Enhanced internal ionic interaction of MFS efflux pump MdfA contributes to its elevated antibiotic export

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Gram-negative bacterial infections pose challenges due to rising antibiotic resistance.
• Efflux pumps are key bacterial defense mechanisms against antibiotics, contributing to resistance.
• The Major Facilitator Superfamily (MFS) efflux pumps, like MdfA, export antibiotics, but their regulation by internal ionic interactions is unclear.

Purpose of the Study:

• To investigate the role of internal ionic interactions in the MFS efflux pump MdfA.
• To understand how these interactions affect antibiotic resistance in *E. coli*.
• To identify potential targets for novel inhibitors against MFS efflux pumps.

Main Methods:

• Identification of MdfA salt bridges and their natural variants in *E. coli*.
• Construction and characterization of MdfA variants to assess antibiotic resistance.
• Molecular dynamics (MD) simulations to analyze the impact of mutations on pump dynamics and interactions.

Main Results:

• Extending a cytoplasmic salt bridge (E136D) in MdfA increased *E. coli* antibiotic resistance.
• Combined mutations (E136D and K346R) further enhanced resistance levels.
• MD simulations showed enhanced ionic interactions without affecting protein flexibility, increasing MdfA protonation sites and activation potential.

Conclusions:

• Internal ionic interactions are crucial for regulating drug export by MFS efflux pumps like MdfA.
• Modulating these interactions can alter bacterial antibiotic resistance.
• Findings provide insights for developing new strategies to combat antibiotic resistance by targeting MFS efflux pumps.