Lipidation of the Acinetobacter baumannii OXA-58 CHDL promotes its secretion in association to outer membrane vesicles

MORAN BARRIO J; BRAMBILLA L; FABBRI C; MARCHIARO P; CAMERANESI MM; LIMANSKY AS; VIALE AM

Sevilla

Congreso; 11th International Symposium on the biology of Acinetobacter; 2017

Objectives. The emergence of multidrug-resistant, carbapenem-resistant Acinetobacter baumannii constitutes a worrisome problem in health-care institutions worldwide. Carbapenem resistance results mainly from the overproduction of acquired carbapenemases such as OXA-type class-D beta-lactamases (CHDLs) with minor contributions of other factors including decreased outer membrane (OM) permeability (1). A. baumannii CHDLs have been characterized at the kinetic and structural levels and the genetic context of blaOXA genes characterized (2),but less attention has been given to the distinct aspects of their biogenesis pathway which also offers potential targets for antimicrobial design. Here, we report that the OXA-58 CHDL undergoes lipidation during transit to the A. baumannii periplasm, and that this post-translational modification is essential for membrane attachment and association to outer membrane vesicles (OMVs).Methods.As a model we employed A. baumannii ATCC 17978 (Ab17978) transformed with plasmid pOXA-58 directing high-level expression of the blaOXA-58 gene (3). Lipidation of OXA-58 was analysed by [3H]palmitate labelling of the cells followed by protein analysis by SDS-PAGE. Presence of OXA-58 in different cell fractions (periplasm, membranes, secreted OMVs) was analyzed with specific antibodies and by spectrophotometric determination of imipenemase activity. Imipenem (IPM) resistance levels in susceptible cells were evaluated byKirby-Bauer agar disk diffusion assays (1) and by the Triton-Hodge test.Results.We predicted the presence of lipoboxes containing the Cys target residue at the N-terminal regions of A. baumannii CHDLs. Differential [3H]palmitate cell labelling followed by SDS-PAGE indicated specific lipidation of OXA-58 in Ab17978/pOXA-58 cells. Cell fractionation analysis indicated that lipidation was necessary for both OXA-58 membrane attachment and stability, as judged by the recovery of the wild-type enzyme mainly in associationto membrane fractions and that of a mutant enzyme lacking the target Cys mainly in soluble periplasmic fractions in largely reduced amounts. Lipidation was found essential for OXA-58 recruitment as cargo into OMVs. The enzyme was associated to the inner side of the vesicles as judged by protease treatment, a result supported by the significant increments of imipenemase activity of the vesicles after Triton X-100 permeabilization. This secreted OXA-58 conferred protection from IPM killing to susceptible strains of A. baumannii and Escherichia coli after surfactant treatment of the OMVs.Conclusions.OXA-58 is a membrane-anchored lipoprotein in A. baumannii, and this post-translational mechanism offers to this enzyme stability and the capability to be secreted as an OMV cargo. The observation that the secreted enzyme is capable of conferring carbapenem protection to accompanying susceptible bacteria suggests roles of this mechanism in the progression of polymicrobial infections.1. Morán-Barrio et al. (2017) Antimicrob Agents Chemother 61(3). pii: e01737-16.2. Potron et al. (2015) Int J Antimicrob Agents 45:568-585.3. Ravasi et al. (2011) Antimicrob Agents Chemother 55:917-920.