterial strains. This research was supported by a Tier 1 Canada Research Chair award, a Foundation grant from the Canadian Institutes of Health Research (CHIR; FRN 143215), and a grant from the Ontario Research Fund (RE09-047) to E.D.B. L.A.C. was supported by a CIHR Canada Graduate Scholarship (CGS-D) scholarship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Lindsey A. Carfrae, Kenneth Rachwalski, Shawn French, Rodion Gordzevich, Caressa N. Tsai, Megan M. Tu, Craig R. MacNair & Eric D. Brown

  2. Institute of Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    Lindsey A. Carfrae, Kenneth Rachwalski, Shawn French, Rodion Gordzevich, Caressa N. Tsai, Megan M. Tu, Craig R. MacNair & Eric D. Brown

  3. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada

    Laura Seidel, Olga G. Ovchinnikova, Bradley R. Clarke & Chris Whitfield

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Contributions

L.A.C. conceived the research, designed and carried out experiments and data analysis and wrote the manuscript. K.R. assisted with the design and creation of CRISPRi strains. S.F. acquired samples for lipidomic analysis and S.F. with R.G. aided in the analysis of the lipidomics dataset. L.S., O.G.O. and B.R.C. performed lipid A purification and mass spec analysis. C.N.T. assisted in the analysis of the RNA-seq dataset. M.M.T. assisted in the animal experiments. C.R.M. acquired motility data and assisted with manuscript editing. C.W. assisted with data interpretation and manuscript editing. E.D.B. conceived the research and assisted with data interpretation and manuscript editing.

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Correspondence to Eric D. Brown.

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Nature Microbiology thanks Eefjan Breukink, Peter Tonge, Judith Steenbergen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Colistin synergizes with biotin and FAS inhibitors against E. coli strains harbouring mcr-1 on natural plasmids.

(a, b) Chequerboard broth microdilution assays showing dose-dependent colistin potentiation by cerulenin, MAC13772, and triclosan against mcr-1 positive E. coli strains (a) N15-02865 and (b) N15-02866. Dark regions represent higher cell density. Chequerboard data are representative of at least three biological replicates.

Source data

Extended Data Fig. 2 Exogenous LPS or Mg2+ abolishes synergy between MAC13772 and colistin against mcr-1 expressing E. coli.

Chequerboard broth microdilution assays between MAC13772 and colistin in M9 minimal media supplemented with (a) purified E. coli LPS (1 mg/mL) or (b) Mg2+ (20 mM). Dark regions represent higher cell density. Chequerboard data are representative of at least 2 biological replicates.

Source data

Extended Data Fig. 3 MAC13772 synergizes with colistin against E. coli expressing mcr-1 by inhibiting BioA.

(a,b) Chequerboard broth microdilution assays between MAC13772 and colistin M9 minimal media supplemented with biotin (10 µg/mL). (c) Expression of bioA suppresses MAC13772’s potentiation of colistin against mcr-1 positive E. coli. Expression of bioA induced by the addition of IPTG (10 mM). (d, e) Chequerboard assay of colistin and anhydrotetracycline against E. coli expressing (d) pfD152 empty or (e) pfD152 bioA. Dark regions represent higher cell density. Chequerboard data are representative of at least two biological replicates.

Source data

Extended Data Fig. 4 Synergy between MAC13772 and colistin is dependent on PEtN modification of lipid A.

Chequerboard broth microdilution assays between colistin and (a) MAC13772, (b) cerulenin, or (c) triclosan against E. coli expressing mcr-1::E246A. Dark regions represent higher cell density. Chequerboard data are representative of at least three biological replicates.

Source data

Extended Data Fig. 5 Synergy between MAC13772 and colistin requires L-Ara4N decoration of lipid A.

(a) Screening of the K. pneumoniae transposon mutant library for sensitivity to colistin. Percent growth was calculated based on growth in the presence of colistin relative to its respective control (untreated strain) growing in M9 minimal media. The blue region indicates genes 3 standard deviations from the mean. Bacterial pathways enriched among these mutants include genes involved in lipid A modification (arnA, arnB, arnC, arnD, arnT, phoPQ), cell division (envC, ftsK, ftsN, ftsX, nlpD, pal, tolA, tolB, tolQ, tolR), and enterobactin biosynthesis (entA, entD, entE, fes). (b) Chequerboard assays of MAC13772 and colistin against wild-type and colistin sensitive transposon mutants. Higher growth is indicated in dark blue, no detectable growth is indicated in white, and results are representative of at least two independent experiments.

Source data

Extended Data Fig. 6 Colistin does not increase intracellular accumulation of cerulenin and triclosan.

(a–c) Accumulation of (a) cerulenin (50 μM; blue, n = 4), (b) triclosan (50 μM; green, n = 5, 4), and (c) rifampicin (50 μM; red, n = 4; p = 0.0143) in mcr-1 expressing E. coli in the absence and presence of colistin (6 μM). Statistical significance was determined by using a one-tailed Mann–Whitney U test relative to the no colistin control, *P < 0.05.

Source data

Extended Data Fig. 7 Inhibiting biotin or FAS does not disrupt the proton motive force.

(a) DiSC3(5) assay, bar plots depict the mean of three biological replicates, error bars indicate standard error. (b, c) Chequerboard broth microdilution assays for (b) cerulenin or (c) triclosan and CCCP. Higher growth is indicated in dark blue; no detectable growth is indicated in white, and results are representative of at least two independent experiments.

