Clinically Relevant Bacterial Outer Membrane Models for Antibiotic Screening Applications

Abstract

Antibiotic resistance is a growing global health concern that has been increasing in prevalence over the past few decades. In Gram-negative bacteria, the outer membrane is an additional barrier through which antibiotics must traverse to kill the bacterium. In addition, outer membrane features and properties, like membrane surface charge, lipopolysaccharide (LPS) length, and membrane porins, can be altered in response to antibiotics and therefore, further mediate resistance. Model membranes have been used to mimic bacterial membranes to study antibiotic-induced membrane changes but often lack the compositional complexity of the actual outer membrane. Here, we developed a surface-supported membrane platform using outer membrane vesicles (OMVs) from clinically relevant Gram-negative bacteria and use it to characterize membrane biophysical properties and investigate its interaction with antibacterial compounds. We demonstrate that this platform maintains critical features of outer membranes, like fluidity, while retaining complex membrane components, like OMPs and LPS, which are central to membrane-mediated antibiotic resistance. This platform offers a non-pathogenic, cell-free surface to study such phenomena that is compatible with advanced microscopy and surface characterization tools like quartz crystal microbalance. We confirm these OMV bilayers recapitulate membrane interactions (or lack thereof) with the antibiotic compounds polymyxin B, bacitracin, and vancomycin, validating their use as representative models for the bacterial surface. By forming OMV bilayers from different strains, we envision that this platform could be used to investigate underlying biophysical differences in outer membranes leading to resistance, to screen and identify membrane-active antibiotics, or for the development of phage technologies targeting a particular membrane surface component.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy

“…This is particularly exciting when we consider the future applications of this method, particularly with respect to antimicrobial screening studies. 14 For instance, if we wish to analyze the interaction of a certain class of antibiotic with a protein target of interest in a bacterial membrane, we can overexpress or indeed delete this protein from our membranes. In this way, we have a platform for tailored pharmacological studies in a reproducible and safe to use manner.…”

“… 13 These bilayers, which contain both bacterial and synthetic lipid fractions, have been shown to retain components such as outer membrane proteins and lipopolysaccharides in the correct orientation. 14 Therefore, the system retains physiological properties that are beneficial to the investigation of protein–protein interactions, protein–ligand interactions, and other lipid membrane properties in vitro . Additionally, because these membrane models do not use live cells, they are much safer when investigating pathogenic bacterial membrane interactions.…”

An Outer Membrane Vesicle‐Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability

“…However, the speed of recovery is slower and in the range of minutes rather than seconds. [ 55 ] In contrast, E. coli BL21 DE3 OMV‐derived membranes recover in a much slower and linear manner indicating that among the components of the OMV‐derived membrane the fluid fraction is virtually non‐existent compared to the liposome‐based model (Figure 8). The low fluidity of the OMV coating is in line with the common imagination of the bacterial OM to be highly rigid.…”

“…The low fluidity of the OMV coating is in line with the common imagination of the bacterial OM to be highly rigid. [ 56 ] However, considering the fluidity of OMV‐derived lipid bilayers from either E. coli [ 57 ] or other Gram‐negative species, [ 55 ] the fluidity of this coating appears particularly low, which gives the impression that the functionality of lipids has been dramatically decreased. This is explainable by the additional presence of LPS and proteins among the OMV PLs.…”

Lipid Nanoparticle Technology for Delivering Biologically Active Fatty Acids and Monoglycerides

“…There is also excellent potential to utilize pathogen-membrane-mimicking SLB platforms and there has been much progress in developing bacterial cell membrane models [110,111]. It is envisioned that these biosensing technologies can serve as a predictive model to evaluate membrane-interacting activities of fatty acids and monoglycerides in free form and within various types of nanostructured assemblies.…”