Mutations in dihydrofolate reductase from Escherichia coli: Impact on aggregation, trimethoprim resistance and fitness

Abstract

Alteration of the structures of proteins due to non-synonymous substitutions can affect their biochemical functions either by perturbation of active sites geometry, decrease in expression level or aggregation and misfolding. These micro-level changes are translated to organism-level phenotypes like growth rate changes, which finally impact the fitness of an individual. The relationship between mutational changes and organism-level changes is referred to as the genotype-phenotype relationship. In this study, we are trying to establish the genotype-phenotype relationship for mutational changes in the metabolic enzyme dihydrofolate reductase (DHFR) in Escherichia coli that confer resistance to the antibiotic trimethoprim. DHFR is expressed as low-copy numbers whose activity is essential for purine and pyrimidine synthesis in bacteria. In this project it is demonstrated that a sub-set of trimethoprim-resistance associated mutations at W30 residue destabilise DHFR leading to its aggregation. Concurrently, some of these mutations are associated with lowered fitness relative to wild type. This decrease in fitness is most likely due to lowered expression and lowered activity of the mutant DHFR enzymes and not due to aggregation-associated cytotoxicity. Using this system, the ultimate aim is to delineate the relationship between expression level, solubility, stability and organismal fitness that would influence the selection dynamics of trimethoprim-resistant bacteria.