Summary of Study ST004466

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002820. The data can be accessed directly via it's Project DOI: 10.21228/M84Z8T This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004466
Study Title	Differential mechanisms of membrane lipid disruption by antibiotic lipopeptides colistin and turnercyclamycins
Study Summary	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. To confirm this analysis, we performed phospholipidomic profiling on whole cells, which identified alterations in bacterial lipid biosynthesis and membrane homeostasis. Phospholipidomics experiments were performed using wild type E. coli BW25113 and a knockout in the same strain in which phospholipid transporter mlaA was deleted. Each strain was treated with a dose of antibiotics that left the majority of cells alive during the time course of the study. Strains were treated with no drug, colistin, turnercyclamycin A, or turnercyclamycin B, collected, and analyzed in an LC-MS phospholipidomics platform in triplicate replicates per condition. The major lipids were analyzed. Colistin and turnercyclamycins exhibited very different effects on the phospholipidome, while turnercyclamycins had a similar impact to deleting mlaA. A decrease in PG lipids was the major change in turnercyclamycin treatment. Results 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.
Institute	University of Utah
Last Name	Schmidt
First Name	Eric
Address	30 South 2000 East Room 307 Salt Lake City UT 84112
Email	ews1@utah.edu
Phone	+1-801-585-5234
Submit Date	2025-12-11
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2026-01-06
Release Version	1

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Project:

Project ID:	PR002820
Project DOI:	doi: 10.21228/M84Z8T
Project Title:	Microbial Ecology-Guided Discovery of Antibacterial Drugs
Project Summary:	The project aims to discover antibiotics and characterize their mechanisms of action. We investigate proteobacteria that live symbiotically with animals as the source of the antibiotics. Specifically, bacteria live in the gills of shipworm mollusks, providing the cellulases needed for the mollusks to consume wood in the sea. The bacteria also contain large numbers of biosynthetic pathways for potential antibiotics, at least some of which are secreted into the animals where they likely act to impact the symbiosis. Because the antibiotics are made and secreted on animals, they are less likely to have direct impacts on animal cells. Among early hit compounds discovered, turnercyclamycins are lipodedpsipeptides that kill many multidrug-resistant, Gram-negative pathogens with clinical relevance. One of the goals of our project is to understand how turnercyclamycins work to kill bacteria that are resistant to other lipopeptide antibiotics, such as colistin. Here, we have studied this by comparing the impacts of colistin and turneryclamycin on bacterial membranes and the phospholipidome. By performing these and related studies on antibiotics produced by symbiotic bacteria, their mechanisms of action are unveiled.
Institute:	University of Utah
Last Name:	Schmidt
First Name:	Eric
Address:	30 South 2000 East Room 307 Salt Lake City UT 84112
Email:	ews1@utah.edu
Phone:	+1-801-585-5234

Subject:

