The Membrane Affinity of Metallo-β-Lactamases Dictates their Sorting Into Outer Membrane Vesicles

LÓPEZ, CAROLINA; GONZÁLEZ, LISANDRO; PRUNOTTO, ALESSIO ; DAL PERARO, MATTEO ; BAHR, GUILLERMO; VILA, ALEJANDRO J.

https://cattendee.abstractsonline.com/meeting/9103/eposters

Congreso; ASM Microbe Online 2020; 2020

American Society For Microbiology (ASM)

Background: The Metallo-β-lactamases, MBLs, with the highest clinical relevance and geographical dissemination are the plasmid-borne NDMs, VIMs and IMPs. While the last two are soluble periplasmic proteins, NDMs are lipoproteins anchored to the bacterial outer membrane (OM). Membrane-anchoring enables secretion of NDM-1 into outer membrane vesicles (OMVs). Instead, a soluble NDM-1 variant (lacking the lipidation site) is not efficiently secreted into OMVs. NDM-containing vesicles can protect nearby populations of otherwise sensitive bacteria, favoring dissemination of resistance. Here we study if this mechanism of secretion extends to other (soluble) MBLs and w identify that interactions of these enzymes with OM modulate vesicle secretion. Methods: Liposome flotation assays, mutagenesis and molecular dynamics (MD) simulations were used to study the interaction of NDM-1, VIM-2 and IMP-1 with the membranes. OMVs were purified from Escherichia coli DH5α expressing NDM-1, IMP-1, VIM-2 and their variants. MBL levels were determined by immunodetection. Results: MD simulations and liposome flotation assays revealed that membrane-anchored NDM-1 interacts with the membrane not only through its lipid group, but also through its globular domain, driven by electrostatic contributions from two Arg residues. Replacement of the two Arg by Glu residues reduced secretion of NDM-1 into OMVs. Regarding soluble MBLs, IMP-1 was much more efficiently secreted into OMVs than VIM-2. These results are due to specific membrane-protein interactions, since bot simulations and flotation assays showed that VIM-2 did not interact with liposomes nor with simulated OM bilayers. This can be attributed to a large region with negative electrostatic potential in the surface of VIM-2. On the contrary IMP-1 strongly interacted with OM bilayers, accounting for its incorporation at higher levels than VIM-2 in the vesicles. MD simulations predict that four Lys residues in IMP-1 sequence may determine its interaction with the membrane. An IMP-1 variant in which these Lys were replaced by Glu residues showed an impaired binding with the OM, confirming this hypothesis. Conclusion:The secretion of MBLs into vesicles is favored by key interactions with the membrane, either by anchoring and/or electrostatic interactions with the soluble domain of MBLs.https://cattendee.abstractsonline.com/meeting/9103/eposters