Abstract
Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.
Highlights
One of the most commonly deployed mechanisms of antimicrobial resistance (AMR) is mediated by bacterial efflux pumps, protein complexes that have evolved to rid cells of toxic compounds [1]
Drug efflux pump component assembly into bacterial outer membranes determine whether the presumptive lateral gate of TamA does engage substrate polypeptides like TolC in vivo, we generated 2 TamA constructs that could be closed and locked to prevent substrate engagement (TamA-G250C/E555C and TamA-G252C/G553C) by introducing a cysteine substitution in the first (G250C or G252C) and last (G553C or E555C) transmembrane β-strands of TamA (Fig 2A)
In the absence of the translocation and assembly machinery (TAM), FimD assembly is mediated solely by the barrel assembly machinery (BAM) complex and proceeds via an assembly intermediate that is instead cleaved by extracellular proteinase K into an approximately 45-kDa fragment
Summary
One of the most commonly deployed mechanisms of antimicrobial resistance (AMR) is mediated by bacterial efflux pumps, protein complexes that have evolved to rid cells of toxic compounds [1]. The fundamental mechanism by which these efflux pumps function in gramnegative bacteria depends on a tripartite architecture, comprised of 3 components that combine to span the inner membrane, periplasm, and outer membrane (Fig 1A), as understood from several landmark structural studies on these tripartite complexes [2,3,4,5]. Most of the structural and functional work has been focussed on the prototypical pumps AcrAB-TolC and MexAB-OprM. These pumps are constitutively expressed in Escherichia coli and Pseudomonas aeruginosa, respectively, and are the workhorses that confer low levels of resistance to clinically relevant drugs before more specialised resistance mechanisms can evolve [6].
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