Bacterial antimicrobial resistance is recognised as one of the greatest challenges currently facing human health, and the threat it poses is continuing to grow. To combat this issue, there is an urgent need for increased research into the wide array of mechanisms through which bacteria resist antimicrobials. One of the key contributors to antimicrobial resistance are multidrug efflux pumps, which are able to recognise a diverse range of antimicrobials and transport them out of the bacterial cell, preventing them from reaching and acting on their targets and leading to resistance. The proteobacterial antimicrobial compound efflux (PACE) family is the most recently discovered group of multidrug efflux pumps, and has been implicated as a contributor to resistance in a number of key multidrug-resistant human pathogens, such as Acinetobacter baumannii.
The Vibrio parahaemolyticus protein VP1155 is a prototypical member of this family that is capable of transporting several chemically distinct antimicrobial compounds and fluorescent dyes. VP1155 was used as a representative PACE protein to investigate the topology and oligomeric state of PACE proteins, as well as the functional importance of charged residues that are highly conserved within the PACE family. The results of native PAGE analysis conducted with protein solubilised in SMA lipid particles suggested that VP1155 forms multimers within the membrane, and that these are most likely to be tetramers. Site-directed mutagenesis and fluorescent transport studies indicated that specific conserved charged residues at the cytoplasmic boundary of the membrane were required for antimicrobial transport function. These results highlight structural and mechanistic features that are unique to the PACE family of transport proteins.