Polymorphisms in a bacterial signalling pathway alter evolutionary routes of antibiotic resistance in Escherichia coli

Antibiotic resistance in bacteria frequently evolves due to mutations in drug-target or detoxifying genes. Pre-existing polymorphisms in these genes are likely to influence the evolvability of drug resistance. However, the role of polymorphisms in driving resistance evolution and the underlying molecular mechanisms are poorly understood. Here, we demonstrate that polymorphisms in a signalling pathway impact the evolvability of trimethoprim (TMP) resistance in Escherichia coli. When challenged with TMP, de-repression of the PhoQ-PhoP two-component signalling system in E. coli transcriptionally upregulates the drug target Dihydrofolate Reductase (DHFR), leading to drug resistance. We identified and characterised naturally occurring polymorphisms in PhoQ and DHFR that modulate intrinsic antibiotic susceptibility. These variants also altered the ability of E. coli to evolve de novo TMP resistance as a result of epistasis with adaptive mutations. Interestingly, a strain harbouring a less-evolvable DHFR variant acquired a novel mutation in PhoQ under TMP pressure that hyperactivated the signalling pathway, conferring high-level resistance but at a large fitness cost. This mutation was not observed in wild type but reached fixation rapidly in the background of the DHFR variant. Using RNA-sequencing we compare how natural variants and adaptive mutations in PhoQ affect the expression of PhoP-target genes and downstream regulatory pathways in E. coli. Finally, we uncouple the roles of resistance level by DHFR overproduction and fitness cost by activation of the RpoS regulon to explain why E. coli more frequently evolves to de-repress PhoQ than hyperactivate it under drug pressure. Our study, thus, demonstrates that pre-existing polymorphisms alter both, evolvability and mutation landscapes during antibiotic adaptation. This work establishes the PhoQ-PhoP-DHFR pathway as an experimental paradigm to understand the evolution of signalling pathways under environmental selection.