Differential membrane lipid disruption by lipopeptide antibiotics, colistin and turnercyclamycins

Domain: chemistry
Published: 2026-01-19
Editorial outcome: major_revision

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.

Introduction
A current estimate is that by 2050, antimicrobial-resistant (AMR) infections will cause 39 million deaths annually1. Pathogenic bacteria are associated with over 550,000 neonate deaths worldwide yearly, of which Gram-negative bacteria Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii are classified as urgent concerns2,3. The lipopeptide colistin is a last-line treatment for these infections, but increasing resistance, dose-limiting toxicity, and a limited antimicrobial spectrum threaten its use4–6. For that reason, there has been significant effort both in discovering new lipopeptide natural products and in making better colistin analogs7–11. Several of these are currently in clinical trials12–14. However, an incomplete understanding of mechanism of action, resistance, and toxicity hinders development efforts.

Turnercyclamycin lipopeptides are similar to colistin in that they require biosynthesis of lipopolysaccharide (LPS) for their mechanism of action, as ΔlpxC knockouts of Acinetobacter, one of the few Gram-negative pathogens that can survive without LPS, are fully resistant to the agents15–17. However, turnercyclamycins retain activity against colistin-resistant Acinetobacter strains without the cytotoxicity and hemolytic activity that is common in lipopeptides. Turnercyclamycins are active against K. pneumoniae, E. coli, and A. baumannii in vitro and controlled infection in an A. baumannii mouse model. Moreover, turnercyclamycins A and B and colistin exhibit very different resistance profiles; turnercyclamycins are active against colistin-resistant mcr-1 strains, while turnercyclamycin B is uniquely blocked by mutations in outer membrane (OM) transporter MlaA. This resistance difference is striking because turnercyclamycins A and B differ simply by the presence of an additional ethylene in the middle of the lipid chain. All three agents are inhibited by aminoarabinosylation of LPS.

Although seemingly straightforward, the colistin/polymyxin mechanism of action remains enigmatic4,5. Polycationic residues in colistin interact with the negatively charged phosphate groups of the outer membrane (OM) lipopolysaccharide (LPS), displacing divalent cations, causing membrane weakness, lysis and cell death5,6,18. Many antimicrobial peptides damage the OM19–22, causing rapid lysis and early onset of cell death23,24. After promoting OM changes, colistin rapidly damages the CM, which is thought to be its major mechanism of action25–27 (among other possible mechanisms28–33). For example, damage to P. aeruginosa was amplified in the presence of murepavadin, a drug that blocks P. aeruginosa LptD25. Without a complete mechanistic understanding, it has been challenging to define the problems facing lipopeptide drug development at the molecular level. We hypothesized that turnercyclamycins would similarly impact CM lipids or proteins. To test this hypothesis, here we measure the impact of colistin and turnercyclamycins A and B on E. coli membrane integrity using cellular dyes. Surprisingly, in contrast to colistin, little CM damage is found with turnercyclamycins, calling the hypothesis into question. Further, we use LPS transport mutants in the non-pathogenic model system Acinetobacter baylyi34–36 and transmission electron microscopy (TEM) to directly observe membrane effects. As expected, significant CM damage is observed with colistin, but again surprisingly, not with turnercyclamycins. To further investigate these observations, we directly measure the interactions of lipopeptides with a variety of lipids and also measure lipopeptide effects on the whole cell phospholipodome. Both colistin and turnercyclamycins A and B differently impact the phospholipid content of the bacterial cell, and the activity of each is differently affected by specific phospholipids. Further, direct binding of LPS to colistin is observed by isothermal titration calorimetry (ITC), but turnercyclamycins do not bind LPS in the assay. These results reveal a substantially different mechanism than other clinically used lipopeptides, emphasizing the promising potential of new lipopeptides as therapeutic agents.

Results
Significant data for polymyxin/colistin activity have been gathered over the course of nearly 80 years of rigorous study. Therefore, all experiments in this study compare the individual actions of test compounds turnercyclamycins A and B against those of colistin, enabling robust conclusions in comparison to prior literature in the field. We used three different bacterial species for this work: E. coli, A. baumannii, and A. baylyi.

Antimicrobial spectrum of action
In previous work, we showed that turnercyclamycins target E. coli, K. pneumoniae, and Acinetobacter strains (including colistin-resistant) at 1, 2, and 4–8 µg mL−1, respectively15. We previously tested turnercyclamycins against E. coli K strains. Here, we treated E. coli B strain BL21(DE3), which is notoriously resistant to colistin37, with colistin and turnercyclamycins A (lipid tail: myristic acid, C14:0) and B (lipid tail: palmitoleic acid, C16:1) (Fig. 1). These results show that turnercyclamycins are broadly effective (MIC90 = 2 µg mL−1) against E. coli, including strains such as mcr-1 and BL21(DE3) known to carry colistin resistance, while colistin was much less effective against BL21(DE3) (MIC90 = 8 µg mL−1) in comparison to E. coli K lines (MIC90 = 0.5 µg mL−1). We also tested the efficacy of turnercyclamycins against Pseudomonas aeruginosa. While colistin was active against P. aeruginosa, turnercyclamycins were not.

Density dependence of antibiotic activity
As found with most lipopeptides, the efficacies of both colistin and turnercyclamycins depend upon the starting density of bacterial culture. While this is sometimes considered to be a disadvantage, it also has an advantage: starting density assays provide a smooth range of conditions that are useful for mechanism of action studies. In initial assays using different concentrations of lipopeptides at different starting densities of culture, we found that applying a concentration of drugs four times higher than the MIC90 seemed potentially ideal. We therefore performed measurements in which lipopeptide concentrations in E. coli were fixed at 4× the MIC90, and the starting density of the inoculum was varied between OD600s of ~0.5 down to «0.01 (Supplementary Fig. 1). Turnercyclamycins and colistin exhibited 90% killing at starting inoculum OD600 0.004, and 0.015, respectively. Turnercyclamycins and colistin showed a gradual increase in percent of dead cells in a concentration range from OD600 0.5 until their 90% kill ODs. This enabled the drugs to be rigorously and reproducibly compared under identical starting OD600s in which the drugs had similar levels of impact on all cell types. Thus, most experiments below use 4× the MIC unless otherwise indicated. In order to ensure that there were still viable cells in experiments, the preceding experiments used starting OD600s that had a substantial population of viable cells treated at 4× MIC.

Colistin causes rapid OM and CM leakage in tandem with cell death, but turnercyclamycins do not
To determine the time-kill curve of turnercyclamycins, a fixed number of E. coli cells were treated with test compounds, incubated, and plated at different time points. Colistin showed the fastest killing curve with significant population reduction as early as 30 min with complete killing at 4 h (Fig. 2a). Turnercyclamycins exhibited a slower killing rate, with turnercyclamycin A significantly reducing cell counts at 6 h with complete inhibition at 10 h, while turnercyclamycin B achieved the lowest counts at 10 h (Fig. 2a).

OM permeability was investigated using N-phenyl-1-naphthylamine (NPN) dye, which increases in fluorescence as the OM becomes permeable38,39. As expected, colistin immediately increased OM permeability (Fig. 2b). Turnercyclamycins also caused increased membrane permeability, but at a slower pace and to a much lesser extent than found with colistin.

Damage to the E. coli CM was assessed by monitoring Sytox Green, whose fluorescence increases upon binding to DNA when the CM has been compromised40,41. The dye reflects direct CM permeabilization and damage, but not other possible effects such as alterations to membrane fluidity, domains, or transport. Colistin caused CM damage in a time course paralleling the killing curve, as shown by a large increase in fluorescence (Fig. 2c). However, turnercyclamycins showed a much lower readout, consistent with the minor fluorescence increase that occurs with delayed cell lysis. This suggested that substantial CM damage may not be a central part of turnercyclamycin mechanism of action (Fig. 2c).

Membrane depolarization in E. coli was also assessed using a potentiometric probe, 3,3’-dipropylthiadicarbocyanine iodide (DiSC3(5))42–44. Fluorescence microscopy of cells treated with DiSC3(5) revealed that, in comparison to controls, lipopeptide-treated cells were more fluorescent and retained fluorescence over a 2-h period (Fig. 2d and Supplementary Fig. 2), suggesting that the dye penetrated the cells, but membrane depolarization did not occur. Differences between turnercyclamycins and colistin are seen at early time points, when less fluorescence is observed in turnercyclamycin-treated cells.

Assessment of changes in Gram-negative bacterial membrane potential remains challenging42. The loss of DiSC3(5) fluorescence was not observed for treated samples, and based on previous observations by Buttress et al.42, the most likely explanation was that treatments caused the OM to become more permeable, allowing more dye to enter the periplasm. As such, further work is required to fully evaluate potential membrane depolarization events.

Unlike colistin, turnercyclamycins do not directly bind to LPS
The actions of colistin on both the OM and CM are thought to be potentiated by binding to LPS and related compounds4–6. We thus sought to determine turnercyclamycin binding affinity with LPS using isothermal titration calorimetry (ITC). Similar to previous reports45–47, we found that colistin binds to LPS with low micromolar affinity (KD 10.17 μM) (Fig. 2e). However, turnercyclamycins did not show any signs of binding to LPS up to the maximum obtainable experimental concentration of 100 μM (Fig. 2f). These data suggested that, rather than binding directly to LPS molecules as does colistin, turnercyclamycins impact earlier steps in the biosynthesis or transport of LPS or other lipids.

