Modeling and Functional Characterization of Reconstituted Efflux Pump Components from Heterologous Gram-Negative...

Efflux pumps operating in bacteria continuously evolve and contribute significantly toward the rising global trends in antimicrobial resistance (AMR). Our earlier studies demonstrated that the expression of tripartite resistance nodulation division (RND) efflux pump containing the outer membrane protein (OMP), membrane fusion protein (MFP), and inner RND pump from different Gram-negative bacteria results in elevated minimum inhibitory concentrations (MICs) of different antibiotics. Interestingly, parts of this complex could be transferred either within the species or across genera. Despite limited sequence homology, we report the existence of significant structural and functional conservation between the distantly related MFP and RND proteins. Following the assembly of MFP components (AcrA, MexA, OqxA) and RND components (AcrB, MexB, OqxB) from E. coli, P. aeruginosa, and K. pneumoniae, respectively, we report evidence of functioning efflux pumps using real-time Nile Red assays and enhanced biofilm formation. Further substantiation of the latter is provided through docking and molecular dynamics (MD) simulation studies, which offer insights about the direct interactions of RND efflux pumps with AI-2, the major quorum-sensing molecule of E. coli. Results described here implicate that after transmission, possibly via horizontal gene transfer or e-DNA within bacteria, the assembled efflux pump components could drive multiple aspects of AMR, including its dissemination and ability to adapt to alternate lifestyles such as biofilms, facilitating better survival.