Subject ID:	SU004642
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Variable
SA531758	neg36	col_treated_for12h_mlaako_neg_mode	E. coli mlaa knockout
SA531759	neg34	col_treated_for12h_mlaako_neg_mode	E. coli mlaa knockout
SA531760	neg35	col_treated_for12h_mlaako_neg_mode	E. coli mlaa knockout
SA531761	pos35	col_treated_for12h_mlaako_pos_mode	E. coli mlaa knockout
SA531762	pos36	col_treated_for12h_mlaako_pos_mode	E. coli mlaa knockout
SA531763	pos34	col_treated_for12h_mlaako_pos_mode	E. coli mlaa knockout
SA531764	neg31	col_treated_for12h_wt_neg_mode	E. coli wild type
SA531765	neg33	col_treated_for12h_wt_neg_mode	E. coli wild type
SA531766	neg32	col_treated_for12h_wt_neg_mode	E. coli wild type
SA531767	pos33	col_treated_for12h_wt_pos_mode	E. coli wild type
SA531768	pos32	col_treated_for12h_wt_pos_mode	E. coli wild type
SA531769	pos31	col_treated_for12h_wt_pos_mode	E. coli wild type
SA531770	neg23	col_treated_for3h_mlaako_neg_mode	E. coli mlaa knockout
SA531771	neg22	col_treated_for3h_mlaako_neg_mode	E. coli mlaa knockout
SA531772	neg24	col_treated_for3h_mlaako_neg_mode	E. coli mlaa knockout
SA531773	pos23	col_treated_for3h_mlaako_pos_mode	E. coli mlaa knockout
SA531774	pos22	col_treated_for3h_mlaako_pos_mode	E. coli mlaa knockout
SA531775	pos24	col_treated_for3h_mlaako_pos_mode	E. coli mlaa knockout
SA531776	neg21	col_treated_for3h_wt_neg_mode	E. coli wild type
SA531777	neg19	col_treated_for3h_wt_neg_mode	E. coli wild type
SA531778	neg20	col_treated_for3h_wt_neg_mode	E. coli wild type
SA531779	pos20	col_treated_for3h_wt_pos_mode	E. coli wild type
SA531780	pos19	col_treated_for3h_wt_pos_mode	E. coli wild type
SA531781	pos21	col_treated_for3h_wt_pos_mode	E. coli wild type
SA531782	neg28	col_treated_for6h_mlaako_neg_mode	E. coli mlaa knockout
SA531783	neg29	col_treated_for6h_mlaako_neg_mode	E. coli mlaa knockout
SA531784	neg30	col_treated_for6h_mlaako_neg_mode	E. coli mlaa knockout
SA531785	pos28	col_treated_for6h_mlaako_pos_mode	E. coli mlaa knockout
SA531786	pos29	col_treated_for6h_mlaako_pos_mode	E. coli mlaa knockout
SA531787	pos30	col_treated_for6h_mlaako_pos_mode	E. coli mlaa knockout
SA531788	neg25	col_treated_for6h_wt_neg_mode	E. coli wild type
SA531789	neg26	col_treated_for6h_wt_neg_mode	E. coli wild type
SA531790	neg27	col_treated_for6h_wt_neg_mode	E. coli wild type
SA531791	pos27	col_treated_for6h_wt_pos_mode	E. coli wild type
SA531792	pos26	col_treated_for6h_wt_pos_mode	E. coli wild type
SA531793	pos25	col_treated_for6h_wt_pos_mode	E. coli wild type
SA531794	neg11	tura_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531795	neg12	tura_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531796	neg10	tura_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531797	pos12	tura_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531798	pos11	tura_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531799	pos10	tura_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531800	neg7	tura_treated_wt_neg_mode	E. coli wild type
SA531801	neg8	tura_treated_wt_neg_mode	E. coli wild type
SA531802	neg9	tura_treated_wt_neg_mode	E. coli wild type
SA531803	pos8	tura_treated_wt_pos_mode	E. coli wild type
SA531804	pos7	tura_treated_wt_pos_mode	E. coli wild type
SA531805	pos9	tura_treated_wt_pos_mode	E. coli wild type
SA531806	neg16	turb_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531807	neg17	turb_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531808	neg18	turb_treated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531809	pos17	turb_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531810	pos16	turb_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531811	pos18	turb_treated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531812	neg14	turb_treated_wt_neg_mode	E. coli wild type
SA531813	neg15	turb_treated_wt_neg_mode	E. coli wild type
SA531814	neg13	turb_treated_wt_neg_mode	E. coli wild type
SA531815	pos15	turb_treated_wt_pos_mode	E. coli wild type
SA531816	pos14	turb_treated_wt_pos_mode	E. coli wild type
SA531817	pos13	turb_treated_wt_pos_mode	E. coli wild type
SA531818	neg6	untreated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531819	neg5	untreated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531820	neg4	untreated_mlaa ko_neg_mode	E. coli mlaa knockout
SA531821	pos5	untreated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531822	pos6	untreated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531823	pos4	untreated_mlaa ko_pos_mode	E. coli mlaa knockout
SA531824	neg1	untreated_wt_neg_mode	E. coli wild type
SA531825	neg3	untreated_wt_neg_mode	E. coli wild type
SA531826	neg2	untreated_wt_neg_mode	E. coli wild type
SA531827	pos2	untreated_wt_pos_mode	E. coli wild type
SA531828	pos3	untreated_wt_pos_mode	E. coli wild type
SA531829	pos1	untreated_wt_pos_mode	E. coli wild type

