Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Gram-negative bacteria utilize tripartite multidrug RND efflux systems for drug expulsion.
• Previous structural studies showed limited contact between outer membrane factors (OMFs) and periplasmic adaptor proteins (PAPs), obscuring OMF recruitment mechanisms.

Purpose of the Study:

• To investigate the role of a MexA Q93R mutation in tripartite efflux complex assembly and function.
• To elucidate the molecular determinants governing OMF recruitment and selectivity.

Main Methods:

• Expression of MexA-MexB-OprM and mutant strains.
• Single-particle electron microscopy for structural analysis.
• Biochemical assays to assess complex formation and function.

Main Results:

• The MexA Q93R mutation increased antibiotic resistance and promoted tripartite complex formation with cognate and non-cognate OMFs.
• MexA_Q93R self-assembled into hexamers, suggesting altered interprotomer interactions.
• OprM C-terminal deletion impaired complex formation with wild-type MexA but not with MexA_Q93R.

Conclusions:

• The Q93R MexA mutation and the OprM C-terminal peptide are critical for modulating tripartite efflux pump assembly.
• These findings provide insights into the mechanisms of OMF selectivity during complex formation, even with components outside the direct interface.