Depletion of LPS biosynthesis, but not transport, abolishes lipopeptide effectiveness
To determine how OM LPS affects the potency of turnercyclamycins, we targeted the Lpt system that transports LPS molecules synthesized in the CM to the OM48,49. lptD knockout mutants are thought to lack LPS in the OM50,51, while lptE encodes a protein complexed with LptD, which disaggregates LPS molecules before OM transport52. We inhibited LPT function in A. baylyi using knockout mutants in the lptD and lptE genes. Colistin and turnercyclamycins retained efficacy against both mutant strains (Fig. 3a and Supplementary Table S2), suggesting that OM LPS is not required for activity of either drug. We previously showed that LPS biosynthetic gene lpxC was required for both colistin and turnercyclamycin efficacy16. Here, we used knockout mutants of lpxA, encoding the first enzyme in LPS biosynthesis53,54. Both colistin and turnercyclamycins lost activity against lpxA mutants (Fig. 3a and Supplementary Table S2). Thus, it is clear that LPS synthesis but not transport is required for colistin and turnercyclamycin potency.

Turnercyclamycins remained potent against single transport and porin mutants
We tested whether single transporter mutants might impact transport of turnercyclamycins and colistin across membranes. A panel of 17 different porin and efflux pump-deficient E. coli and A. baumannii mutants were tested but had minimal effects on drug potency (Supplemental Figs. 3 and 4). This result indicates that the porin and efflux pump mutants tested were not solely responsible for lipopeptide drug transport.

TEM reveals cellular and membrane damage over a treatment time course
TEM was used to observe morphological changes caused by the lipopeptide antibiotics in A. baylyi wild type and ΔlptD cells over 24 h (Fig. 4 and Supplementary Fig. 5). Colistin damaged wild type cells within 5 h, a time point consistent with the time-kill curves shown in Fig. 2a. Observable damage included extensive cell lysis, increased membrane vesiculation, and membrane breaking. Colistin-induced damage was exacerbated in ΔlptD cells, with heavy damage observed as early as 1 h. Colistin treatment led to visibly damaged OM and CM. In ΔlptD cells, colistin caused obvious and extensive CM damage in cells that still had largely intact OM. Overall, these results provide further support to the literature in the field demonstrating that OM LPS is not central to colistin action.

When treated with turnercyclamycins A and B, cells lysed after 10 h and 12 h, respectively (Fig. 4 and Supplementary Fig. 5). This timing is much later than observed with colistin and is consistent with the time-kill curves in Fig. 2a. Unlike what was observed with colistin, CM damage could not be visualized under any condition over the time course, and instead, modest OM damage followed by complete cell lysis at later time points were observed. These differences in action reflect our findings using cell permeability dyes, in which colistin caused extensive CM permeability while turnercyclamycins did not. Additionally, it should be noted that only a relatively major disruption of membranes is easily visualized by this method.

Also noteworthy was that treated cells exhibited darkly stained granules associated with the CM. While these were found in all treatments, they were more noticeable in turnercyclamycin-treated cells. We cannot speculate from the data as to the identity of these granules.

Diverse phospholipid species and membrane constituents differentially modulate lipopeptide activity
In previous work, addition of LPS to culture media greatly decreased the efficacy of turnercyclamycins A and B, while lipid A had a lesser impact (both lipids added at 100 μg mL−1)16. By contrast, colistin was equally impacted by both lipid species. Above, we showed that LPS binds to colistin, but not to turnercyclamycins. Moreover, mutations of lipid transporter MlaA, which cycles mislocalized phospholipids out of the OM, caused resistance only to turnercyclamycin B, but not turnercyclamycin A or colistin16. Nonetheless, by multiple measures in this study, we showed that all three agents cause OM damage. Therefore, we sought to determine how exogenous lipids might more broadly impact the activity of the agents.

We first used outer membrane vesicles (OMV) as a proxy for OM lipids and proteins55. We isolated OMVs from a ΔtolA E. coli mutant, which produces high levels of OMVs that contain mostly OM content56,57. A fixed amount (109 mL−1) of OMVs was mixed with the compounds for periods derived from their time-kill kinetics. The resulting compound-OMV mixture was used as a test agent for the preceding antimicrobial assay using A. baumannii as the test pathogen. Both turnercyclamycins exhibited reduced potency after OMV exposure, with MIC90s increased by more than four-fold to >32 μg mL−1 (Fig. 3b and Supplementary Table S3). Colistin retained its potency in the presence of OMVs at the fixed concentration (Fig. 3b and Supplementary Table S3). Because these experiments required extensive amounts of limited compounds, we then switched to using variable OMV concentrations at fixed antibiotic concentrations. OMVs were used at concentrations between 0.16-4.0×109 mL−1. Colistin was inactivated in the presence of 3×109 mL−1, indicating that at higher concentrations, OMVs blocked colistin (Fig. 3c). By contrast, turnercyclamycins A and B were relatively inactive above an OMV concentration of 0.25×109 mL−1 (Fig. 3d), indicating that OMVs much more potently block turnercyclamycins (and especially turnercyclamycin B) in comparison to colistin. Similarly, a previous study showed that E. coli OMVs block colistin action58.

To determine whether turnercyclamycins were directly bound to vesicles, we treated ΔtolA E. coli mutants with both turnercyclamycin analogs and isolated the resulting OMVs. Both turnercyclamycin analogs were associated with the OMVs as determined by mass spectrometry (Fig. 3e). These data suggested that lipids in the OMV bind to turnercyclamycins.

Since turnercyclamycins did not bind to LPS as gauged by ITC, these findings prompted us to investigate the impact of other phospholipids on antibiotic efficacy. Phospholipids cardiolipin (CL) and phosphatidylglycerol (PG) impact the efficacy of colistin59–61. Moreover, the lipopeptide brevicidine B also binds to CL and PG, with nanomolar affinity62. Data showing that only turnercyclamycin B was affected by ΔmlaA mutation16 led us to test phosphatidylethanolamine (PE), a major lipid recycled from the OM by MlaA63,64. However, addition of PE to the culture media did not change the MICs of colistin or turnercyclamycins against E. coli C600 (Fig. 3f).

We combined commercially available phospholipids with antibiotics. Some of these phospholipids were pure compounds with defined acyl groups, while others were mixtures isolated from biological sources. We then used the resulting phospholipid/drug mixture in antimicrobial assays against E. coli C600 (Fig. 3f). Neither phosphatidylcholine (PC) nor hexanoylceramide (HC) impacted any compound. CL affected the potency of colistin but not turnercyclamycins, while phosphatidic acid (PA) reduced the effectiveness of both turnercyclamycins but not that of colistin. Interestingly, PG greatly decreased the potency of turnercyclamycin B specifically, with detectable but comparatively minimal impact on colistin. This implies that PG specifically interacts with turnercyclamycin B, which might be related to the sensitivity of the compound to ΔmlaA.

Lipidomic profiles of turnercyclamycin-treated cells resemble those of ΔmlaA mutants
To further define the impact of lipids on turnercyclamycins, we treated E. coli wild type and ΔmlaA cells with the drugs and compared them to untreated controls using a mass spectrometry-based phospholipidomics protocol. Partial least squares discriminant analysis (PLS-DA) clustering resulted in the triplicate replicates for each condition being correlated, reflecting the reproducibility of the experiment. Colistin-treated cells, both wild type and ΔmlaA, clustered separately from the remaining conditions (Fig. 5a). The much different growth and death characteristics of colistin- versus turnercyclamycin-treated cells made it difficult to interpret this difference. However, lipidomics has been performed previously using colistin-treated bacteria, revealing many significant differences reflecting the wide impact of colistin on bacterial lipid homeostasis65–68.

Remarkably, conditions lacking colistin were more closely clustered in the PLS-DA analysis (Fig. 5a). Among these, turnercyclamycin A- and B-treated WT cells and untreated ΔmlaA cells occupied a relatively similar space, with turnercyclamycin B-treated wild type cells overlapping the untreated ΔmlaA cell cluster, indicative of their strikingly similar lipidomic profiles. Colistin and turnercyclamycins modify lipid homeostasis in different ways, leading to different lipid profiles over the course of treatment. This reflects findings for turnercyclamycins and colistin, which each bind to and are inhibited by a different set of lipids, and each interacts differently with lipid-associated proteins such as MlaA.

Decrease in PG and diacylated phospholipids as a consequence of turnercyclamycin treatment
Upon closer comparison using volcano plots, three key trends were apparent (Fig. 5b–f and Supplementary Tables 4–8). First, the smallest number of significant differences was observed when comparing turnercyclamycin A- and turnercyclamycin B-treated samples, suggesting that both molecules have a similar impact on lipid homeostasis (Fig. 5b, c and Supplementary Tables 4 and 5). Second, in untreated ΔmlaA samples, monoacylated lipids were generally increased, while diacylated lipids were decreased when compared to untreated wild type cells (Fig. 5d and Supplementary Table 6). We speculate that this may be caused by a change in the regulation of lipid biosynthesis; for example, in completely acylated phospholipids that end up in the OM may no longer be recycled into the periplasm for further synthetic modification. The same trend, although to a lesser degree, was observed when turnercyclamycin-treated wild-type cells were compared with untreated ΔmlaA cells (Fig. 5e, f and Supplementary Tables 7 and 8). Third, PG was significantly decreased in turnercyclamycin-treated wild type cells compared with untreated ΔmlaA cells (Fig. 5e, f and Supplementary Tables 7 and 8), implying that PG might play a direct role in turnercyclamycin potency.

Methods
Bacterial strains
The bacterial strains used in this study are listed in Supplementary Table 1. Transposon mutant strains were generated in a previous study74. All strains were cryopreserved as single-use cultures at −80 °C.

