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. 2026 Jan 19;17(1):1880.
doi: 10.1038/s41467-026-68681-0.

Differential membrane lipid disruption by lipopeptide antibiotics, colistin and turnercyclamycins

Albebson L Lim  1 , Bailey W Miller  1 , Mark A Fisher  2 , Margo G Haygood  1 , Louis R Barrows  3 , Eric W Schmidt  4
Affiliations

Differential membrane lipid disruption by lipopeptide antibiotics, colistin and turnercyclamycins

Albebson L Lim et al. Nat Commun. .

Abstract

Lipopeptide natural products are essential agents against multidrug-resistant bacteria, but their clinical utility is often constrained by toxicity and resistance. Here, we compare the mechanisms of action of two superficially similar lipopeptide antibiotics: colistin, a last-line treatment for Gram-negative infections, and turnercyclamycins, a new class active against certain colistin-resistant strains. Both antibiotics require lipopolysaccharide (LPS) biosynthesis, even when LPS transport to the outer membrane (OM) is impaired. Colistin rapidly disrupts both the OM and the cytoplasmic membrane (CM), causing swift bacterial death. Turnercyclamycins, by contrast, act independently of the CM, with delayed OM disruption. Unlike colistin, which binds LPS directly to damage membranes, turnercyclamycins show no measurable LPS binding by calorimetry. Instead, their activity is modulated by different phospholipids, as confirmed by phospholipidomic profiling on whole cells, which identifies alterations in bacterial lipid biosynthesis and membrane homeostasis. These findings support a mechanistically distinct mode of action for turnercyclamycins, which we propose to correlate with their different pharmacological properties and potential therapeutic applications. Our results highlight how subtle structural differences between lipopeptides can lead to major functional divergence, offering a framework for the rational design of next-generation antibiotics with improved safety and efficacy profiles.

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Conflict of interest statement

