Gonococcal infections, caused by the obligate human pathogen Neisseria gonorrhoeae, are the cause of increasing concern among clinicians as gonococcal strains are developing higher levels of tolerance to current antibiotic therapies. Bacterial multidrug efflux proteins are major contributors to drug resistance and thus, reduced efficacy of treatments. The MtrD efflux protein of N. gonorrhoeae provides resistance to a broad spectrum of structurally-diverse antimicrobial agents, including human antimicrobial peptides. The aim of this study is to identify and characterise the amino acid residues of MtrD involved in binding and translocation of these antimicrobial drugs and peptides.

Using molecular modelling of MtrD against high resolution crystal structures of related efflux proteins, a number of target residues were identified. In a fully-functional Escherichia coli MtrD cysteine-less derivative clone, these residues were individually replaced with cysteine. Resulting mutants were confirmed by sequencing and each construct was integrated into the N. gonorrhoeae chromosome. Western blot analysis confirmed expression of the MtrD mutant proteins and the resistance profile of cells expressing these MtrD mutants was determined to a range of compounds.

Substitution of the MtrD amino acid residues changed the activity of the efflux pump for selected antimicrobial compounds. All mutations led to a considerable reduction of resistance to the spermicide non-oxynol 9, Triton X100, rifampicin, novobiocin, polymyxin B, crystal violet, cholic acid, benzalkonium, ethidium, acriflavin, triclosan, and chlorhexidine. Mutations had different levels of impact on the susceptibility of cells to an individual compound. Taken together, the results suggest that the amino acids F136, F176, I605, F610, F612 and F623 play an important role in the interaction of the MtrD efflux pump with substrates. Identification of such functionally-important residues will help us to gain a better understanding of the transport mechanism(s) of this family of multidrug transporters.