Periodic Reporting for period 1 - PROBYDE (Probing the bypassability of genetic constraints in drug-resistance enzymes)
Periodo di rendicontazione: 2021-07-01 al 2023-06-30
In brief, we uncovered the potential adaptive pathways available via single step mutations for several drug-resistance enzymes to improve their activity. This information enabled making predictions about antibiotic resistance evolution based on single-gene (in-vitro) observations of repeatability. I also conducted experimental adaptation to antibiotics with a model of E. coli clinical strain carrying the different drug-resistance enzymes. Comparison of the potential versus realized adaptive pathways provided insight into the bypassability of genetic constraints in the drug-resistance enzymes and into the role of mutation biases and GC content in this process. If we were able to predict the most probable new mutants, we could anticipate evolution and design new antimicrobials or inhibitors to tackle the expected new variants.
During the three-month secondment, I conducted directed mutagenesis experiments on the chromosome expressed penicillin binding protein PBP3, a protein essential for cell growth and division and are therefore critical targets for β-lactam antibiotics. Several mutations in PBP3 conferring resistance to cephalosporins were described previously. Our objective was to evaluate if there were presented epistatic constrains in the mutations controlling the system. Using CRISPR/Cas9-mediated error prone genome editing methodology (CREPE), a technique that allows the high-throughput generation of mutants, I generated a library of mutants within a targeted gene in its native genomic context. Moreover, I performed reconstruction experiments demonstrating selective effects of candidate mutations separately and in combination.