OXA β-lactamases fromAcinetobacterspp. are membrane-bound and secreted into outer membrane vesicles
Authors: Capodimonte Lucia , Meireles Fernando Teixeira Pinto , Bahr Guillermo , Bonomo Robert A. , Dal Peraro Matteo , López Carolina , Vila Alejandro J. 
Published: November 2024

ABSTRACTβ-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the class D OXAs. Namely, 60% of the OXA class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (> 99%) putative lipidated OXAs are present inAcinetobacterspp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins inAcinetobacter baumannii. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) fromAcinetobacter baumanniicells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protectA. baumannii, Escherichia coliandPseudomonas aeruginosacells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in whichAcinetobacter spp.are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of class D OXA β-lactamases (all fromAcinetobacterspp) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40 are membrane-bound proteins inA. baumannii. This cellular localization favors secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated byAcinetobacterspp., particularly in polymicrobial infections, being a potent evolutionary advantage.