Metallo-beta-lactamases: a Tug of War between Bacteria and the Immune System for the available Zn(II)

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

Simposio; 32nd Annual Symposium of The Protein Society; 2018

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 alsoto stabilize the protein scaffold. During infection, the immune system elicits a response that scavenges the available Zn(II), impacting in the activity of stability of these proteins, thus compromising bacterial survival. However, the activity and stability of these proteins in vitro does not necessarily correlates with those in the periplasm. Thus, the whole picture must bedescribed by means of an integrated approach. 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 to endure the action of the immune system response. 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. Thus,natural NDM variants with enhanced Zn(II) binding affinity have been selected,overriding the most common evolutionary pressure acting on catalytic efficiency.