Competing interests: The authors declare the following competing interests: E.W.S., B.W.M., and M.G.H. have submitted a patent application for turnercyclamycins. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Chemical structures of colistin and turnercyclamycins,, with MIC90 values against various Gram-negative bacteria.
nt is not tested.
Fig. 2
Fig. 2. Effect of turnercyclamycins on membrane permeabilization and depolarization, and interaction with LPS molecules.
All experiments were performed with duplicate replicates. A single legend at the top right is used for (a–c) Col, colistin (2 μg/mL); TurA, turnercyclamycin A (8 μg/mL); Tur B, turnercyclamycin B (8 μg/mL); DMSO, 1% v/v; compound + dye ctrl, compounds tested with the dye but without cells. a Turnercyclamycins are slower to kill E. coli in comparison to colistin. Time-kill curves are shown for E. coli C600-treated cells; y-axis, total bacterial counts (CFU/mL); x-axis, incubation time. b Colistin causes almost immediate OM permeabilization, while turnercyclamycins permeabilize the OM more slowly. NPN fluorescence (y-axis) increases as the OM is permeabilized over time (x-axis). Starting OD600 inoculum = 0.5. c Turnercyclamycins cause significantly less inner membrane damage compared to colistin. Inner membrane damage assessment using Sytox Green for E. coli C600 cells treated with 2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B, and 1% DMSO v/v. y-Axis shows Sytox green fluorescence and x-axis shows incubation time. Starting OD600 inoculum = 0.5. d Representative fluorescent microscopy images of E. coli C600 cells untreated and treated with 1% DMSO v/v, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B, and 2 μg/mL colistin, for 10 min, and stained with DiSC3(5). Scale bar indicates 10 µm. e, f Micro-ITC profile of turnercyclamycin A, turnercyclamycin B, and colistin binding to LPS analyzed by MicroCal’s ORIGIN software. e Curve generated for colistin showing computed KD values. f Raw titration curves from both colistin and turnercyclamycin interaction experiment with LPS. Curve shown is for colistin while turnercyclamycin A and B binding curve could not be generated. The highest concentrations titrated to 100 µM turnercyclamycin A, turnercyclamycin B, and colistin is 100 µM LPS.
Fig. 3
Fig. 3. Effects of LPS, lipids, and OMVs on lipopeptide activity.
a MIC90s of colistin, turnercyclamycin A, and turnercyclamycin B against A. baylyi WT, ∆lptD, ∆lptE, and ∆lpxA strains. *indicates concentration ≥32 µg/mL. Darker shades of blue show a higher MIC90 value while lighter blue shows a lower MIC90 value. Experiment was done with two biological replicates. b OMVs decrease the potency of turnercyclamycins, but not colistin. MIC90s of colistin, turnercyclamycin A, and turnercyclamycin B combined with 9.62 × 108 OMV particles/mL/well against A. baumannii ATCC 19606. Light red indicates lower MIC90 and dark red indicates higher MIC90. *indicates concentration ≥32 µg/mL. Experiment was done with two biological replicates. In contrast to the other panels in this figure, the dose of compounds is varied in these experiments. c Percent inhibition of colistin at 4 μg/mL upon interaction with an increased amount of OMVs, against A. baumannii ATCC 19606. 1× is 9.62 × 108 OMV particles/mL/well. Experiment was done with three biological replicates. * indicates p < 0.05; ** indicates p < 0.01. d Percent inhibition of turnercyclamycin A and turnercyclamycin B at 8 μg/mL upon interaction with a reduced amount of OMVs, against A. baumannii ATCC 19606. 1× is 9.62 × 108 OMV particles/mL/well. Experiment was done with three biological replicates. e Turnercyclamycin analogs are detected in isolated OMVs. Extracted ion chromatogram highlighting the presence of turnercyclamycin signal in OMVs isolated from turnercyclamycin A and turnercyclamycin B treated ∆tolA E. coli BW25113. f MIC90 fold change of turnercyclamycin A, turnercyclamycin B, and colistin after combination with various phospholipids against E. coli C600. The phospholipids used are as follows: cardiolipin (CL); hexanoylceramide (HC); phosphatidic acid (PA); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylglycerol (PG). A * indicates fold change ≥8. Experiment was done with two biological replicates.
Fig. 4
Fig. 4. Turnercyclamycins and colistin induce cellular damages to both WT and ΔlptD A. baylyi.
Representative transmission electron micrographs of untreated, colistin, turnercyclamycin A, and turnercyclamycin B-treated WT and ΔlptD A. baylyi. Images shown are either magnified at ×6000 or ×12,000. The experiment was done once using duplicates for each sample.
Fig. 5
Fig. 5. Lipopeptide antibiotics alter phospholipid homeostasis differently.
a Partial least squares-discriminant analysis (PLS-DA) plot of phospholipid composition of untreated, colistin, turnercyclamycin A, and turnercyclamycin B-treated WT and ∆mlaA E. coli BW25113 taken from analysis by MetaboAnalyst 6.0. A red line divides the colistin treatments from turnercyclamycin treatment and ΔmlaA. Turnercyclamycins A and B treatment is at 8 µg/mL and colistin is at 2 µg/mL. b–f Volcano plot analysis showing significant lipids at a cut off value fold change (FC) = 1.5 and p-value = 0.05. P-value was calculated via MetaboaAnalyst 6.0 statistical package using two-sided t-test. Groups compared are b turnercyclamycin A-treated WT cells vs. turnercyclamycin B-treated WT cells; c turnercyclamycin A-treated ΔmlaA cells vs. turnercyclamycin B-treated ΔmlaA cells; d untreated ΔmlaA cells vs. untreated WT cells; e turnercyclamycin A-treated WT cells vs. untreated ΔmlaA cells; f turnercyclamycin B-treated WT cells vs. untreated ΔmlaA cells.
Fig. 6
Fig. 6. Schematic of cellular changes induced in Gram-negative bacteria after treatment with turnercyclamycins.
(Created in BioRender. Lim, A. (2025) https://BioRender.com/sfza2um.).
See this image and copyright information in PMC

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