OXAs class D Beta-lactamases “get greasy” in Acinetobacter: selective advantage of OMVs

CAPODIMONTE, LUCIA; LÓPEZ, CAROLINA; BONOMO, ROBERT A.; VILA, ALEJANDRO J.

Conferencia; 14th Beta-lactam and Beta-lactamase meeting; 2023

University of L Aquila, Italy

β-lactamases from Gram-negative bacteria have been historically regarded as mainly solubleperiplasmic enzymes. A few membrane-bound enzymes, such as BRO from M. catarralis, wereconsidered an exception to this rule. This preconception changed upon the characterization ofthe NDM family as lipid-anchored proteins located in the inner leaflet of the outer membrane.Lipid anchoring can be predicted in silico by the presence of a lipobox sequence, a short aminoacid motif located in the signal peptide that contains a cysteine residue responsible for lipidation.Here we report that more than 50% of the OXA class D β-lactamases contain these lipoboxsequences. We then selected two paradigmatic OXA β-lactamases with a lipobox, OXA-23 andOXA-24. We found that both enzymes are membrane-bound proteins in Acinetobacter baumanniiby cell fractionation and immunodetection. The membrane fractions containing OXAs weresubjected to differential solubilization, and we found that OXAs were only solubilized upontreatment with Triton X-100, confirming that they are lipid-anchored proteins.We then isolated outer membrane vesicles (OMVs) from Acinetobacter baumannii cells expressingeach OXA enzyme. OMVs are spherical portions of the outer membrane enclosing periplasmiccontent released during the normal cell cycle and have many and varied roles, such as traffic ofvirulence factors and regulation of interactions within bacterial communities. OXA-23 and OXA-24were detected in OMVs, which may impart a variety of benefits to them, including protectionfrom proteolytic degradation and enhancement of long-distance delivery. We found that OMVscarrying OXAs have a protective effect on susceptible bacteria against 8-fold MIC piperacillin. Thischaracteristic could be crucial in polymicrobial infections, in which Acinetobacter spp are usuallyinvolved. These findings reveal that membrane binding is much more common that expected, andprompt for the elucidation of the benefits of this cellular localization for OXA enzymes.