Studying genetic and enzymatic constraints driving evolution of antibiotic resistance

Official URL: http://risc01.sabanciuniv.edu/record=b1589544 (Table of Contents)

World is heading towards a post-antibiotic era due to emergence of antibiotic resistance. Several fatal infectious diseases caused by antibiotic resistant bacteria cannot be treated anymore using the existing antibiotic surplus. Novel antibiotics or novel strategies to use antibiotic more efficiently are therefore crucial to combat against resistance. However, both of these approaches require a clear understanding of antibiotic resistance at molecular and genetic levels. Here in this study, we studied evolutionary dynamics of trimethoprim resistance under dynamically sustained drug selection. Using a custom made continuous culture device that we call the Morbidostat; we evolved drug sensitive Escherichia coli cells against increasing levels of trimethoprim adapting strong or mild dilution rates. First, using Illumina whole genome sequencing and Sanger sequencing, we identified trimethoprim resistance conferring mutations in dihydrofolate reductase (folA) gene and the order that these mutations appear in the population. Our results suggest that clonal interference between different genotypes is common and longer under strong dilution where trimethoprim stress is applied in shorter and steeper pulses. Second, we cloned and purified dihydrofolate reductase (DHFR) enzymes with single mutations and carried out biochemical assays to quantify mutant enzymes’ enzymatic activities. Our preliminary results showed that DHFR mutants have slightly worse substrate affinity (higher km values) but up to ~20 fold elevated catalysis rate (kcat/km) compared to their wild type ancestor. We conclude that trimethoprim-resistance-conferring DHFR mutations decrease affinity to both enzyme’s substrate and competing drug molecules, yet enzymatic activity, which is essential for folic acid synthesis, is still adequately efficient to maintain bacterial fitness.