Antibiotics
Colistin (J660915.03) was obtained from Thermo Scientific. Turnercyclamycins A and B were produced by fermentation in the shipworm symbiotic bacterial strain Teredinibacter turnerae T7901 and purified to homogeneity as previously described15. Their chemical identities and high levels of purity were demonstrated using HPLC, mass spectrometry, NMR, and other experiments as previously reported.

Antimicrobial assay (broth microdilution)
Assessment of antimicrobial activity by broth microdilution assay was performed following the Clinical and Laboratory Standards Institute (CLSI) M100 method with some modifications75. Cryopreserved strains were streaked onto Luria-Bertani (LB) agar for E. coli and Mueller–Hinton (MH) agar for A. baylyi, A. baumannii, and P. aeruginosa. The plates were then incubated at 37 °C for 8–12 h. Single colonies were subsequently transferred into LB or MH broth and incubated for 6–8 h at 37 °C with shaking at 150 rpm. For A. baylyi ΔlptD mutants, glycerol stocks were thawed and immediately suspended in M9 medium supplemented with 0.2% succinate and 10 μg/mL kanamycin and incubated for 18–24 h at 37 °C, 150 rpm before assay use. Cultures were adjusted to achieve a density corresponding to the 0.5 McFarland standard (approximately 1×108 cells/mL), diluted 200-fold, and used as the inoculum for the assays. Each well of a 96-well plate (Costar® 3474) received 200 µL of the test organisms, followed by the addition of compounds dissolved in 100% DMSO at a two-fold dilution scheme starting at 32 µg/mL, with eight dilutions per compound (for colistin, the solvent is 1× Phosphate buffered saline (PBS) and the highest dilution starts at 8 μg/mL). For E. coli knockouts and A. baylyi mutants, 10 μg/mL kanamycin was added, while for A. baumannii mutants, 10 μg/mL tetracycline was supplemented. The solvent (DMSO and 1× PBS) was maintained at 1%. The plates were then incubated at 37 °C, 150 rpm for 18–20 h. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, InvitrogenTM M6494, 10 µL of a 5 mg/mL solution in 1× PBS) was added to each well and incubated for 2 h. MTT formazan crystals were solubilized in 100 µL of DMSO for 1 h, and absorbance at 570 nm was measured using a Biotek-Synergy 2 Microplate Reader (Biotek). Each dilution was done in triplicate with three biological replicates unless stated otherwise. Percent inhibition was computed using the formula below:
% inhibition = (1 - (A570 test agent - A570 media) / (A570 solvent - A570 media)) x 100

Density dependence of drug effectiveness
Density dependence of drug effectiveness was assessed via the previously discussed broth microdilution antimicrobial assay with some modifications. The compound concentrations that were used were at 4× MIC against E. coli C600 (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B) and the starting inoculum was set at decreasing OD600 values, with the densest inoculum at OD600 = 0.5.

Time-kill assay
An overnight culture of E. coli C600 was diluted to ~106 CFU/mL. The resulting bacterial suspension was then treated with 4× MIC90 of the compounds (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B). They were then incubated for 0.5, 1, 2, 4, 6, 8, 10, 12, 17, and 24 h at 37 °C, 150 rpm, of which 400 µL aliquots were collected for each time point, spun down at 12,000×g for 2 min and resuspended in sterile 1× PBS. Aliquots (100 µL) of 10-fold serial dilutions were then made and plated on LB agar plates, followed by overnight incubation at 37 °C. The resulting colonies were then counted, and the resulting CFU/mL per experimental treatment was recorded. The experiment was performed in two biological replicates.

Outer membrane vesicle isolation and quantification
E. coli ΔtolA was grown in LB supplemented with 10 μg/mL kanamycin for 18–24 h. Afterward, outer membrane vesicles were isolated using SBI System Biosciences ExoBacteriaTM OMV Isolation Kit (EXO-BAC100A-1) following the manufacturer’s protocols using 25 mL of the bacterial suspension at OD600 = 0.8. Isolated OMVs were quantified using the SBI System Biosciences ExoELISA-ULTRA Complete Kit (EXEL-ULTRA-GroEL-1) following the manufacturer’s protocols.

OMV-test compounds co-treatment antimicrobial assay
Various OMV concentrations (1× = 9.62×108 particles/mL/well) were combined with various concentrations of the test compounds, followed by incubation at room temperature. For colistin, the incubation time was 2 h and for turnercyclamycin was 6 h. The mixture was used as a test treatment agent for the antimicrobial assay described above with A. baumannii ATCC 19606 as the test pathogen.

Turnercyclamycin detection in OMVs via mass spectrometry
An overnight culture of ΔtolA E. coli BW25113 grown in LB broth with 10 μg/mL kanamycin at 37 °C, 150 rpm, was diluted to an OD600 = 0.5. Afterward, 8 μg/mL turnercyclamycin A and B were added to the cell suspension and further incubated at 37 °C, 150 rpm for 20 h. Upon incubation, an OD600 = 0.8 suspension (10 mL) was used for OMV isolation and quantification following protocols described above. The isolated OMVs were lyophilized overnight. The lyophilized solids were extracted with 100 µL of methanol with gentle sonication for 1 h. Samples were centrifuged (13,000×g) for 10 min to remove particulates, and the sample was diluted 1:10 in methanol for LC-HRMS analysis. MS spectra were collected on a Waters Xevo G2-XS Q-ToF MS with an Acquity UPLC system using an Acquity UPLC BEH C18 column (1.8 µm, 2.1×50 mm). The column flow rate was 0.6 µL/min with mobile phase A as water with 0.1% formic acid and mobile phase B as acetonitrile with 0.1% formic acid. The gradient method consisted of a 0.1 min hold at 5% B, a linear gradient to 100% B over 7.4 min, a hold at 100% B for 1 min, a linear gradient back to 5% B over 0.4 min, and a re-equilibration at 5% B for 0.6 min. One microlitre of each sample was injected for analysis.

Membrane depolarization assay
Assessment of membrane depolarization was done following methods adapted from Buttress et al.42. E. coli C600 was grown in LB broth at 37 °C, 150 rpm until it reached an OD600 of 0.3. These were then transferred to 2 mL Eppendorf tubes with perforated lids and test compounds (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B) and 1% DMSO was added prior to incubation for 5 min, 55 min and 115 min, at 37 °C, 150 rpm. Once the chosen time point was reached, 1 µM of DiSC3(5) (InvitrogenTM D306) was added to the sample and incubated for 5 min. Five hundred microlitres of the sample was then placed on Teflon-coated multi-spot microscope slides (EprediaTM FIS 9991090) covered with 1.2% agarose in H2O. Imaging was done immediately on a Nikon Automated Widefield CCD Fluorescence Microscope. Resulting raw images were viewed and processed using Nikon NIS-Elements Viewer. Each treatment was tested in duplicates and two biological replicates. Fluorescence microscopy was done at the University of Utah Cell Imaging Core Facility.

Membrane permeability assay
E. coli C600 was cultured in LB broth at 37 °C with shaking at 150 rpm until reaching an OD600 of 0.5. The culture was subsequently centrifuged at 12,000×g for 5 min and washed twice with 1× PBS. The resulting pellet of cells was then suspended in M9 medium. NPN (Thermo Scientific 147160250) dye (10 µM) was then added to the suspension, followed by dispensation in a black, clear-bottom, 96-well plate. Afterwards, 4× MIC (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B) of the test compounds was added to the plate. Fluorescence was then read at 350/420 nm, at 2-min intervals for 5 h at 37 °C using a Molecular Devices SpectraMax M5 plate reader. One percent DMSO was used as a solvent control. Each test compound was tested in quadruplicate wells and two biological replicates.

Sytox green assay
E. coli C600 was cultured in LB broth at 37 °C with shaking at 150 rpm until it reached an OD600 of 0.5. The culture was then centrifuged at 12,000×g for 5 min and washed twice with 1× PBS. The resulting cell pellet was suspended in an M9 medium. Next, Sytox Green (InvitrogenTM S7020) (2 µM) was added to the suspension, which was transferred to a black, clear-bottom 96-well plate. Following this, the plate received test compounds at 4× MIC (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B). Fluorescence was measured at 488/523 nm at 2-min intervals over 20 h at 37 °C using a Molecular Devices SpectraMax M5 plate reader, with 1% DMSO serving as the solvent control. Each test compound was tested in quadruplicate wells and two biological replicates.

Transmission electron microscopy (TEM)
WT and ΔlptD A. baylyi were grown in LB broth and M9 medium supplemented with 0.2% succinate and 10 μg/mL kanamycin, respectively at 37 °C, 150 rpm overnight. Afterward, the culture was diluted with the appropriate media to an OD600 = 0.3. Test compounds were at 4× MIC (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B). This was then incubated at 37 °C, 150 rpm. Aliquots (2.0 mL) were then collected after 1, 2, 5, and 8 h for colistin-treated cells, 2, 5, 10, and 24 h for turnercyclamycin A-treated cells, and 2, 7, 12, and 24 h for turnercyclamycin B-treated cells. The aliquots were then spun down at 12,000×g for 15 min and washed thrice with 1× PBS. The resulting cell pellets were resuspended in 1.5 mL of fixative agent (2.5% glutaraldehyde, 1.0% paraformaldehyde). Upon fixation, the samples were repelleted, removing the fixative agent, and rinsed with 1× PBS. They were then postfixed with 2% osmium tetroxide for 1 h, followed by an H2O rinse and en bloc staining for 1 h. After staining, they were dehydrated with multiple washes of ethanol and acetone before infiltration with plastic. The blocks were then cut using a microtome with a diamond edge and placed in TEM grids. Finally, the samples were viewed and imaged using a JEOL JEM-1400 electron microscope. All electron microscopy and sample preparation were done at the University of Utah Electron Microscopy Core Laboratory. Imaging was done on two sets of samples treated with the compounds.

