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

Abstract

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound-and bacteria-specific passive uptake pathways.

Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

“…The lipid bilayer arrangement in our giant vesicle system is a unique feature compared to PAMPA-like OM platforms where antibiotic transport is estimated; however, AMPs could not be tested due to the lower amount of lipids in the system. 11 Electrophysiology-based OM models 17,69 provide high-resolution quantitative information on antibiotic interaction with membrane proteins but cannot distinguish between binding and transport events. One of the major drawbacks of our platform is the fluorescence detection system, which limits the range of antibiotics that can be investigated, unlike mass spectrometry-based detection.…”

“…In-vitro membrane models provide a handy tool to comprehend membrane interface processes with significant implications in drug discovery platforms . Several models have been developed to mimic the Gram-negative OM, like supported membrane assays from OM vesicles (OMVs) of bacteria. , OMV-based permeation assays employ mass-spectrometry based detection of antibiotics; however, the underlying membrane structure (in terms of lamellarity) coated on the surface remains unclear . The bilayer structure of an alternative OMV-based OM model is very accurate in investigating AMP activity .…”

“…10,11 OMV-based permeation assays employ massspectrometry based detection of antibiotics; however, the underlying membrane structure (in terms of lamellarity) coated on the surface remains unclear. 11 The bilayer structure of an alternative OMV-based OM model is very accurate in investigating AMP activity. 10 Another OMV-based assay employs the swelling technique to determine mainly relative permeability rates.…”

Proteomic Profiling of Outer Membrane Vesicles Released by Escherichia coli LPS Mutants Defective in Heptose Biosynthesis

“…TEM figures were visualized using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and analyzed using Student’s t -test. Diagrams were generated using the GraphPad Prism software (version 8.0, GraphPad software, San Diego, CA, USA) [ 28 , 29 ].…”

Functional Materials to Overcome Bacterial Barriers and Models to Advance Their Development

“…However, care must be taken that such modifications may not affect the affinity to the drug's intracellular targets leading to other limitations. [ 161,162 ]…”