Membrane-Disruptive Effects of Fatty Acid and Monoglyceride Mitigants on E. coli Bacteria-Derived Tethered Lipid Bilayers

Abstract

We report electrochemical impedance spectroscopy measurements to characterize the membrane-disruptive properties of medium-chain fatty acid and monoglyceride mitigants interacting with tethered bilayer lipid membrane (tBLM) platforms composed of E. coli bacterial lipid extracts. The tested mitigants included capric acid (CA) and monocaprin (MC) with 10-carbon long hydrocarbon chains, and lauric acid (LA) and glycerol monolaurate (GML) with 12-carbon long hydrocarbon chains. All four mitigants disrupted E. coli tBLM platforms above their respective critical micelle concentration (CMC) values; however, there were marked differences in the extent of membrane disruption. In general, CA and MC caused larger changes in ionic permeability and structural damage, whereas the membrane-disruptive effects of LA and GML were appreciably smaller. Importantly, the distinct magnitudes of permeability changes agreed well with the known antibacterial activity levels of the different mitigants against E. coli, whereby CA and MC are inhibitory and LA and GML are non-inhibitory. Mechanistic insights obtained from the EIS data help to rationalize why CA and MC are more effective than LA and GML at disrupting E. coli membranes, and these measurement capabilities support the potential of utilizing bacterial lipid-derived tethered lipid bilayers for predictive assessment of antibacterial drug candidates and mitigants.

Keywords: antimicrobial; critical micelle concentration; electrochemical impedance spectroscopy; fatty acid; monoglyceride; tethered bilayer lipid membrane.

Figure 1

Overview of EIS-based antibacterial-mitigant-testing strategy. (A) Molecular structures of medium-chain fatty acids and monoglycerides (CA, LA, MC, GML) and surfactant control (SDS). These amphipathic molecules are dispersed as free monomers below their respective CMC values but begin self-assembling to form micelles at and above their respective CMC values. (B) Schematic illustration of tBLM platform consisting of reconstituted E. coli lipid extract that contains CL, PG, and PE lipids among various components. (C) Measurement concept based on (I) E. coli tBLM fabrication and (II) subsequent addition of test compound at defined bulk concentration. Time-resolved EIS measurements were performed to track changes in the electrical conductance (G_m) and capacitance (C_m) properties of the E. coli tBLM platform during the interaction process. The presented graphs are schematic illustrations for conceptual purposes and, thus, unitless without dimensions.

Figure 2

Time-resolved EIS measurements tracking effects of capric acid (CA) treatment on E. coli lipid-derived tBLM platform. (A) Conductance (G_m) and capacitance (C_m) signals are reported as a function of time for the addition of 16,000 μM CA (4× CMC) to tBLM platform at t = 10 min (arrow 1) and subsequent buffer rinsing step at t = 40 min (arrow 2). The baseline signals depict the tBLM platform prior to CA addition. (B) Bode plot snapshots for tBLM platform prior to CA addition and during CA treatment. (C–F) Corresponding data for 8000 μM CA (2× CMC) and 2000 μM CA (0.5× CMC) treatment cases. Graphs are representative from three independent runs.

Figure 3

Time-resolved EIS measurements tracking effects of lauric acid (LA) treatment on E. coli lipid-derived tBLM platform. (A) Conductance (G_m) and capacitance (C_m) signals are reported as a function of time for the addition of 4000 μM LA (4× CMC) to tBLM platform at t = 10 min (arrow 1) and subsequent buffer rinsing step at t = 40 min (arrow 2). The baseline signals depict the tBLM platform prior to LA addition. (B) Bode plot snapshots for tBLM platform prior to LA addition and during LA treatment. (C–F) Corresponding data for 2000 μM LA (2× CMC) and 500 μM LA (0.5× CMC) treatment cases. Graphs are representative from three independent runs.

Figure 4

Time-resolved EIS measurements tracking effects of monocaprin (MC) treatment on E. coli lipid-derived tBLM platform. (A) Conductance (G_m) and capacitance (C_m) signals are reported as a function of time for the addition of 2000 μM MC (4× CMC) to tBLM platform at t = 10 min (arrow 1) and subsequent buffer rinsing step at t = 40 min (arrow 2). The baseline signals depict the tBLM platform prior to MC addition. (B) Bode plot snapshots for tBLM platform prior to MC addition and during MC treatment. (C–F) Corresponding data for 1000 μM MC (2× CMC) and 250 μM MC (0.5× CMC) treatment cases. Graphs are representative from three independent runs.

Figure 5

Time-resolved EIS measurements tracking effects of glycerol monolaurate (GML) treatment on E. coli lipid-derived tBLM platform. (A) Conductance (G_m) and capacitance (C_m) signals are reported as a function of time for the addition of 250 μM GML (4× CMC) to tBLM platform at t = 10 min (arrow 1) and subsequent buffer rinsing step at t = 40 min (arrow 2). The baseline signals depict the tBLM platform prior to GML addition. (B) Bode plot snapshots for tBLM platform prior to GML addition and during GML treatment. (C–F) Corresponding data for 125 μM GML (2× CMC) and 31 μM GML (0.5× CMC) treatment cases. Graphs are representative from three independent runs.

Figure 6

Effects of fatty acid and monoglyceride mitigant treatment on the electrochemical properties of reconstituted tethered E. coli membranes. The summarized maximum shifts in electrical conductance (ΔG_m) are presented due to mitigant treatment at (A) 4× CMC, (B) 2× CMC, and (C) 2000 µM fixed concentration. The measurement data are reported as mean ± standard deviation from three independent replicates.