Micro-isothermal titration calorimetry
Micro-ITC used the Malvern MicroCal iTC system at 25 °C. Colistin, turnercyclamycin A, and turnercyclamycin B, dissolved in 1× PBS with 1.8% DMSO at a final concentration of 100 µM was placed in the sample cell chamber. LPS (Sigma-Aldrich L2018) dissolved in 1× PBS with 1.8% DMSO at 1000 µM was then placed in the injector and titrated at a rate of 1.8 µL at 3-min intervals. All samples were degassed prior to the start of interaction. The KD values were calculated using MicroCal’s ORIGIN software.

Sample preparation for lipidomics analysis
Wild type E. coli BW25113 and ΔmlaA E. coli strains were grown in LB broth supplemented with 10 μg/mL kanamycin at 37 °C, 150 rpm overnight. The bacterial suspension was adjusted to an OD600 = 0.3. Afterwards, 4× MIC (2 μg/mL colistin, 8 μg/mL turnercyclamycin A, 8 μg/mL turnercyclamycin B) was added to the bacterial suspensions and incubated at 37 °C, 150 rpm. Aliquots amounting to an OD600 = 0.5 was collected for each treated sample at the following time points: untreated, 20 h; turnercyclamycin A and B treated, 20 h; colistin treated, 3, 6, and 12 h. The suspension was pelleted at 12,000×g for 15 min and washed thrice with 1× PBS.

Sample extraction procedures followed the protocol from Matyash et al.76. Briefly, extraction was performed using a biphasic solvent system comprising cold methanol, methyl tert-butyl ether (MTBE), PBS, and water, with some adjustments. Each sample received 225 µL of methanol (MeOH) containing internal standards, followed by 750 µL of MTBE. The samples were sonicated for 60 s and then kept on ice with periodic vortexing for 1 h. To induce phase separation, 188 µL of PBS was added, followed by a 10-s vortex, a 15-min rest at room temperature, and centrifugation at 15,000×g for 10 min at 4 °C. The upper organic layer was collected, while the lower aqueous layer was re-extracted using 1 mL of a mixture containing 10:3:2.5 (v/v/v) MTBE/MeOH/dd-H2O. After brief vortexing, incubation at room temperature, and centrifugation under the same conditions, the upper phases were combined and dried in a SpeedVac. The lipid extracts were then reconstituted in 600 µL of 4:1:1 (v/v/v) IPA (isopropyl alcohol)/ACN (acetonitrile)/water and transferred into LC-MS vials for analysis. Simultaneously, a procedural blank sample was prepared, and pooled quality control (QC) samples were generated by combining equal volumes of each sample after final resuspension. The experiment was done on three technical replicates for each sample with one biological replicate.

Mass spectrometry and lipidomic analysis
Lipid extracts were separated using an Acquity UPLC CSH C18 column (2.1×100 mm; 1.7 µm) along with an Acquity UPLC CSH C18 VanGuard precolumn (5×2.1 mm; 1.7 µm) (Waters, Milford, MA). The system was maintained at 65 °C and linked to an Agilent HiP 1290 Sampler, Agilent 1290 Infinity pump, and an Agilent 6545 Accurate Mass Q-TOF dual AJS-ESI mass spectrometer (Agilent Technologies, Santa Clara, CA). Samples were analyzed in a random order in separate experiments for both positive and negative ionization modes, with a scan range of m/z 100–1700. In positive mode, the source gas temperature was set to 225 °C with a drying gas flow of 11 L/min, a nebulizer pressure of 40 psi, a sheath gas temperature of 350 °C, and a sheath gas flow of 11 L/min. The VCap voltage was 3500 V, the nozzle voltage 500 V, the fragmentor 110 V, the skimmer 85 V, and the octopole RF peak 750 V. In negative mode, the source gas temperature was 300 °C with a drying gas flow of 11 L/min, a nebulizer pressure of 30 psi, a sheath gas temperature of 350 °C, and a sheath gas flow of 11 L/min. The VCap voltage was 3500 V, the nozzle voltage 75 V, the fragmentor 175 V, the skimmer 75 V, and the octopole RF peak 750 V. Mobile phase A was ACN (60:40, v/v) with 10 mM ammonium formate and 0.1% formic acid, while mobile phase B was IPA:ACN (90:9:1, v/v/v) with 10 mM ammonium formate and 0.1% formic acid. For negative mode analysis, the modifiers were switched to 10 mM ammonium acetate. The chromatography gradient for both modes began at 15% mobile phase B, increased to 30% B over 2.4 min, then to 48% B from 2.4 to 3.0 min, then to 82% B from 3 to 13.2 min, then to 99% B from 13.2 to 13.8 min, held until 16.7 min, and then returned to the initial conditions for a 5-min equilibration. The flow rate was 0.4 mL/min throughout, with injection volumes of 5 µL for positive mode and 10 µL for negative mode. Tandem mass spectrometry used iterative exclusion with the same LC gradient and collision energies of 20 V and 27.5 V in positive and negative modes, respectively.

For data processing, Agilent MassHunter (MH) B.07.00 Workstation, along with the MH Qualitative and MH Quantitative software packages, was used. To ensure the reliability of the lipidomics data, pooled QC samples (n=8) and process blanks (n=4) were injected throughout the sample queue. Lipid annotation was performed using accurate mass and MS/MS matching with LipidMatch 3.177. Data exported from MH Quantitative was examined in Excel, where initial lipid targets were filtered based on specific criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples were included in the data analysis. Additionally, only lipids with background AUC counts in process blanks that were less than 30% of the QC counts were considered. The filtered Excel data tables were then normalized to the ratio of class-specific internal standards, adjusted to tissue mass, and summed prior to statistical analysis. Multivariate analysis was conducted using MetaboAnalyst 6.078. Statistical models were developed for the normalized data following logarithmic transformation (base 10) and Pareto scaling. The initial pass for the volcano plot applied a fold change (FC) cut-off of 1.5, an adjusted p-value cut-off of 0.05. The experiment was done on three replicates of each treatment.

Phospholipid-test compound co-treatment antimicrobial assay
Phospholipids cardiolipin (100 μg/mL) (Sigma CAR-201), phosphatidic acid (100 μg/mL) (Sigma P4013), phosphatidylglycerol (100 μg/mL) (Sigma P6412), phosphatidylcholine (100 μg/mL) (Sigma P3556), phosphatidylethanolamine (50 µg/mL) (European Pharmacopeia Y0001953), and hexanoylceramide (10 μg/mL) (Larodan 56-1049) were combined with varying concentrations of colistin, turnercyclamycin A, and turnercyclamycin B for 1 h. The resulting mixture was used as test compounds for the antimicrobial assay. Afterwards, the broth microdilution antimicrobial assay proceeded as described in the “Antimicrobial assay (broth microdilution)” section of “Methods” above, using E. coli C600 as the test pathogen. The experiment was done on two biological replicates.

