Hitch a ride: cargo sorting of metallo-b-lactamases (MBLs) into outer membrane vesicles (OMVs) is determined by protein-membrane interactions

LÓPEZ, CAROLINA; PRUNOTTO, ALESSIO; BAHR, GUILLERMO; CAPODIMONTE, LUCÍA; BONOMO, ROBERT A.; GONZALEZ, LISANDRO J.; DAL PERARO, MATTEO; VILA, ALEJANDRO J.

Ibaraki

Workshop; EMBO Workshop "Bacterial membrane vesicles: Biogenesis, functions and medical applications"; 2021

EMBO

Background: The role of OMVs in the dissemination of antimicrobial resistance (AMR) is linked to their ability to transport β-lactamases and related plasmids. MBLs hydrolyze nearly all β-lactam antibiotics. MBLs of major clinical importance are NDM, VIM and IMP. NDMs are lipoproteins that are anchored to the outer membrane (OM), while VIM and IMP are soluble periplasmic enzymes. The secretion of lipidated and soluble MBLs into OMVs and the molecular bases determining the differential packaging of MBLs into OMVs was studied. Methods: liposome flotation, mutagenesis and molecular dynamics simulations were employed to study the interaction of NDM-1, VIM-2 and IMP-1 and variants with the bacterial membranes. OMVs were purified from E. coli expressing the MBLs. Protein levels were determined by immunodetection. Results & Conclusions: NDM-1 is selectively packaged into vesicles by being anchored to the OM. A soluble variant (NDM-1 C26A) is packaged in smaller amounts, indicating that membrane anchoring is not the only molecular feature determining cargo selection. We studied the protein-membrane interactions of NDM-1 (both lipidated and soluble), IMP-1 and VIM-2. Attractive electrostatic interactions between the membrane and NDM-1 or IMP-1 were demonstrated that favor their packaging into OMVs. In contrast, the lack of interaction of VIM-2 with the membrane results in lower levels in OMVs. Mutagenesis of the residues involved in protein-membrane interactions in NDM-1 and IMP-1 disrupts the interaction with the membrane and reduces the MBL levels into vesicles, confirming that these interactions play a role in selecting the protein cargo into vesicles.