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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
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Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

Aarshi N Singh1, Meishan Wu2, Tiffany T Ye1

  • 1Department of Chemistry, Lehigh University, Bethlehem, PA, USA.

Biorxiv : the Preprint Server for Biology
|September 4, 2024
PubMed

Insights

Researchers developed a new method to create artificial bacterial outer membranes from vesicles. This platform aids in studying antibiotic effectiveness against gram-negative bacteria, accelerating drug development.

Area of Science:

  • Biochemistry
  • Microbiology
  • Materials Science

Background:

  • Antibiotic resistance poses a significant threat to global health.
  • The outer membrane of gram-negative bacteria presents a barrier to antibiotic penetration, complicating drug development.
  • Novel methods are needed to mimic this barrier for effective high-throughput antibiotic screening.

Purpose of the Study:

  • To develop a method for creating planar supported bilayers that mimic the gram-negative bacterial outer membrane.
  • To establish a platform for studying antibiotic interactions with bacterial outer membranes.

Main Methods:

  • Modification of outer membrane vesicles (OMVs) from *Aggregatibacter actinomycetemcomitans* using a freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids).
  • Spontaneous rupture of OM-Hybrids on SiO2 surfaces to form planar outer membrane supported bilayers (OM-SBs).
  • Characterization using dynamic light scattering, fluorescence quenching, quartz crystal microbalance with dissipation monitoring (QCM-D), and fluorescence recovery after photobleaching (FRAP).

Main Results:

  • Successful formation of OM-Hybrids and subsequent generation of OM-SBs.
  • Demonstrated detection of surface-associated DNA and proteins on OM-SBs.
  • Assessed the interaction of polymyxin B with the OM-SBs, validating the model's utility.

Conclusions:

  • The developed platform effectively generates planar bacterial outer membrane surfaces.
  • This approach provides a valuable tool for streamlining antibiotic development and research.
  • Facilitates high-throughput studies of antibiotic transport and efficacy against gram-negative bacteria.
Keywords:
Aggregatibacter actinomycetemcomitansGram-negative bacteriaantibiotic developmentmodel membraneouter membrane vesicles

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