Antimicrobial resistance in Neisseria gonorrhoeae, the causative agent of gonorrhoea, has reached an alarming level, severely impacting on effective treatment. A key mechanism it uses to achieve this is the expulsion of structurally different antimicrobials by the MtrD membrane protein, the only member of the resistance-nodulation-division (RND) family of H+/drug exporters in this bacterium. Although MtrD shares homology with the well-known Escherichia coli AcrB and Pseudomonas aeruginosa MexB multidrug RND exporters, distinctive features are also apparent. One of these is an unique sequence (N917-P927) in MtrD, which is not present in closely-related RND proteins, and was the subject of this study.
To assess the importance of this sequence, a ∆N917-P927 MtrD derivative was constructed in addition to a set of MtrD site-directed mutants where each of these eleven amino acids were individually replaced by cysteine. These constructs were integrated into the chromosome of N. gonorrhoeae KH15ΔmtrDΔnorM, a highly discriminatory strain which overexpresses the other components of this tripartite pump that are mandatory for full function. Once sequence and expression of the constructed MtrD mutant proteins were confirmed, the resistance profile of this set of mutants was determined by minimum inhibition concentration analysis on agar media to a panel of eight structurally-distinct antimicrobials.
Removal of N917-P927 from the MtrD protein significantly reduced resistance to six antimicrobials with >128-fold reduction of resistance to the spermicide nonoxynol-9, however resistance to cholic acid and polymyxin-B was not affected. Three single replacement MtrD mutants (N917C, G924C and P927C) resulted in altered resistance profiles similar to ∆N917-P927, while six mutants showed wild-type resistance pattern and one (E920C) had lowered resistance to three compounds.
These findings indicate that N917-P927 region is crucial for MtrD-mediated multidrug resistance in N. gonorrhoeae and within this region, four residues play a crucial role in translocation of specific substrates.