Quantification and statistical analysis
MICs were plotted using GraphPad Prism 10.1.2. Error bars indicate mean ± SD of three replicate wells, with experiments conducted in either two or three biological replicates. In-panel text and axes were adjusted for readability in figures using Adobe Illustrator 2025.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Lim et al. present a comparative mechanistic study of the established lipopeptide drug colistin and two new lipopeptides, turnercyclamycin A and B. They set out to determine mechanistic differences between these lipopeptides with respect to outer and inner membrane damage. The authors found clearly distinct behavior of the turnercyclamycins in comparison with colistin and convincingly argue for distinct mechanisms of action of this new lipopeptide class. While the study is in principle interesting, it leaves many questions unanswered and does not provide a conclusive model for the mechanism of turnercyclamycins. Unfortunately, the paper also has several critical issues regarding study design and experimental execution that hamper the interpretability of the findings.\n\nMajor issues\n1. How were the antibiotic concentrations chosen? It seems that 4xMIC was selected arbitrarily. The authors show killing curves, but these are not sufficient for guiding concentration and treatment time selection as even just one log unit in CFU reduction will result in 90% dead cells in every assay. For studying antibiotic mechanisms, it is not appropriate to look at dead cells, as they only show the final result and do not allow assessment of the complex processes leading to cell death, i.e., the mode of action. Instead, the authors should have done acute shock assays, using growth experiments conducted under the same conditions used for their mechanistic experiments (here, log phase cells). Based on those, they should have selected a concentration (and treatment time) that visibly inhibits bacterial growth without leading to excessive killing and cell lysis. With lipopeptides, it is even often necessary to examine two concentrations as their window for growth inhibition without lysis is typically extremely narrow. Similarly, the treatment times, at least for colistin, are far too long to see the effects leading to inhibition/death, as evidenced by the killing kinetics shown in Figure 2A. At the very least, the authors need to perform acute shock assays with the chosen concentrations and up to the chosen treatment times. If these reveal that the majority of the cell population is already dead, the whole study is called into question and experiments would have to be repeated with suitable concentrations and timeframes to allow interpreting the findings correctly.\n2. The DiSC assay has multiple issues. Most importantly, it lacks essential controls and was not performed as would be appropriate for Gram-negative bacteria. In general, spectroscopic measurements of the membrane potential do not work well in Gram-negative organisms, at least not without eliminating the OM barrier first. Obviously, in most cases this is not the method of choice for OM-permeabilizing lipopeptides. Therefore, the assay should have been performed microscopically. The authors should read Buttress et al. (https://doi.org/10.1099/mic.0.001227) and perform DiSC microscopy accordingly. In brief, the uptake of DiSC is limited by the OM, which gives wild artifacts depending on the efficiency and kinetics of OM permeabilization by the tested drugs. Given the clear differences between colistin and turnercyclamicins in that regard, the DiSC assay as performed by the authors is inconclusive.\nAdditionally, the compounds were added before the baseline was stable. This is not correct. Also, the fact that the baseline does not stabilize is further evidence of the issues that Gram-negative bacteria pose in the DiSC assay.\nThe assay lacks vital controls for interaction between the compounds and the DiSC dye. This is very common, especially with peptides, and should always be assessed by recording fluorescence in solution with and without compounds.\nThe timeline of the assay is also problematic. DiSC is highly toxic upon prolonged exposure and assays should typically not exceed 30 min for that reason. In E. coli, the OM protects from this toxicity but upon OM disruption, the dye does become toxic, importantly at concentrations below those used by the authors. It is therefore likely that what the authors observe are combined effects of the lipopeptides with DiSC toxicity. What the authors should do, is a microscopic assay where they treat the cells, then withdraw aliquots at different timepoints, stain with DiSC for 10 min, and then examine fluorescence under the microscope (note that the readout in the microscope is opposite of the spectroscopic readout, see Buttress et al.).\nFurther, the timeline for colistin makes no sense. It appears that the cells are dying faster than they depolarize, which would indicate that depolarization is a secondary effect of cell death. This would go against what is know about colistin and is further evidence that there are issues with the DiSC assay as performed here.\nFurther, given the concentration issue pointed out before, it is pivotal to gain a population overview and not just a batch measurement, another argument for performing the assay microscopically instead of spectroscopically.\n3. The NPN results do not align with the DiSC results, likely due to the issues pointed out above. The turnercyclamycins do not even permeabilize the OM at the timepoint where they seem to depolarize the IM. This could fit with the authors’ interpretation, but could also be based on artifacts, as outlined above. Properly performing DiSC assays is therefore pivotal for the interpretation of the mechanism.\nLike the DiSC assay, also the NPN assay lacks peptide interaction controls. Just like DiSC, NPN is prone to interacting with different types of drugs, especially peptides, making these controls essential.\nAdditionally, the timelines of the DiSC and NPN assays do not line up. According to NPN, the turnercyclamycins reach their maximum OM permeabilization at 3-4 h, while the DiSC was only performed up to 2.5 h. It would be good to include measurements up to 4 h when repeating DiSC.\n4. The TEM results with ΔlptD are problematic. It is obvious from the untreated controls that the mutant strain has a generally reduced fitness. Additionally, Figure 4A shows differences in susceptibility compared to the WT. Yet, it does not seem that the treatment concentrations were adjusted accordingly. This can skew the results in either direction. Again, the authors should have performed acute shock assays with this strain to determine appropriate concentrations for this assay.\n5. Single transporter mutants do not say much as the compounds could travel through more than one and thus still have an available route in a single mutant. To make a reliable claim that the compounds are not taken up by porins, systematic testing of multiple mutant strains should have been done. I am aware that this sounds easier than it is in practice, but at the very least, the results need to be put into the right perspective by acknowledging this limitation. As for efflux pumps, however, this can be relatively easily tested by simply using de-energized cells (incubated in glucose-free buffer or treated with a depolarizing drug like CCCP).\n6. In OMV and lipid supplementation experiments, it seems that the authors have not included controls with only the antibiotics incubated under the same conditions as the antibiotic-OMV/lipid mixtures. This would be essential to truly attribute the observed effects to the presence of the different additives.\n7. The conclusions drawn from the lipidomics are very superficial, essentially just descriptions of similarities between groups without biological insight. Is it really not possible to go deeper into the analysis and provide a more thorough discussion and testable hypotheses?\n8. The discussion is too superficial and fails to address several interesting points. I would encourage the authors to think a bit further on their study. For example, a few thoughts that came to me:\nDo turnercyclamycins bind to LPS that is bound to the IM? Maybe they bind to intermediates or they need to form a complex with IM components that then allows binding to IM-bound LPS, or if not mediating direct binding, indirectly affects how it is processed and transported.\nHow would turnercyclamicins achieve any of the proposed mechanisms, if they neither interact with LPS nor use porins to enter the periplasm? How would they cross the OM/the outer OM leaflet to interact with the inner OM leaflet or enter the periplasm? Would that not be an argument that the porin single mutant screen is unreliable?\nIf the compounds would bind to the inner leaflet of the OM, would they not be effectively attenuated by pre-incubation with OMVs? Similarly, if they cross the OM to enter the periplasm, would they not enter OMVs? Would it not make sense to measure whether they are taken up into OMVs? Similarly, is it possible to make protein-free OM vesicles and compare them with native OMVs? This would give more conclusive insight into a possible uptake through porins.\nOther comments\n1. The title is not specific enough and suggests a higher degree of generality than the paper provides. It would be good to specify which lipopeptides were studied.\n2. In the second paragraph of the introduction, it would be good to mention that lpxC can be deleted in Acinetobacter and that is where the data are derived from. This information comes a little late in the paper and may cause confusion right off the bat with the non-expert reader.\n3. I would suggest being more careful with phrases stating that turnercyclamycins do not affect the IM, act IM-independently, etc. If there is initial depolarization, this is an effect, even if transient. Also, there are many other effects the compounds could have that the assays used in this paper would not capture and it would be an overstatement of the authors’ results to generalize their findings too much. I would advise to be more precise and only state what was really observed in this study (transient depolarization, no large-scale IM disruption visible in Sytox assays and TEM).\n4. Similarly, there are effects in the Sytox assay and, while the authors logically explain in the results that this is likely due to increased permeability after cell death/lysis, in other instances in the paper it sounds as if no effect was observed at all. I would suggest being more careful and using precise language to avoid misleading the reader.\n5. It would be good to clearly specify early on (abstract, introduction) whether lipidomics was performed on IM, OM, or whole cells.\n6. Throughout the text, please always specify whether you are talking about the inner or outer membrane to avoid unclarities.\n7. In the legend of Figure 1, please specify what the numbers in parentheses mean.\n8. The lines in Figure 2A and F are too thin and colors are virtually invisible. In 1F, I cannot see what is happening at all. Please use thicker lines.\n9. Figure 4 does not allow reading actual MIC values. It would be very useful to supply these as supplementary tables.\n10. In the section describing TEM, it would be appropriate to acknowledge that not all types of membrane damage can be seen in TEM (in fact only the most extreme can be seen).\n11. Likewise, it should be noted in the discussion that the IM assays performed here only inform about IM permeabilization. Any other effects (fluidity changes, phase separation, domain formation, protein delocalization, impairment of membrane-bound processes like respiration or membrane transport, etc.) remain elusive.\n12. I am curious about the finding that the compounds do not act on P. aeruginosa. Can the authors speculate on why that may be in the discussion?\n13. In the next to last paragraph of the discussion, correct ‘phosopholipid’ to phospholipid.", "reviewer_2": "I read the article \"Differential membrane lipid disruption by lipopeptide antibiotics\" with interest. I found the manuscript well written and detailed, with no fundamental flaws. The strength of the contribution lies in the combination of whole cell work, combined with some standard biophysical methods, such as isothermal titration calorimetry (ITC).\nThe study, however, raises some questions, some of them minor, that I think the authors might want to address before publication.