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

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

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

Protein evolution can be described as a walk on sequence space where a fitness value is assigned toeach particular sequence. A major challenge to understand the biological mechanisms of proteinevolution is the attempt to correlate genotype with phenotype and fitness at a molecular level. This is acomplex challenge,involving the assessment of biochemical, biophysical and structural features to manyprotein variants. Antibiotic resistance mediated by β-lactamases is an ideal system to study proteinevolution, since the chances of a whole organism to survive depend on the availability of a folded, stableand active protein in the proper cellular compartment (the bacterial periplasm). Metallo-β-lactamases(MBLs) are Zn(II)-dependent β-lactamases that constitute the latest resistance mechanism ofpathogenic 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. Wedeveloped a strategy aimed to correlate the biochemical and biophysical features in purified enzymeswith those in the bacterial periplasm,ultimately leading to the selected phenotype, i.e., resistance toantibiotics. This strategy allows us to dissect the molecular features that are tailored by accumulatingmutations 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 theepistatic interactions between mutations at a structural level. We have also studied the naturalevolutionary landscape of allelic variants of a clinically relevantlactamase (NDM), that has been shapedby 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 mostcommon evolutionary pressure acting on catalytic efficiency. Finally, mechanistic studies based on theseevolutionary criteria have allowed the design of novel inhibitors able to restore the antibacterial activityof carbapenems against otherwise resistant clinical strains.