Showing results 1 to 72 of 72

Showing results 1 to 72 of 72

Collection:

Collection ID:	CO004635
Collection Summary:	This study used compounds colistin, turnercyclamycin A, and turnercyclamycin B; and bacteria Escherichia coli BW25113 ("wild type") and Escherichia coli BW25113 ∆mlaA ("knockout"). Compound source and storage. Colistin was purchased from Thermo (J660915.03). Turnercyclamycins A and B were obtained by fermentation of Teredinibacter turnerae T7901 and purified to homogeneity using chemical and multistep chromatographic methods. The purity of compounds was assessed using LC coupled to diode array and high-resolution mass spec detectors, as well as by nuclear magnetic resonance experiments. Compounds were stored as dried powders at -20 C, while dilutions in DMSO were stored at -20 C. Stability of compounds was assessed by HPLC analysis, demonstrating long-term stability under storage conditions. Bacterial source and storage. Bacteria were obtained from the Keio Collection, stored as glycerol stocks at -80 C, and freshly plated the night before use in experiments. Cell growth and collection for phospholipidomics experiments. Growth of E. coli strains in the presence or absence of antibiotics was maintained for no more than 20 hours. Cells were pelleted by centrifiguation at the termination of the experiment and stored at -80 C until analysis.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR004651
Treatment Summary:	Wild type and mlaA knockout E. coli were treated either with DMSO control, colistin, turnercyclamycin A, or turnercyclamycin B. MlaA is a protein that transports phospholipids in the Gram-negative bacterial outer membrane. In this work, we employed mlaA knockouts, sometimes referred to here as "mlaa" or "mlaako". In turn, the antibiotics are referred to as "col" (colistin), "tura" (turnercyclamycin A), and "turb" (turnercyclamycin B).

Treatment ID:

TR004651

Treatment Summary:

Wild type and mlaA knockout E. coli were treated either with DMSO control, colistin, turnercyclamycin A, or turnercyclamycin B. MlaA is a protein that transports phospholipids in the Gram-negative bacterial outer membrane. In this work, we employed mlaA knockouts, sometimes referred to here as "mlaa" or "mlaako". In turn, the antibiotics are referred to as "col" (colistin), "tura" (turnercyclamycin A), and "turb" (turnercyclamycin B).

Sample Preparation:

Sampleprep ID:	SP004648
Sampleprep Summary:	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 sec 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-sec 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.

Sampleprep ID:

SP004648

Sampleprep Summary:

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 sec 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-sec 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.

Combined analysis:

Analysis ID	AN007487	AN007488
Chromatography ID	CH005678	CH005678
MS ID	MS007183	MS007184
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity pump	Agilent 1290 Infinity pump
Column	Acquity UPLC CSH C18 column (2.1 x 100 mm; 1.7 µm)	Acquity UPLC CSH C18 column (2.1 x 100 mm; 1.7 µm)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	retention time	retention time

Chromatography:

Chromatography ID:	CH005678
Instrument Name:	Agilent 1290 Infinity pump
Column Name:	Acquity UPLC CSH C18 column (2.1 x 100 mm; 1.7 µm)
Column Temperature:	22 C
Flow Gradient:	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
Flow Rate:	0.4 mL/min
Solvent A:	ACN (60:40, v/v) with 10 mM ammonium formate and 0.1% formic acid
Solvent B:	IPA:ACN (90:9:1, v/v/v) with 10 mM ammonium formate and 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS007183
Analysis ID:	AN007487
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	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.
Ion Mode:	POSITIVE

MS ID:	MS007184
Analysis ID:	AN007488
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	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.
Ion Mode:	NEGATIVE