Source data

Extended Data Fig. 8 Bacterial lysis assays with colistin and MAC13772 against empty and mcr-1 expressing E. coli.

(a) Killing of E. coli expressing the empty vector in the presence of water (n = 3), colistin (0.4 μg/mL; n = 4), and colistin (2 μg/mL; n = 5). (b, c) Killing of mcr-1 expressing E. coli in the presence of (b) water (n = 2), colistin (2 μg/mL; t = 4 and 8: n = 4, t = 0, 4, 24: n = 5), colistin (4 μg/mL; t = 0, 2, 4: n = 5, t = 8 and 24: n = 4), colistin (8 μg/mL; n = 2), colistin (32 μg/mL; n = 2) or (c) DMSO, MAC13772 (32 μg/mL; n = 4), MAC13772 (64 μg/mL; n = 2), MAC13772 (128 μg/mL; t = 0, 2, 4, 24: n = 2), MAC13772 (256 μg/mL; t = 0, 2, 4, 24: n = 2), MAC13772 (512 μg/mL; n = 2). The initial cell density is ~5 × 107 CFU/mL. Shown is the mean of a minimum of two biological replicates. Bars denote standard error.

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Extended Data Fig. 9 Addition of exogenous fatty acids suppresses synergy between colistin and cerulenin or triclosan.

(a) Diagram depicting the path for the incorporation of exogenous and endogenous fatty acids in LPS and phospholipids. E. coli synthesizes phosphatidic acid (PA), the major precursor to all phospholipid species, through two acylation reactions. First glycerol-3-phosphate (G3P) is acylated to lysophosphatidic acid (LPA) by PlsB, which is subsequently acylated by PlsC to form phosphatidic acid. The acyltransferases (PlsB and PlsC) can use both acyl-CoA, generated by FadD from exogenous fatty acids, and acyl-ACP from FAS as acyl donors in the synthesis of phosphatidic acid. LPS biosynthesis requires endogenous β-hydroxyacyl-ACP for acylation reactions as there is not a pathway in E. coli to generate β-hydroxyacyl-ACP from exogenous fatty acids. (b–d) Chequerboard broth microdilution assays between cerulenin, MAC13772, and triclosan with colistin in media supplemented with (b) 2-hexadecenoic acid, (c) linoleic acid, or (d) oleic acid. Higher growth is indicated in dark blue, no detectable growth is indicated in white, and results are representative of at least three independent experiments.

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Extended Data Fig. 10 Depletion of phosphatidylethanolamine biosynthesis genes sensitize mcr-1 expressing E. coli to colistin.

Chequerboard assay of colistin and anhydrotetracycline against E. coli expressing (a) mcr-1 or (b) empty vector and with a CRISPR knockdown of cdsA, pssA, psd, or pgsA (left to right). Dark regions represent higher cell density. Chequerboard data are representative of at least three biological replicates.

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Supplementary information

Supplementary Information

Supplementary figs. 1–15, tables 2, 3, 5, 6 and 9–11, and unprocessed scans for supplementary figures.

Reporting Summary

Supplementary Table

Table 1. Interactions of cerulenin, MAC13772 and triclosan with colistin against a broad spectrum of bacteria. Minimum inhibitory concentration represents the median of at least two biological replicates. FICi were calculated with colistin using chequerboard assays and are the mean of at least two biological replicates. The fold change indicates the change in colistin MIC in the presence of ¼ MIC of partner compound and represents the median of at least two biological replicates (when an even number of replicates was performed, the mean of the two median values is shown). Short forms, Col, Cer, MAC and Tri indicate colistin, cerulenin, MAC13772 and triclosan. Results corresponding to the grey shaded squares were not assessed. Table 4. Screening data for K. pneumoniae MKP103 transposon library grown in M9 supplemented with amino acids and subinhibitory colistin. Normalized growth data associated with genetic screen in M9 and colistin. Mutants that exhibited growth <3 s.d. from the mean in the presence of colistin, but not M9 minimal media, were considered as exclusively defective for growth in the presence of colistin. Table 5. Gene ontology (GO) term enrichment for K. pneumoniae MKP103 transposon library grown in M9 supplemented with amino acids and subinhibitory colistin. The table depicts the top GO terms enriched by biological function. Enrichment was tested using pathway-tools software (Grossmann’s parent-child-union variation of the Fisher-exact test with the Benjamini-Hochberg correction). Table 8. Impact of mcr-1 expression and MAC13772 treatment on E. coli transcriptome. Raw values used to generate RNA sequencing heat maps and volcano plots. A Wald test corrected for multiple testing using the Benjamini and Hochberg method was used to calculate differentially expressed genes. Table 9. Impact of mcr-1 expression and MAC13772 treatment on E. coli lipidome. A moderated t statistic was used to identify significantly differential lipids between biological conditions applying a multiple hypothesis testing correction. Table 10. Identified mutations in an E. coli mutant resistant to MAC13772-colistin synergism.

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Carfrae, L.A., Rachwalski, K., French, S. et al. Inhibiting fatty acid synthesis overcomes colistin resistance. Nat Microbiol 8, 1026–1038 (2023). https://doi.org/10.1038/s41564-023-01369-z

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