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Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-β-Naphthylamide from Random Mutagenesis

Sabine Schuster1, Jürgen A Bohnert2, Martina Vavra3

  • 1Division of Infectious Diseases, Department of Medicine II, University Hospital and Medical Center, 79106 Freiburg, Germany. sabine.schuster@uniklinik-freiburg.de.

Molecules (Basel, Switzerland)
|February 1, 2019
PubMed

Insights

Phe-Arg-β-naphthylamide (PAβN) resistance arises from mutations in the lpxM gene, not efflux pumps. This indicates PAβN targets the lipopolysaccharide (LPS) layer in bacteria.

Area of Science:

  • Microbiology
  • Bacterial Physiology
  • Drug Resistance

Background:

  • Phe-Arg-β-naphthylamide (PAβN) is known as an efflux pump inhibitor (EPI) targeting major multidrug resistance transporters like AcrB in Gram-negative bacteria.
  • Understanding PAβN's mechanism of action is crucial for developing strategies to combat bacterial multidrug resistance.

Purpose of the Study:

  • To elucidate the mechanism of action of Phe-Arg-β-naphthylamide (PAβN).
  • To identify the molecular targets responsible for PAβN resistance and sensitizing activity.

Main Methods:

  • In vitro random mutagenesis was employed to evolve bacterial resistance to PAβN.
  • Next-generation sequencing was used to identify mutations in resistant strains.
  • An engineered lpxM knockout strain was created to assess the role of LpxM in PAβN activity.
  • Molecular modeling was used to analyze structural changes in lipid A.

Main Results:

  • Mutations conferring PAβN resistance were found in the lpxM gene, not in efflux pump genes like acrB.
  • lpxM deficiency resulted in significantly decreased PAβN activity (up to 16-fold) against large lipophilic drugs.
  • The efflux capacity and efficacy of other EPIs remained unaffected in lpxM mutants.
  • lpxM deficiency leads to underacylated lipid A, causing steric conformational changes in the bacterial membrane.

Conclusions:

  • PAβN's target is located in the lipopolysaccharide (LPS) layer, specifically involving lipid A structure.
  • The drug-sensitizing potency of PAβN is linked to its interaction with the bacterial membrane and LPS.
  • PAβN resistance can arise from alterations in LPS synthesis, independent of efflux pump activity.
Keywords:
PAβNefflux pump inhibitorlpxM (msbB)penta-acylated lipid Apermeabilizerrandom mutagenesis

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