\n1. The most striking of these is the observation that ΔlpxC knockouts lose sensitivity to turnercyclamycins. While this observation is not new, the authors specifically test for interaction with LPS by ITC – and find none. While it is possible that the free energy of binding to LPS is dominated by entropy and not enthalpy and thus not detectable by ITC, it is unlikely. Which leaves us with the observation that in the absence of lpxC, cells lose sensitivity to the agent, while not interacting with its product, LPS.\nOn line 63 in the MS, the authors state (and repeat throughout the MS) that turnercyclamycins require LPS for action. I don’t think that conclusion is warranted. The authors have shown that the turnercyclamycins require the presence of enzymes in the biosynthetic pathway to LPS for action, but not LPS, per se. It has been shown that lpxC interacts with a number of other proteins in the biosynthetic pathways of membrane lipids, including phospholipids (https://doi.org/10.3389/fmicb.2018.03285). The observation that that turnercyclamycins alter the cell phospholipid composition may be linked to this point. While a resolution of this question is obviously beyond the current MS, a discussion is warranted, I believe.\n2. Throughout the article, including in Fig 6, the authors assume that turnercyclamycins act on the OM. However, the membrane potential is established across the IM. While it is necessary for the lipopeptide to penetrate the OM, it must act on the IM to depolarize the cell, however transiently (many antimicrobial peptides are known to do this).\n3. This would also account for the clear binding of turnercyclamycins to PG, which is abundant in both the IM and the inner leaflet of the OM. In this context the attenuation of turnercyclamycin activity in the presence of PG vesicles and OMV is simply explained by a partitioning of the peptides between exogenous vesicles and the CM, leading to a smaller fraction of cell-bound peptide.\n4. L. 52: Minor point: it would be clearer to state that turnercyclamycin A is N-terminally amidated by myristic acid (C14:1), and turnercyclamycin B by palmitoleic acid (C16:1).", "reviewer_3": "Summary/key results\nThe manuscript represents a continuation of Dr. Schmidt’s work the two new cyclic lipopeptides, turnercyclamycins, previously isolated and structurally elucidated by members of the research group, which was found to have potent gram-negative activity. This is a comprehensive paper that describes an intriguing mode of action for a lipopeptide with structural features similar to colistin. This paper is well written and methodologically sound.\nMajor and minor issues:\n• Antibiotics section: I recommend specifying the stock concentrations and solvents used for each antibiotic, along with the final working concentrations and the percentage of DMSO present in the assays. This information is currently unclear, and since higher DMSO levels can significantly affect bacterial viability, it’s important to clarify these details. Indicate percentage purity of lipopeptides.\n• Antimicrobial assay: Please revise the term microbroth dilution to the correct and widely accepted term broth microdilution. Additionally, I suggest clearly listing the controls used in this assay—for example: sterile broth control, untreated cell control, and a DMSO-only control to account for any solvent-related effects on the cells. This clarification should be applied consistently throughout the Methods section.\n• Materials section: Consider a materials section in the methods (or combine with the antibiotics section) to specify which 96 well plates were used (as colistin generally required specific plates that the antibiotic won’t stick to) and which company the various reagents (such as MTT, NPN, DiSC3(5)) were purchased from.\n• Referencing standards: Please ensure that appropriate references are provided in the Methods section, for example for the broth microdilution assay, time-kill assays, and membrane depolarization assays.\n• Membrane depolarization assay: The initial incubation time of the cell suspension with DiSC₃(5) appears relatively short (10 minutes). Have you considered a longer pre-incubation period before adding the antibiotic? The initial decrease in fluorescence observed—even in untreated controls—may be due to incomplete dye equilibration, as DiSC₃(5) may still be intercalating into the membrane. In my experience, an incubation time of around 40 minutes has yielded more stable baseline fluorescence and clearer results.\n• Figures:\no Figure 1: Ensure that references 15 and 16 in the table are formatted as superscripts to avoid confusion with numerical data.\no Figure 2A and 2B: It is difficult to distinguish between the treatment and control lines—particularly between turB and the untreated samples. Consider using more distinct colors or line styles to improve clarity.\no Figure Order: Switch the graphs currently labeled as Figures 2D and 2B, as well as Figures 3 and 4, so that they follow the order in which they are discussed in the manuscript.\no Figure Quality: Improve the resolution of all figures. This may have been an issue during the submission process, but as it stands, the lower resolution makes it hard to differentiate the plotted lines, especially in Figures 2A, 2B, and 2F."}, "author_response": "Overview: We are grateful to the reviewers for their thorough and helpful reviews. All requests have been accommodated, and as a result we believe we have provided a high-quality product that will help to push the lipopeptides field forward. A summary of major changes is as follows. We now:\n• more fully describe the rigorous and exacting experiment that we performed to select assay OD and drug concentration conditions. This experiment defined a reproducible, 600 robust method, which is now described in its own results and methods sections and shown in a new Extended Data Figure 1;\n• more clearly show or describe controls in a revised Figure 2 and in the methods section, demonstrating that good controls were performed supporting this work;\n• describe the performance of newly requested DISC3(5) assays, causing us to revise the membrane depolarization section, adding new Figure 2D and new Extended Data Figure 2. We modified this section in the results, methods, discussion, and Figure 6;\n• clarify some misconceptions about our work by adding key sentences, modifying part of the introduction, rewriting a stretch of the discussion to be more concrete, and dividing the phospholipidomics results into two sections; and\n• add requested underlying data showing MIC90s for heat map panels in Extended Data Tables.\n\nReviewer #1 (Remarks to the Author):\nLim et al. present a comparative mechanistic study of the established lipopeptide drug colistin and two new lipopeptides, turnercyclamycin A and B. They set out to determine mechanistic differences between these lipopeptides with respect to outer and inner membrane damage. The authors found clearly distinct behavior of the turnercyclamycins in comparison with colistin and convincingly argue for distinct mechanisms of action of this new lipopeptide class. While the study is in principle interesting, it leaves many questions unanswered and does not provide a conclusive model for the mechanism of turnercyclamycins. Unfortunately, the paper also has several critical issues regarding study design and experimental execution that hamper the interpretability of the findings.\nMajor issues\n1. How were the antibiotic concentrations chosen? It seems that 4xMIC was selected arbitrarily. The authors show killing curves, but these are not sufficient for guiding concentration and treatment time selection as even just one log unit in CFU reduction will result in 90% dead cells in every assay. For studying antibiotic mechanisms, it is not appropriate to look at dead cells, as they only show the final result and do not allow assessment of the complex processes leading to cell death, i.e., the mode of action. Instead, the authors should have done acute shock assays, using growth experiments conducted under the same conditions used for their mechanistic experiments (here, log phase cells). Based on those, they should have selected a concentration (and treatment time) that visibly inhibits bacterial growth without leading to excessive killing and cell lysis. With lipopeptides, it is even often necessary to examine two concentrations as their window for growth inhibition without lysis is typically extremely narrow.\nWe did indeed run many experiments in order to choose the concentrations that we used in the paper, but these were not explicitly described in the first submission. In part because of the narrow window and resulting problems described by the reviewer, we exploited a different property of lipopeptides: their dependence upon the starting culture density. Using this property enabled us to provide very robust comparisons across compounds and cell types.\nIn response to review, we have now added those experiments to new sections in Results and Methods, and we show the results in in a new Extended Data Fig. 1. These experiments demonstrate that—at the selected antibiotic concentrations and the selected starting density (OD ) of bacterial cells—the OD s that were chosen for 600 600 these experiments did not kill a majority of cells over their time frames.\nSimilarly, the treatment times, at least for colistin, are far too long to see the effects leading to inhibition/death, as evidenced by the killing kinetics shown in Figure 2A. At the very least, the authors need to perform acute shock assays with the chosen concentrations and up to the chosen treatment times. If these reveal that the majority of the cell population is already dead, the whole study is called into question and experiments would have to be repeated with suitable concentrations and timeframes to allow interpreting the findings correctly.\nAs above, most cells were not dead under the conditions shown in Figures 2b-d. As a note added in proof, this phenomenon is very clearly observed as well in the new DiSC experiments performed in response to review, and we are grateful for your suggestion to try this (point 2 below).\n2. The DiSC assay has multiple issues. Most importantly, it lacks essential controls and was not performed as would be appropriate for Gram-negative bacteria. In general, spectroscopic measurements of the membrane potential do not work well in Gram-negative organisms, at least not without eliminating the OM barrier first. Obviously, in most cases this is not the method of choice for OM-permeabilizing lipopeptides. Therefore, the assay should have been performed microscopically. The authors should read Buttress et al. (https://doi.org/10.1099/mic.0.001227) and perform DiSC microscopy accordingly. In brief, the uptake of DiSC is limited by the OM, which gives wild artifacts depending on the efficiency and kinetics of OM permeabilization by the tested drugs. Given the clear differences between colistin and turnercyclamicins in that regard, the DiSC assay as performed by the authors is inconclusive.\nAdditionally, the compounds were added before the baseline was stable. This is not correct. Also, the fact that the baseline does not stabilize is further evidence of the issues that Gram-negative bacteria pose in the DiSC assay.\nThe assay lacks vital controls for interaction between the compounds and the DiSC dye. This is very common, especially with peptides, and should always be assessed by recording fluorescence in solution with and without compounds.\nThe timeline of the assay is also problematic. DiSC is highly toxic upon prolonged exposure and assays should typically not exceed 30 min for that reason. In E. coli, the OM protects from this toxicity but upon OM disruption, the dye does become toxic, importantly at concentrations below those used by the authors. It is therefore likely that what the authors observe are combined effects of the lipopeptides with DiSC toxicity. What the authors should do, is a microscopic assay where they treat the cells, then withdraw aliquots at different timepoints, stain with DiSC for 10 min, and then examine fluorescence under the microscope (note that the readout in the microscope is opposite of the spectroscopic readout, see Buttress et al.).\nFurther, the timeline for colistin makes no sense. It appears that the cells are dying faster than they depolarize, which would indicate that depolarization is a secondary effect of cell death. This would go against what is know about colistin and is further evidence that there are issues with the DiSC assay as performed here.\nFurther, given the concentration issue pointed out before, it is pivotal to gain a population overview and not just a batch measurement, another argument for performing the assay microscopically instead of spectroscopically.