title: 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.
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Liao
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
authors.forename: Shu-Chen
authors.initials: SC
authors.lastname: Kuo
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Ming-Hsien
authors.initials: MH
authors.lastname: Chiang
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan Department of Emergency Department, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Yi-Tzu
authors.initials: YT
authors.lastname: Lee
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
authors.forename: Wang-Chou
authors.initials: WC
authors.lastname: Sung
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
authors.forename: You-Hsuan
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan tecklayyy@gmail.com.
authors.forename: Te-Li
authors.initials: TL
authors.lastname: Chen
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan Institute of Emergency and Critical Care Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Chang-Phone
authors.initials: CP
authors.lastname: Fung
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D021381:Protein Transport; D012395:Rose Bengal / Q000494:pharmacology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D022142:Secretory Vesicles / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D015780:Carbapenems; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D012395:Rose Bengal; D001618:beta-Lactamases; C534889:beta-lactamase OXA-58, Acinetobacter baumannii; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins