The resistance–nodulation–cell division (RND) superfamily includes the tripartite efflux pump system, which mediates antibiotic resistance in gram-negative bacteria, including Escherichia coli, through elimination of toxic compounds.

The E coli CusCBA efflux system expels Cu(I) and Ag(I) from the bacteria. E coli also has RND efflux transporters—categorized into HAE (hydrophobic and amphiphilic efflux)-RND and HME (heavy-metal efflux)-RND subfamilies. The HME-RND transporter CusA confers antibacterial resistance to Cu(I) and Ag(I) ions. In this study, the structure of CusA was detailed in the presence and absence of Cu(I) and Ag(I) ions, providing information about the HME-RND CusA transporter. The multiple-isomorphous replacement technique with anomalous scattering (MIRAS) described the structure of CusA as a trimeric molecular pump, allowing metal uptake from the bacterial periplasm and cytoplasm. Methionine pairs were significant in metal binding and transport; mutants that targeted methionine, charged residues, or methionine pairs did not expel Ag(I) and Cu(I) ions. Structural analysis suggested that metal-binding sites were formed by a methionine cluster located in a cleft in the periplasmic domain. It appeared that Cu(I) and Ag(I) binding resulted in conformational changes in periplasmic and transmembrane domains, modeling an ion export pathway for the bacteria. In addition, study results suggested that CusA functions as a trimeric pump that picks up metal ions from the periplasm and cytoplasm, supporting the hypothesis that the pump uses methionine pairs/clusters to bind and expel metal ions through a transport pathway.

Commentary: Bacterial resistance is an ongoing problem in both the human and veterinary fields. There are many causes for resistance, including injudicious and subtherapeutic (dose or duration) antibiotic use. Antibiotics typically induce heavy metal (silver, copper, or manganese) toxicity in bacteria. However, bacteria have developed efflux protein pumps that remove heavy metals, decreasing antimicrobial efficacy and resulting in resistance. The CusA efflux pump described in this paper is a protein pump that removes heavy metals from E coli. Determining the structure of this pump allowed elucidation of mechanisms of action, which can potentially assist in the design of drugs or substances that could inactivate or alter pump structure, halting heavy metal expulsion from bacteria and enhancing antibiotic efficacy. Future studies might focus on how to block such pathways and confer heavy metal toxin susceptibility to pathogenic bacteria.—Indu Mani, DVM, DSc

Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Long F, Su C-C, Zimmermann MT, et al. NATURE 467:484-488, 2010.