Adaptation Through Lifestyle Switching Sculpts the Fitness Landscape of Evolving Populations: Implications for the Selection of Drug-Resistant Bacteria at Low Drug Pressures.

Novel genotypes evolve under selection through mutations in pre-existing genes. However, mutations have pleiotropic phenotypic effects that influence the fitness of emerging genotypes in complex ways. The evolution of antimicrobial resistance is mediated by selection of mutations in genes coding for antibiotic-target proteins. Drug-resistance is commonly associated with a fitness cost due to the impact of resistance-conferring mutations on protein function and/or stability. These costs are expected to prohibit the selection of drug-resistant mutations at low drug pressures. Using laboratory evolution of rifampicin resistance in Escherichia coli, we show that when exposed intermittently to low concentration (0.1 × minimal inhibitory concentration) of rifampicin, the evolution of canonical drug resistance was indeed unfavorable. Instead, these bacterial populations adapted by evolving into small-colony variants that displayed enhanced pellicle-forming ability. This shift in lifestyle from planktonic to pellicle-like was necessary for enhanced fitness at low drug pressures, and was mediated by the genetic activation of the fim operon promoter, which allowed expression of type I fimbriae. Upon continued low drug exposure, these bacteria evolved exclusively into high-level drug-resistant strains through mutations at a limited set of loci within the rifampicin-resistance determining region of the rpoB gene. We show that our results are explained by mutation-specific epistasis, resulting in differential impact of lifestyle switching on the competitive fitness of different rpoB mutations. Thus, lifestyle-alterations that are selected at low selection pressures have the potential to modify the fitness effects of mutations, change the genetic structure, and affect the ultimate fate of evolving populations.

Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes.

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.