The rise of multidrug-resistant bacterial pathogens, especially in healthcare environments, presents an urgent public health threat. Among the primary resistance mechanisms in these pathogens are multidrug efflux pumps (MDEPs), which can severely limit antibiotic efficacy. Targeting MDEPs has promising potential to restore drug sensitivity and curb bacterial virulence.
This study focuses on the newly identified proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. They confer resistance to various biocides, including disinfectants and antiseptics, and are encoded by many critical human pathogens, including WHO-priority organisms. Despite their importance, the full functional scope and transport mechanisms of PACE proteins remain poorly understood. Interestingly, genes encoding PACE pumps are highly conserved in bacterial core genomes rather than being found on recently acquired mobile genetic elements, suggesting their essential roles beyond antimicrobial resistance.
To facilitate structural studies, we designed mutants of two PACE homologs using computational methods, introducing 5–20% amino acid substitutions to enhance protein yield and stability. One such mutant, Fb4.1, achieved a six-fold increase in yield and significantly higher thermal stability than the wild type. Our analyses - including detergent screenings that revealed distinct oligomeric states in different environments and extensive crystallisation trials using vapour diffusion and lipidic cubic phase (LCP) techniques - provide insights into optimising conditions for high-quality crystal formation. This work lays the groundwork for achieving the first 3D structure of the PACE family, an essential step for developing targeted inhibitors to counter multidrug-resistant pathogens, ultimately enhancing hospital infection control and improving patient outcomes.