AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.The incidence of multidrug-resistant (MDR, also used herein for “multidrug resistance”) bacterial infections is increasing, and the 2013 World Economic Forum Global Risks report listed antibiotic-resistant bacteria as one of the greatest threats to human health (1). Resistance-nodulation division (RND) efflux pumps of Gram-negative bacteria confer intrinsic and acquired MDR in clinically relevant infections by exporting antibiotics out of the bacterial cell, allowing bacteria to survive at increased drug concentrations.To date the best-characterized efflux pump is AcrAB-TolC of Escherichia coli, composed of the inner membrane RND antiporter AcrB that functions in a tripartite assembly with a periplasmic adaptor protein, AcrA, and the outer membrane channel, TolC. The AcrB pump is an asymmetric homotrimer whose monomers undergo a functional rotation through three states: access, binding, and extrusion (labeled A, B, and E, respectively) to pump substrates from the periplasm, or outer leaflet of the inner membrane, to outside of the cell (2, 3). Drug binding within AcrB is complex. The distal binding pocket has a phenylalanine-rich region that binds low-molecular-weight drugs and a proximal binding pocket and vestibule bind larger compounds such as erythromycin (4, 5).To date, MDR in clinically relevant infections mediated by RND efflux pumps, including AcrB, has been documented to be due to overexpression of the efflux pump and concomitant increased efflux of antibiotics from the bacterial cell (6). Overproduction of efflux pumps can occur via four mechanisms: (i) mutation of the local repressor gene (7, 8), (ii) mutation in a global regulatory gene (9, 10), (iii) mutation of the promoter region of the efflux pump gene (11), or (iv) insertion elements upstream of the transporter gene (12, 13).This study focused on elucidating the mechanism of resistance in a unique set of clinical isolates collected over the course of a complex Salmonella infection (Fig. S1) (14, 15). The isolates were obtained from a 52-y-old male patient admitted for repair of a leaking abdominal aortic aneurysm graft. Salmonella Typhimurium (L3) was isolated before ciprofloxacin treatment and was susceptible to ampicillin, sulphonamide, trimethoprim, cefuroxime, chloramphenicol, gentamicin, and ciprofloxacin. Over the course of the infection the patient received i.v. ciprofloxacin, oral ciprofloxacin, i.v. ceftazidime, and i.v. aztreonam. Isolates were taken throughout infection, and during treatment MDR strains were isolated. L18, the last MDR strain to be isolated, was from wound drainage fluid when the patient had received no antibiotics for 2 wks. The patient died soon afterward with the infection unresolved. Compared with the pretherapy isolate L3, the MDR posttherapy isolate, L18, was less susceptible to numerous agents, including ciprofloxacin and β-lactams, and accumulated less ciprofloxacin and Hoechst dye (16). This set of isolates has provided a unique opportunity to investigate in vivo evolution of MDR in response to clinically validated courses of antimicrobial treatment.Here, we report that whole genome sequencing revealed a new mechanism of clinically significant MDR selected during therapy: substitution in a transporter protein, which altered the specificity of the efflux pump for antibacterial drugs. The structural impact of the substitution was investigated by molecular dynamics (MD) simulations.