\nThanks for this suggestion. In response to review, we repeated the assay in the manner requested by the reviewer. The result was interesting and unexpected, and we have modified the text accordingly.\n3. The NPN results do not align with the DiSC results, likely due to the issues pointed out above. The turnercyclamycins do not even permeabilize the OM at the timepoint where they seem to depolarize the IM. This could fit with the authors’ interpretation, but could also be based on artifacts, as outlined above. Properly performing DiSC assays is therefore pivotal for the interpretation of the mechanism.\nLike the DiSC assay, also the NPN assay lacks peptide interaction controls. Just like DiSC, NPN is prone to interacting with different types of drugs, especially peptides, making these controls essential.\nAdditionally, the timelines of the DiSC and NPN assays do not line up. According to NPN, the turnercyclamycins reach their maximum OM permeabilization at 3-4 h, while the DiSC was only performed up to 2.5 h. It would be good to include measurements up to 4 h when repeating DiSC.\nThe membrane depolarization experiments were redone as requested and added to the manuscript (see above).\nIn addition, we did indeed previously perform the requested peptide interaction experiments, and in response to review have now added them to Figures 2b and 2c.\n4. The TEM results with ΔlptD are problematic. It is obvious from the untreated controls that the mutant strain has a generally reduced fitness. Additionally, Figure 4A shows differences in susceptibility compared to the WT. Yet, it does not seem that the treatment concentrations were adjusted accordingly. This can skew the results in either direction. Again, the authors should have performed acute shock assays with this strain to determine appropriate concentrations for this assay.\nThe lptD experiments were based upon a hypothesis that the CM would be damaged, leading to cell death (as was previously observed with colistin-treated Pseudomonas). We thus selected the concentrations and the starting cell densities for these experiments based upon the need to observe accruing cell damage over a period of hours, using a dense cell culture to perform the experiments as done in Figs 2b-d. Indeed, in Figure S4, this accruing damage can be readily observed over a period of hours; it is not immediate, and it accumulates. Moreover, the MIC determinations with these strains were normalized to DMSO-treated cells, accurately assessing the difference in survival.\nIn response to review, we have now more explicitly described the approach and the goals in the introduction and in the results sections, instead of merely describing our observations as in the original document. In addition, one thing that in retrospect was a bit confusing about this manuscript is that we assessed the CM hypothesis basically simultaneously using the E. coli dye experiments shown in Figs 2a and b and via TEM with A. baylyi. This is now more clearly explained early on in the introduction to try to avoid this misunderstanding.\n5. Single transporter mutants do not say much as the compounds could travel through more than one and thus still have an available route in a single mutant. To make a reliable claim that the compounds are not taken up by porins, systematic testing of multiple mutant strains should have been done. I am aware that this sounds easier than it is in practice, but at the very least, the results need to be put into the right perspective by acknowledging this limitation. As for efflux pumps, however, this can be relatively easily tested by simply using de-energized cells (incubated in glucose-free buffer or treated with a depolarizing drug like CCCP).\nWe did not make the claim attributed to us in this comment. We simply did the experiments and pointed out that none of the single mutants that we had access to affect the MIC90 of the compounds. This will be useful information for other work in the field.\nIn response to review, we explicitly acknowledge this limitation in the relevant results section.\n6. In OMV and lipid supplementation experiments, it seems that the authors have not included controls with only the antibiotics incubated under the same conditions as the antibiotic-OMV/lipid mixtures. This would be essential to truly attribute the observed effects to the presence of the different additives.\nIn response to review, we have run the requested OMV experiment and included that in the methods section. In terms of the lipid additive experiment, the control is that many different lipids were run under identical conditions, yet only 1-2 block the activity of any individual agent, demonstrating a lack of systematic issue.\n7. The conclusions drawn from the lipidomics are very superficial, essentially just descriptions of similarities between groups without biological insight. Is it really not possible to go deeper into the analysis and provide a more thorough discussion and testable hypotheses?\nOur analysis was exceptionally detailed and included a careful look at all changes indicated in the data. The reviewer missed our description of individual lipid classes that were modified during turnercyclamycin treatment, and that were highly relevant to potential mechanisms of action. In the initial submission, we presented the major, most significant findings, which revealed a similarity in effects by turnercyclamycins, with the most significant changes being consistent decrease in phosphatidylglycerol and in the diacylated:monoacylated lipid profiles.\nTo remedy this situation, we broke the results section into two: 1) comparison between subgroups; and 2) the analysis of individual changes provoked by turnercyclamycins into another. Further, we significantly altered the discussion section to highlight why those changes matter to the mechanism, with testable hypotheses.\n8. The discussion is too superficial and fails to address several interesting points. I would encourage the authors to think a bit further on their study. For example, a few thoughts that came to me:\nDo turnercyclamycins bind to LPS that is bound to the IM? Maybe they bind to intermediates or they need to form a complex with IM components that then allows binding to IM-bound LPS, or if not mediating direct binding, indirectly affects how it is processed and transported.\nWe have previously thought deeply about these areas and are engaged in testing the next hypotheses. We have tried in the revised discussion to give a more precise indication of what we are currently thinking.\nHow would turnercyclamicins achieve any of the proposed mechanisms, if they neither interact with LPS nor use porins to enter the periplasm? How would they cross the OM/the outer OM leaflet to interact with the inner OM leaflet or enter the periplasm? Would that not be an argument that the porin single mutant screen is unreliable?\nAs mentioned above, we did not intend the porin mutant data to be interpreted in this way. The results simply do not imply, at all, that porins are not involved in transport. What they rigorously and reproducibly demonstrate is that individual porin and transporter mutants that were available to us have virtually unchanged MICs in comparison to wild type, which is interesting data relevant to the field.\nWe wanted to report this conclusive data, which will be useful to future studies by others, but we cannot draw the further conclusions wished by the reviewer because of the limitations described by that reviewer.\nIf the compounds would bind to the inner leaflet of the OM, would they not be effectively attenuated by pre-incubation with OMVs? Similarly, if they cross the OM to enter the periplasm, would they not enter OMVs? Would it not make sense to measure whether they are taken up into OMVs? Similarly, is it possible to make protein-free OM vesicles and compare them with native OMVs? This would give more conclusive insight into a possible uptake through porins.\nThis is at the crux of what we want to do. We reported in our original submission that the compounds are attenuated by and found bound to OMVs during those incubation experiments, which is similar to what we might expect for the OM itself. Our idea to further test possibilities has been to make fluorescent turnercyclamyin analogs and to track them, which is work in progress (the idea is not as simple as it seems). In response to review, we now mention the fluorescent analogs in the discussion.\nOther comments\n1. The title is not specific enough and suggests a higher degree of generality than the paper provides. It would be good to specify which lipopeptides were studied.\nTitle have been changed to include turnercyclamycins and colistin.\n2. In the second paragraph of the introduction, it would be good to mention that lpxC can be deleted in Acinetobacter and that is where the data are derived from. This information comes a little late in the paper and may cause confusion right off the bat with the non-expert reader.\nAdded the lpxC comment immediately into the introduction.\n3. I would suggest being more careful with phrases stating that turnercyclamycins do not affect the IM, act IM-independently, etc. If there is initial depolarization, this is an effect, even if transient. Also, there are many other effects the compounds could have that the assays used in this paper would not capture and it would be an overstatement of the authors’ results to generalize their findings too much. I would advise to be more precise and only state what was really observed in this study (transient depolarization, no large-scale IM disruption visible in Sytox assays and TEM).\nSuch phrases have been omitted or changed to be more precise.\n4. Similarly, there are effects in the Sytox assay and, while the authors logically explain in the results that this is likely due to increased permeability after cell death/lysis, in other instances in the paper it sounds as if no effect was observed at all. I would suggest being more careful and using precise language to avoid misleading the reader.\nSame as above.\n5. It would be good to clearly specify early on (abstract, introduction) whether lipidomics was performed on IM, OM, or whole cells.\nAdded statement that lipidomics was done on whole cells.\n6. Throughout the text, please always specify whether you are talking about the inner or outer membrane to avoid unclarities.\nStatements regarding membrane have been specified and clarified.\n7. In the legend of Figure 1, please specify what the numbers in parentheses mean.\nFigure 1 has been changed to the proper citation format to indicate that the MIC values are from a former reference.\n8. The lines in Figure 2A and F are too thin and colors are virtually invisible. In 1F, I cannot see what is happening at all. Please use thicker lines.\nWe have improved the figures.\n9. Figure 4 does not allow reading actual MIC values. It would be very useful to supply these as supplementary tables.\nSupplementary tables have been added showing actual MIC values for Figure 4.\n10. In the section describing TEM, it would be appropriate to acknowledge that not all types of membrane damage can be seen in TEM (in fact only the most extreme can be seen).\nAcknowledgment of this has been added to the TEM result section.\n11. Likewise, it should be noted in the discussion that the IM assays performed here only inform about IM permeabilization. Any other effects (fluidity changes, phase separation, domain formation, protein delocalization, impairment of membrane-bound processes like respiration or membrane transport, etc.) remain elusive.\nAcknowledgment of SYTOX green limitations were added.\n12. I am curious about the finding that the compounds do not act on P. aeruginosa. Can the authors speculate on why that may be in the discussion?\nDiscussion on why the compounds are not killing P. aeruginosa has been added.\n13. In the next to last paragraph of the discussion, correct ‘phosopholipid’ to phospholipid.\nCorrection has been made.\n\nReviewer #2 (Remarks to the Author):\nI read the article \"Differential membrane lipid disruption by lipopeptide antibiotics\" with interest. I found the manuscript well written and detailed, with no fundamental flaws. The strength of the contribution lies in the combination of whole cell work, combined with some standard biophysical methods, such as isothermal titration calorimetry (ITC).\nThe study, however, raises some questions, some of them minor, that I think the authors might want to address before publication.\n1. The most striking of these is the observation that ΔlpxC knockouts lose sensitivity to turnercyclamycins. While this observation is not new, the authors specifically test for interaction with LPS by ITC – and find none. While it is possible that the free energy of binding to LPS is dominated by entropy and not enthalpy and thus not detectable by ITC, it is unlikely. Which leaves us with the observation that in the absence of lpxC, cells lose sensitivity to the agent, while not interacting with its product, LPS.