Evolutionary Mechanisms for Metallo-Beta-Lactamases

MARÍA ROCÍO MEINI; MARIANO M. GONZÁLEZ; PABLO E. TOMATIS; ALEJANDRO J. VILA

San Francisco

Congreso; ICAAC 2012- Interscience Conference on Antimicrobial Agents and Chemotherapy; 2012

American Society for Microbiology

Background: Metallo-beta-lactamases (MBLs) constitute a resistance mechanism of bacteria towards beta-lactams antibiotics. In contrast to serine-beta-lactamases, they are able to hydrolyze all beta-lactams. MBLs are rapidly spreading and new evolved variants are continually discovered while there is no therapeutic inhibitor available for them. This situation turns MBLs into a worrisome clinical threat.Methods: Directed Molecular Evolution strategies simulate natural evolution process in the laboratory. We have employed these techniques to generate evolved variants of the metallo-β-lactamase BcII from Bacillus cereus. The capacity to confer resistance of these variants was characterized by MICs towards different beta-lactams. The isolated proteins were characterized by using biochemical and biophysical tecniques, including Nuclear Magnetic Resonance (NMR) studies to assess their structural flexibility. Results: Four mutations were responsible of the enzyme evolution. Two of them, G262S and N70S are located under the active site floor. The other two, V112A and L250S, are located far away from the active site. We identified the possible pathways that could lead to the acquisition of larger levels of resistance and we have studied the interactions between these mutations. Interestingly, only one third of the 24 possible evolutionary pathways are accessible. This restriction is due to the fact that mutation G262S acts as a bottleneck in the evolutionary mechanism, providing a more evolvable genetic background than wt BcII. We have found that the hydrolytic efficiency (which becomes the limiting property), is first optimized by mutation G262S. The other mutations play different roles affecting the enzyme stability, the affinity for the essential Zn(II) cofactor and the flexibility of the loops flanking the active site (as revealed by NMR studies). Conclusions: This work highlights key ?hotspots? for evolvability in the protein sequence which improve MBL performance in vivo. We also show the contribution and interplay of different protein properties that are under selection pressure: catalytic activity, stability, affinity for Zn(II) and flexiblity around the active site.