Potent activity of polymyxin B is associated with long-lived super-stoichiometric accumulation mediated by...

Area of Science:

• Microbiology
• Biochemistry
• Pharmacology

Background:

• Polymyxins are vital antibiotics for treating gram-negative bacterial infections.
• Their precise molecular mechanisms of action, particularly interactions with lipid A, are not fully understood.
• Lipid A is the essential membrane anchor of lipopolysaccharide in the outer membrane of gram-negative bacteria.

Purpose of the Study:

• To elucidate the kinetic and mechanistic details of polymyxin interactions with lipid A.
• To develop a phenomenological model describing polymyxin binding and activity.
• To identify key molecular events that lead to polymyxin's bactericidal effects.

Main Methods:

• Surface plasmon resonance (SPR) was employed to kinetically analyze polymyxin-lipid A interactions.
• A phenomenological model was derived from the kinetic data.
• Interactions of brevicidine and polymyxin-resistant lipid A mutants were also studied.

Main Results:

• Polymyxins interact with lipid A via a three-state mechanism: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with long residence times.
• Brevicidine, another lipid A-targeting molecule, also showed accumulation.
• Polymyxin resistance mutations and a non-bactericidal derivative impaired the transition to long-lived species.

Conclusions:

• The proposed three-state mechanism, involving transient binding for outer membrane permeabilization and cluster accumulation for bactericidal activity, explains polymyxin action.
• These findings provide a framework for designing novel lipid A-targeting antibiotics.
• The study offers a generalizable method for investigating interactions with the gram-negative outer membrane.