Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D β-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.