Assessing Barriers for Antimicrobial Penetration in Complex Asymmetric Bacterial Membranes: A Case Study with Thymol

Abstract

The bacterial cell envelope is a complex multilayered structure evolved to protect bacteria in hostile environments. An understanding of the molecular basis for the interaction and transport of antibacterial therapeutics with the bacterial cell envelope will enable the development of drug molecules to combat bacterial infections and suppress the emergence of drug-resistant strains. Here we report the successful creation of an in vitro supported lipid bilayer (SLB) platform of the outer membrane (OM) of E. coli, an archetypical Gram-negative bacterium, containing the full smooth lipopolysaccharide (S-LPS) architecture of the membrane. Using this platform, we performed fluorescence correlation spectroscopy (FCS) in combination with molecular dynamics (MD) simulations to measure lipid diffusivities and provide molecular insights into the transport of natural antimicrobial agent thymol. Lipid diffusivities measured on symmetric supported lipid bilayers made up of inner membrane lipids show a distinct increase in the presence of thymol as also corroborated by MD simulations. However, lipid diffusivities in the asymmetric OM consisting of only S-LPS are invariant upon exposure to thymol. Increasing the phospholipid content in the LPS-containing outer leaflet improved the penetration toward thymol as reflected in slightly higher relative diffusivity changes in the inner leaflet when compared with the outer leaflet. Free-energy computations reveal the presence of a barrier (∼6 kT) only in the core-saccharide region of the OM for the translocation of thymol while the external Oantigen part is easily traversed. In contrast, thymol spontaneously inserts into the inner membrane. In addition to providing leafletresolved penetration barriers in bacterial membranes, we also assess the ability of small molecules to penetrate various membrane components. With rising bacterial resistance, our study opens up the possibility of screening potential antimicrobial drug candidates using these realistic model platforms for Gram-negative bacteria.

A Systematic Approach: Molecular Dynamics Study and Parametrisation of Gemini Type Cationic Surfactants

“…Our positive control indicates that decreasing effect on lateral diffusion should be desirable however, the antimicrobial effect of OCT is based on membrane disruption and cannot be considered as a factor in this case. Results obtained for model membrane are in strong agreement with the GUVs E. coli mimicking studies, i.e., experimental diffusion coefficient equals D = 6.09 µm 2 /s [59]. Interestingly, different antimicrobial agent-thymol induced growth in lipids mobility, supporting the agent translocation [59].…”

Interactions of surfactants with the bacterial cell wall and inner membrane: Revealing the link between aggregation and antimicrobial activity

“…Surfactant-incorporated IM models were obtained from CHARMM-GUI web server 27 . Inner membrane model was procured from our previous work 15 . The membranes containing laurate and oleate have been studied at surfactant molar concentrations 20% and 40%, as summarized in Table S2.…”

“…Whether surfactants solubilize the OM or penetrate the OM through channels to access the IM are open questions. Recent in vitro studies on model bacterial membrane platforms, coupled with super resolution microscopy methods indicate that model membrane constructs can potentially be used to assess the barrier characteristics of bacterial membranes 15–17 . However, these experiments are challenging both from the point of constructing the reliable bacterial cell wall mimics and using appropriate microscopic tools to interrogate the membrane in the presence of external agents 15,18 .…”

“…In recent years, molecular dynamics (MD) simulations have evolved as a powerful tool to study bacterial membranes and assess their interactions and free energy barriers with small molecules and antibiotics. MD simulations have provided a molecular understanding of the barriers offered by different regions of the complex OM 15,19 highlighting the asymmetric free energy landscape for molecule translocation 19 which is quite distinct from the IM 15 . The barrier properties of the PGN layer have only recently been investigated in our laboratory 20 .…”

Evaluating Coarse-Grained MARTINI Force-Fields for Capturing the Ripple Phase of Lipid Membranes

“…1,2 Lipid bilayers are important constituents of biomembranes which are ubiquitous in living organisms 3 , and nanoparticle pharmaceuticals such liposomal drugs [4][5][6] . Specifically, lipid membrane phases influence: (i) biological processes at the cellular level such as membrane fusion and budding 7 , permeation of drugs 8,9 , effect of anaesthetics 10 , vulnerability to pathogenic toxins 11,12 , lipid flip-flop 13,14 , neural signalling 15 , and the function of membrane proteins 3,7 , and (ii) industrial applications such as the design, storage and usage of liposomal drugs [4][5][6]16 , cosmetic formulations 1 , and lipid-rich foods 1 . Molecular simulations of membranes at various levels of detail have yielded atomistic insights into the rich phase behaviour of phospholipid membranes while complementing experimental observations 1,17 .…”