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

Abstract

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.

Keywords: AcrB; antimicrobial resistance; efflux; whole genome sequencing.

Fig. 1.

Binding of doxorubicin to the AcrB monomer. (A) Overall view of the AcrB monomer bound to doxorubicin (shown in space fill) as per PDB ID code 4DX7 (22). (B) A close-up view of the doxorubicin binding pocket in the PDB ID code 4DX7, highlighting the principal residues involved in the drug binding. (C) Close view of the binding site where important residues are shown (blue, wild type; red, mutant). Owing to the presence of the side chain of the aspartate residue in the G288D mutant, the side chain of F178 tilts away in the G288D mutant compared with its orientation in the wild-type protein (denoted by the arrow and labeled as 1). In turn, the side chain orientation of the residue Q176 is also altered in the mutated protein (denoted by 2) with respect to the wild-type form. Finally, the side chain of F136 also changes orientation (denoted by 3).

Fig. 2.

RDF profiles indicating the distribution of water molecules near the D288 residue in the mutant (solid lines) protein and near the G288 residue in the wild-type protein (dashed lines). Differently colored lines (solid or dashed) represent the RDF profiles calculated from two different simulations and for each of the monomers of the AcrB protein. RDF profiles are calculated based on the distance between the water oxygen to center of mass of G288/D288 residue’s side chain. (Inset) A representative snapshot of the presence of water molecules in the binding pocket of the G288D mutant protein. Water molecules at the corresponding positions are absent in the wild-type protein.

Fig. 3.

Predicted effects of the mutation on the binding of the doxorubicin (DOXO) and ciprofloxacin (CIP) in the distal binding pocket. DOXO and CIP are rendered with transparent and solid thick sticks, respectively, colored by atom type. The Cα atom of G288 is rendered in green and the side chain of D288 with yellow sticks. Side chains of other most relevant residues are shown with thinner sticks, colored by residue type and red in wild type and G288D, respectively. The orientation of CIP is derived from the MD simulations (27), and that of DOXO is based on the experimental X-ray structure as from PDB ID code 4DX7 (22).

Fig. 4.

The effect of the G288D mutation on the accumulation of ciprofloxacin (Cip), doxorubicin (Dxr), and minocycline (Min) and on the efflux of doxorubicin in Salmonella (A and B) and E. coli (C and D). A and C show accumulation of Cip, Dxr, and Min in Salmonella and E. coli, respectively. Black bars show Cip accumulation, gray bars show Dxr accumulation, and white bars show Min accumulation. Data presented are the mean of the steady-state values for three biological replicates ± SD. Asterisk denotes values that are significantly different from the corresponding wild-type parental strain (SL1344 or MG1655). Lines denote values, for strains carrying the G288D mutation, which are significantly different to the strain carrying the wild-type AcrB. B and D show the efflux of Dxr by Salmonella SL1344 and MG1655, respectively. In each case diamonds denote the wild type, squares denote ΔacrB, triangles denote the acrB mutant complemented with wild-type acrB, and crosses denote the acrB mutant complemented with G288D acrB.