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

ANTONELA R. PALACIOS; CAROLINA LÓPEZ; A. J. VILA; GUILLERMO BAHR; MA. AGUSTINA ROSSI; TOMATIS PABLO E.; ESTEFANIA GIANNINI; JULIANA DELMONTI; GONZÁLEZ LISANDRO J

Simposio; Symposium on Protein Biophysics at the end of the World, Santiago de Chile, 2018.; 2018

Protein evolution can be describedas a walk on sequence space where a fitness value is assigned to eachparticular sequence. A major challenge to understand thebiological mechanisms of protein evolution is the attempt to correlate genotypewith 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-lactamasesis an ideal system to study protein evolution, since the chances of a whole organismto 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 resistancemechanism of pathogenic and opportunistic bacteria against carbapenems,considered as last resort drugs. Zn(II) binding is critical in the bacterialperiplasm, not only to activate these enzymes and provide resistance, but alsoto stabilize the protein scaffold. This phenomenon is not paralleled by invitro studies. We developed a strategy aimed to correlate the biochemical andbiophysical 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 dissectthe molecular features that are tailored by accumulating mutations duringevolution. We have applied this approach to invitro evolved protein in the laboratory, as well as to natural allelicvariants selected in clinical strains. This has allowed us to account for theepistatic interactions between mutations at a structural level. We have also studied thenatural evolutionary landscape of allelic variants of a clinically relevantlactamase (NDM), that has been shaped by Zn(II) deprivation conditions as thoseinduced by the host immune response. As a consequence, natural NDM variantswith enhanced Zn(II) binding affinity have been selected, overriding the mostcommon evolutionary pressure acting on catalytic efficiency. Finally, mechanisticstudies based on these evolutionary criteria have allowed the design of novelinhibitors able to restore the antibacterial activity of carbapenems againstotherwise resistant clinical strains.