Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• AcrB is the primary multidrug exporter in Escherichia coli, crucial for bacterial survival against toxic compounds.
• The precise mechanisms and substrate specificities of AcrB's multiple identified transport pathways remain incompletely understood.

Purpose of the Study:

• To elucidate the functional role and substrate specificity of a newly identified substrate channel (channel 3) in the AcrB multidrug exporter.
• To investigate how different substrate entry channels contribute to the broad substrate recognition and transport capabilities of AcrB.

Main Methods:

• Utilized targeted mutagenesis to create AcrB variants with altered channel functionalities.
• Investigated substrate transport using specific drug molecules like ethidium, minocycline, and erythromycin.
• Analyzed drug efflux patterns in wild-type and mutant strains to determine channel preference and competition.

Main Results:

• Identified and characterized channel 3, a direct pathway from the AcrB central cavity to the deep pocket, bypassing the switch-loop and proximal pocket.
• Demonstrated that planar aromatic cations, exemplified by ethidium, preferentially utilize channel 3.
• Showed that mutations enhancing channel 3 activity do not interfere with drug export via channels 1 and 2, and that a switch-loop mutant is restricted to channel 3 substrates.

Conclusions:

• The AcrB multidrug exporter employs multiple, distinct substrate entry channels, including the newly defined channel 3.
• Channel 3 provides a specific route for planar aromatic cations, contributing to the exporter's broad substrate range.
• The coordinated use of multiple entry pathways allows AcrB to efficiently recognize and transport diverse drugs with varying physicochemical properties.