Abstract:
Evolutionary trajectories and mutational landscapes of drug-resistant bacteria are influenced by cell-intrinsic and extrinsic factors. In this study, I demonstrated that loss of the Lon protease altered susceptibility of Escherichia coli to trimethoprim and that these effects were strongly contingent on the drug concentration and genetic background. Lon, an AAA+ ATPase, is a bacterial master regulator protease involved in cytokinesis, suppression of transposition events, and clearance of misfolded proteins. I show that Lon deficiency enhances intrinsic drug tolerance at sub-MIC levels of trimethoprim. As a result, loss of Lon, though disadvantageous under drug-free conditions, has a selective advantage at low concentrations of trimethoprim. At high drug concentrations, however, Lon deficiency is detrimental for E. coli. I show that the former is explained by suppression of drug efflux by Lon, while the latter can be attributed to SulA-dependent hyperfilamentation. On the other hand, deletion of lon in a trimethoprim-resistant mutant E. coli strain (harboring the Trp30Gly dihydrofolate reductase [DHFR] allele) directly potentiates resistance by enhancing the in vivo stability of mutant DHFR. Using extensive mutational analysis at 3 hot spots of resistance, I show that many resistance-conferring mutations render DHFR prone to proteolysis. This trade-off between gaining resistance and losing in vivo stability limits the number of mutations in DHFR that can confer trimethoprim resistance. Loss of Lon expands the mutational capacity for acquisition of trimethoprim resistance. This paper identifies the multipronged action of Lon in trimethoprim resistance in E. coli and provides mechanistic insight into how genetic backgrounds and drug concentrations may alter the potential for antimicrobial resistance evolution.