\nOn line 63 in the MS, the authors state (and repeat throughout the MS) that turnercyclamycins require LPS for action. I don’t think that conclusion is warranted. The authors have shown that the turnercyclamycins require the presence of enzymes in the biosynthetic pathway to LPS for action, but not LPS, per se. It has been shown that lpxC interacts with a number of other proteins in the biosynthetic pathways of membrane lipids, including phospholipids (https://doi.org/10.3389/fmicb.2018.03285). The observation that that turnercyclamycins alter the cell phospholipid composition may be linked to this point. While a resolution of this question is obviously beyond the current MS, a discussion is warranted, I believe.\nWe removed the statement about LPS requirements. See response to reviewer 1 which addresses the rest of the concern.\nAs far as the cited reference, we actually came to a similar conclusion and have been working on that for some time, but you are right resolution is beyond the current manuscript. We more exactly described what we are thinking in a revised discussion.\n2. Throughout the article, including in Fig 6, the authors assume that turnercyclamycins act on the OM. However, the membrane potential is established across the IM. While it is necessary for the lipopeptide to penetrate the OM, it must act on the IM to depolarize the cell, however transiently (many antimicrobial peptides are known to do this).\nWe have revised this section (see response to reviewer 1) to repair this issue.\n3. This would also account for the clear binding of turnercyclamycins to PG, which is abundant in both the IM and the inner leaflet of the OM. In this context the attenuation of turnercyclamycin activity in the presence of PG vesicles and OMV is simply explained by a partitioning of the peptides between exogenous vesicles and the CM, leading to a smaller fraction of cell-bound peptide.\nThis is definitely possible and something that we are thinking about. We tried to put in as precise a statement as possible given the current data in the revised discussion. We hesitated to make such an exact statement in the absence of further data, as there are still other possiblities.\n4. L. 52: Minor point: it would be clearer to state that turnercyclamycin A is N-terminally amidated by myristic acid (C14:1), and turnercyclamycin B by palmitoleic acid (C16:1).\nStatements regarding the name of the acyl tail have been added.\n\nReviewer #3 (Remarks to the Author):\nSummary/key results\nThe manuscript represents a continuation of Dr. Schmidt’s work the two new cyclic lipopeptides, turnercyclamycins, previously isolated and structurally elucidated by members of the research group, which was found to have potent gram-negative activity. This is a comprehensive paper that describes an intriguing mode of action for a lipopeptide with structural features similar to colistin. This paper is well written and methodologically sound.\nMajor and minor issues:\n• Antibiotics section: I recommend specifying the stock concentrations and solvents used for each antibiotic, along with the final working concentrations and the percentage of DMSO present in the assays. This information is currently unclear, and since higher DMSO levels can significantly affect bacterial viability, it’s important to clarify these details. Indicate percentage purity of lipopeptides.\nConcentrations of both solvent and compounds have been added in the methodology section.\n• Antimicrobial assay: Please revise the term microbroth dilution to the correct and widely accepted term broth microdilution. Additionally, I suggest clearly listing the controls used in this assay—for example: sterile broth control, untreated cell control, and a DMSO-only control to account for any solvent-related effects on the cells. This clarification should be applied consistently throughout the Methods section.\n\"Broth microdilution\" is now used throughout. The formula used to compute for percent inhibition has been added to reflect all the necessary controls that were used for the experiments.\n• Materials section: Consider a materials section in the methods (or combine with the antibiotics section) to specify which 96 well plates were used (as colistin generally required specific plates that the antibiotic won’t stick to) and which company the various reagents (such as MTT, NPN, DiSC3(5)) were purchased from.\nMaterials section has been updated to indicate the origin of materials.\n• Referencing standards: Please ensure that appropriate references are provided in the Methods section, for example for the broth microdilution assay, time-kill assays, and membrane depolarization assays.\nReferences for the various assays have been added.\n• Membrane depolarization assay: The initial incubation time of the cell suspension with DiSC₃(5) appears relatively short (10 minutes). Have you considered a longer pre-incubation period before adding the antibiotic? The initial decrease in fluorescence observed—even in untreated controls—may be due to incomplete dye equilibration, as DiSC₃(5) may still be intercalating into the membrane. In my experience, an incubation time of around 40 minutes has yielded more stable baseline fluorescence and clearer results.\nMembrane depolarization experiments were now done using fluorescence microscopy as requested by reviewer 1. This led to a revision of related manuscript sections.\n• Figures:\no Figure 1: Ensure that references 15 and 16 in the table are formatted as superscripts to avoid confusion with numerical data.\nReferences have now been updated to match Nature Communications formatting.\no Figure 2A and 2B: It is difficult to distinguish between the treatment and control lines—particularly between turB and the untreated samples. Consider using more distinct colors or line styles to improve clarity.\nLines have been made thicker for better viewing. 2B has been changed to a fluorescence microscopy method.\no Figure Order: Switch the graphs currently labeled as Figures 2D and 2B, as well as Figures 3 and 4, so that they follow the order in which they are discussed in the manuscript.\nFigures have been switched to follow the order they were mentioned in the manuscript.\no Figure Quality: Improve the resolution of all figures. This may have been an issue during the submission process, but as it stands, the lower resolution makes it hard to differentiate the plotted lines, especially in Figures 2A, 2B, and 2F.\nHigh resolution figures are available and will be present in the final version."}, {"round": 2, "reviewer_comments": {"reviewer_3": "The manuscript has been greatly improved compared to the first submission. Valid assays were done to confirm phospholipid alteration and interaction, as well as membrane permeabilization. Indeed, there are still many unknowns in the MOA of turnercyclamycins with regards to what happens to the cells after outer membrane changes and permeability (i.e. the period between 5-10 h) that leads to cell death for future research.\n\nMain commnets:\nFigure 2c. You indicate ‘Turnercyclamycins do not cause inner membrane damage’. However, there is some increase in fluorescence over time compared to time 0 and the controls – is there a % limit or cut-off for this analysis that it is considered as ‘no damage’?\n\nThe DiSC(3)5 depolarization assay displays more accurate baseline/controls, and controls of other assay were included into the manuscript methods section. However, the results are somewhat contradictory, given that despite inner membrane damage, the depolarization assay indicated no change. In general, if the inner and outer membranes are damaged (causing permeabilization) as suggested in the TEM analysis, NPN and Sytox green assays, there should be a loss of the electrical potential (Δψ) across the membrane, especially for the control colistin that had significant inner membrane damage (Sytox assay).\n\nThe following statement in the discussion needs clarification: ‘DiSC3(5) assays suggest permeabilization of the OM as a potential common feature of the drugs.’ DiSC3(5) assay indicates depolarization rather than permeabilisation – please update this in the discussion and clarify what is meant.\n\nMinor comments:\nIn the discussion, ‘models’ should not be italized after P. aeruginosa in the second paragraph.\nResults section for figure 3f – include mentioning in text which strain the compounds were tested against to help the reader check track [i.e. ‘We then used the resulting phospholipid/drug mixture in antimicrobial assays against E. coli C600 (Fig. 3f)]. Neither phosphatidylcholine (PC) nor hexanoylceramide (HC) impacted the potency of any compound’]."}, "author_response": "Overview: We are grateful for the thoughtful review and the chance to further improve the manuscript. We have positively and completely addressed all remaining comments in modest changes that are highlighted on manuscript lines 148, 150, 252, 256, 321, 325, and 902. These changes clarify the meaning (we agree with the reviewer in all respects) but do not alter the interpretations or conclusions. Finally, this submission is consistent with all editorial requests/requirements.\n\nReviewer #3 (Remarks to the Author):\nComments for Author\nThe manuscript has been greatly improved compared to the first submission. Valid assays were done to confirm phospholipid alteration and interaction, as well as membrane permeabilization. Indeed, there are still many unknowns in the MOA of turnercyclamycins with regards to what happens to the cells after outer membrane changes and permeability (i.e. the period between 5-10 h) that leads to cell death for future research.\n\nMain commnets:\nFigure 2c. You indicate ‘Turnercyclamycins do not cause inner membrane damage’. However, there is some increase in fluorescence over time compared to time 0 and the controls – is there a % limit or cut-off for this analysis that it is considered as ‘no damage’?\n• This statement has been corrected to “Turnercyclamycins cause significantly less inner membrane damage compared to colistin”.\nThe DiSC(3)5 depolarization assay displays more accurate baseline/controls, and controls of other assay were included into the manuscript methods section. However, the results are somewhat contradictory, given that despite inner membrane damage, the depolarization assay indicated no change. In general, if the inner and outer membranes are damaged (causing permeabilization) as suggested in the TEM analysis, NPN and Sytox green assays, there should be a loss of the electrical potential (Δψ) across the membrane, especially for the control colistin that had significant inner membrane damage (Sytox assay).\n• Buttress and co-workers (the same manuscript referenced by reviewers) point out that, in the context of OM permeabilization, CM hyperpolarization cannot be ruled out, although they emphasized the likelihood of increased disc3(5) diffusion. This idea influenced our interpretation of the data.\n• In response to review, we have added the following: \"Assessment of changes in Gram-negative bacterial membrane potential remains challenging. The loss of DiSC3(5) fluorescence was not observed for treated samples, and based on previous observations by Buttress et al42, the most likely explanation was that treatments caused the OM to become more permeable, allowing more dye to enter the periplasm. As such, further work is required to fully evaluate potential membrane depolarization events.\"\nThe following statement in the discussion needs clarification: ‘DiSC3(5) assays suggest permeabilization of the OM as a potential common feature of the drugs.’ DiSC3(5) assay indicates depolarization rather than permeabilisation – please update this in the discussion and clarify what is meant.\n• This statement has been clarified to read: “Increased NPN and DiSC3(5) fluorescence upon drug treatment, most likely due to increased uptake after OM damage, suggests permeabilization of the OM as a potential common feature of the drugs.”\nMinor comments:\nIn the discussion, ‘models’ should not be italized after P. aeruginosa in the second paragraph.\n• Fixed.\nResults section for figure 3f – include mentioning in text which strain the compounds were tested against to help the reader check track [i.e. ‘We then used the resulting phospholipid/drug mixture in antimicrobial assays against E. coli C600 (Fig. 3f)]. Neither phosphatidylcholine (PC) nor hexanoylceramide (HC) impacted the potency of any compound’].\n• Fixed."}]

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