Escherichia coli aerobic respiratory complexes as modulators of aminoglycoside susceptibility and targets for drug discovery

Antibiotic resistance is an increasing problem making the treatment of infectious bacterial diseases more difficult. This leads to higher mortality rates and a greater economic burden in the future. Consequently, there is a pressing demand for new antimicrobials. Bacterial aerobic respiration is linked to antibiotic susceptibility in Gram-negative pathogens. Susceptibility to aminoglycosides has previously been linked to antibiotic uptake related to the proton motive force (PMF) and reactive oxygen species production (ROS) but the relative contributions of each was poorly understood. A combination of ROS assays and PMF disruption via uncoupler were used to study the relative ROS vs. PMF contributions to gentamicin toxicity in a pathogenic E. coli clinical isolate. The data gathered supported the hypothesis that maintenance of the PMF rather that the production of ROS underpins the mechanism of how the aerobic respiratory chain potentiates the toxicity of aminoglycosides.

Cytochrome bd-I is a terminal oxidase of the E. coli aerobic respiratory chain and an interesting new target for the development of new antimicrobials. To investigate the structure and phylogenetics of cytochrome bd-I a hhsearch pipeline was used to infer phylogenetic relationships in cytochrome bd-I that could be linked to structural features. Phylogenetic evidence suggested that the presence/absence of amino acid residue 101 (in the E. coli sequence) provides information on the position of the haem d cofactor: absence of residue 101 supports a haem d cofactor close to the periplasm (as in the Geobacillus thermodenitrificans cytochrome bd structure), whereas the presence of residue 101 was consistent with the haem b595 in this position and the haem d closer to the cytoplasmic side (as in E. coli cytochrome bd-I). Potential drug targeting sites on E. coli cytochrome bd-I were also investigated, aside from the quinol binding pocket, with the oxygen entry tunnel being the most promising site.

A drug repurposing approach was then used to target E. coli cytochrome bd-I to search for new inhibitors as potential antimicrobials. An in silico molecular docking pathway was developed to screen FDA approved drugs for their ability to bind in the quinol binding site of cytochrome bd-I. Experimental methods were also developed to screen the top in

silico hits generating several interesting new potential inhibitors with the prodrug quinestrol being the most promising. Isolated E. coli membranes containing cytochrome bd-I as the sole respiratory oxidase were assayed for respiratory activity in the presence of various concentrations of Quinestrol, and an IC50 in the nM range was obtained, which was consistent with subsequent respiratory experiments with whole cells. This work has paved the way for future structural, drug-derivatisation and mutagenesis studies, and the drug repurposing pipeline can readily be adapted for other target sites on a range of respiratory complexes and expanded drug libraries.