RESUMEN
Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.
Asunto(s)
Acinetobacter/patogenicidad , Proteínas de la Membrana Bacteriana Externa/análisis , Membrana Celular/química , Proteoma/análisis , Vesículas Secretoras/química , Factores de Virulencia/análisis , Membrana Celular/metabolismo , Cromatografía Liquida , Vesículas Secretoras/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10(-6) to 10(-8), with transfer efficiencies of approximately 10(3) and 10(2) per µg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.
Asunto(s)
Acinetobacter/genética , ADN Bacteriano/análisis , Transferencia de Gen Horizontal , Vesículas Secretoras/química , Transformación Bacteriana , ADN Bacteriano/genética , Escherichia coli/genética , Plásmidos/análisisRESUMEN
The role of vesicle-mediated gene transfer in Acinetobacter baumannii populations has been investigated in the last decade. Importantly, outer membrane vesicles (OMVs) secreted from A. baumannii cells have proven to be efficient agents of transfer of antimicrobial resistance genes to other bacterial species. However, the measurement of vesicle-mediated transfer depends on many experimental parameters. Here, we describe an experimental method useful to study transfer of DNA via membrane vesicles of A. baumannii in various bacterial populations.