Host Defense Peptide Piscidin and Yeast-Derived Glycolipid Exhibit Synergistic Antimicrobial Action through Concerted Interactions with Membranes

Abstract

Developing new antimicrobials as alternatives to conventional antibiotics has become an urgent race to eradicate drug-resistant bacteria and to save human lives. Conventionally, antimicrobial molecules are studied independently even though they can be cosecreted in vivo. In this research, we investigate two classes of naturally derived antimicrobials: sophorolipid (SL) esters as modified yeast-derived glycolipid biosurfactants that feature high biocompatibility and low production cost; piscidins, which are host defense peptides (HDPs) from fish. While HDPs such as piscidins target the membrane of pathogens, and thus result in low incidence of resistance, SLs are not well understood on a mechanistic level. Here, we demonstrate that combining SL−hexyl ester (SL-HE) with subinhibitory concentration of piscidins 1 (P1) and 3 (P3) stimulates strong antimicrobial synergy, potentiating a promising therapeutic window. Permeabilization assays and biophysical studies employing circular dichroism, NMR, mass spectrometry, and X-ray diffraction are performed to investigate the mechanism underlying this powerful synergy. We reveal four key mechanistic features underlying the synergistic action: (1) P1/3 binds to SL-HE aggregates, becoming α-helical; (2) piscidin−glycolipid assemblies synergistically accumulate on membranes; (3) SL-HE used alone or bound to P1/3 associates with phospholipid bilayers where it induces defects; (4) piscidin−glycolipid complexes disrupt the bilayer structure more dramatically and differently than either compound alone, with phase separation occurring when both agents are present. Overall, dramatic enhancement in antimicrobial activity is associated with the use of two membrane-active agents, with the glycolipid playing the roles of prefolding the peptide, coordinating the delivery of both agents to bacterial surfaces, recruiting the peptide to the pathogenic membranes, and supporting membrane disruption by the peptide. Given that SLs are ubiquitously and safely used in consumer products, the SL/peptide formulation engineered and mechanistically characterized in this study could represent fertile ground to develop novel synergistic agents against drug-resistant bacteria.

Antiviral activity of the host defense peptide piscidin 1: investigating a membrane-mediated mode of action

“…The disordering effect of P1 is also demonstrated through the 2 H SS-NMR experiments that establish the destabilization of the L o domains in the L o /L d mixture. Since P1 is able to interact with the L o , its inhibitory effect on demixing of the L o /L d could be due to its inherent ability to partition in the region between the headgroups and hydrocarbon core, where it can induce disorder in the acyl chains of the lipids ( Perrin et al, 2014 ; Hayden et al, 2015 ; Perrin et al, 2015 ; Mihailescu et al, 2019 ; Comert et al, 2021 ; Liu et al, 2023 ). Interestingly, P1 is more active on the L o than L d when each of them is used alone.…”

“…The permeabilization assays were performed as previously described ( Mihailescu et al, 2019 ; Liu et al, 2023 ). Briefly, for each lipid composition, either L o or L o /L d , a film was made in round bottom flask using DPPC, POPC, DPPE, POPE, and Chol (4 µmol total of lipids) mixed at a desired molar ratio in chloroform.…”

“…As part of this disordering effect, the lipid acyl chains form “baskets” to wrap around the peptide and fill the gap under it. An important consequence of this conformational rearrangement of the acyl chains is a pronounced thinning of the membrane ( Perrin et al, 2014 ; Hayden et al, 2015 ; Perrin et al, 2015 ; Mihailescu et al, 2019 ; Comert et al, 2021 ; Liu et al, 2023 ). Prior work has also revealed that P1 induces and leverages membrane heterogeneity to disrupt homogeneous bilayers containing Chol ( Comert et al, 2019 ).…”

Presentation1.pdf

“…The disordering effect of P1 is also demonstrated through the 2 H SS-NMR experiments that establish the destabilization of the L o domains in the L o /L d mixture. Since P1 is able to interact with the L o , its inhibitory effect on demixing of the L o /L d could be due to its inherent ability to partition in the region between the headgroups and hydrocarbon core, where it can induce disorder in the acyl chains of the lipids (Perrin et al, 2014;Hayden et al, 2015;Perrin et al, 2015;Mihailescu et al, 2019;Comert et al, 2021;Liu et al, 2023). Interestingly, P1 is more active on the L o than L d when each of them is used alone.…”

“…As part of this disordering effect, the lipid acyl chains form "baskets" to wrap around the peptide and fill the gap under it. An important consequence of this conformational rearrangement of the acyl chains is a pronounced thinning of the membrane (Perrin et al, 2014;Hayden et al, 2015;Perrin et al, 2015;Mihailescu et al, 2019;Comert et al, 2021;Liu et al, 2023). Prior work has also revealed that P1 induces and leverages membrane heterogeneity to disrupt homogeneous bilayers containing Chol (Comert et al, 2019).…”