The Last Frontier of Antibiotic Resistance: At the Heart of Protein Evolution

BAHR, GUILLERMO; GIANNINI, ESTEFANÍA; PALACIOS, ANTONELA R.; ROSSI, AGUSTINA; DELMONTI, JULIANA; LÓPEZ, CAROLINA; TOMATIS, PABLO E.; GONZÁLEZ, LISANDRO J.; VILA, ALEJANDRO J.

Simposio; 3rd Protein Biophysics at the End of the World (PBATEOTW); 2018

Sociedad de Bioquímica y Biología Molecular de Chile

Protein evolution can be described as a walk on sequence space where a fitness value is assigned to each particular sequence. A major challenge to understand the biological mechanisms of protein evolution is the attempt to correlate genotype with phenotype and fitness at a molecular level. This is a complex challenge, involving the assessment of biochemical, biophysical and structural features tomany protein variants. Antibiotic resistance mediated by β-lactamases is an ideal system to study protein evolution, since the chances of a whole organism to survive depend on the availability of a folded, stable and active protein inthe proper cellular compartment (the bacterial periplasm). Metallo-β-lactamases (MBLs) are Zn(II)-dependent β-lactamases that constitute the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. Zn(II) binding is critical in the bacterial periplasm, not only to activate these enzymes and provide resistance, but also to stabilize the protein scaffold. This phenomenon is not paralleled by in vitro studies. We developed a strategy aimed to correlate the biochemical and biophysical features in purified enzymes with those in the bacterial periplasm, ultimately leading to the selected phenotype, i.e., resistance to antibiotics. This strategy allows us to dissect the molecular features that are tailored by accumulating mutations during evolution. We have applied this approach to in vitro evolved protein in the laboratory, as well as to natural allelic variants selected in clinical strains. This has allowed us to account for the epistatic interactions between mutations at a structural level. We have also studied the natural evolutionary landscape of allelic variants of a clinically relevant lactamase (NDM), that has been shaped by Zn(II) deprivation conditions as those induced by the host immune response. As a consequence, natural NDM variants with enhanced Zn(II) binding affinity have been selected, overriding the most common evolutionary pressure acting on catalytic efficiency. Finally, mechanistic studies based on these evolutionary criteria have allowed the design of novel inhibitors able to restore the antibacterial activity of carbapenems against other wise resistant clinical strains.