IMPACT OF THE INTERACTION BETWEEN THE BACTERIAL MEMBRANE AND CARBAPENEMASES ON THEIR TRANSPORT INTO OUTER MEMBRANE VESICLES

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

Congreso; REUNIÓN CONJUNTA SAIB-SAMIGE 2020 - ONLINE; 2020

SAIB-SAMIGE

The rise in carbapenem-resistant bacteria is particularly concerning, since this class of β-lactam antibiotics is reserved as a last resort option for life-threatening infections. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Among MBLs, the plasmid-borne NDMs, VIMs and IMPs are the enzymes with the highest clinical relevance and geographical dissemination. 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). These spherical lipid bilayer nanostructures are released by all Gram-negative bacteria. The role of OMVs in transporting β-lactamases is a growing paradigm within the field of antimicrobial resistance, and the molecular features that define the incorporation of β-lactamases into vesicles are not known. We have shown that membrane anchoring favors secretion of NDM-1 into vesicles by constructing the soluble mutant C26A, which lacks the lipidated Cys residue that enables membrane anchoring. C26A NDM-1 is secreted in smaller quantities than the native enzyme. This suggests that in addition to the role of membrane anchoring, others interactions between MBLs and membranes would also have an impact on cargo selection in vesicles. Here we study and identify the interactions of MBLs with bacterial membrane that modulate their incorporation in vesicles. 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 expressing NDM-1, IMP-1, VIM-2 and their variants. MD simulations and liposome flotation assays revealed that membrane anchored NDM-1 interacts with the membrane through its lipid group and its globular domain, the latter 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 the assays showed that VIM-2 did not interact with liposomes nor with simulated membrane 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 model membrane 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. We conclude that the transport 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.