Proteomic Characterization of the Outer Membrane Vesicle of Pseudomonas putida KT2440

Abstract

Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, Acinetobacter baumannii DU202

“…Transmission electron microscopy (TEM) of OMVs was performed as described previously [20]. Briefly, OMV fractions were diluted with PBS, centrifuged at 150,000× g for 3 h, resuspended in PBS, applied to 400-mesh copper grids, stained with 2% uranyl acetate and visualized on a TEM instrument (FEI, USA) operating at 120 kV.…”

“…C18 reversed-phase column at a flow rate of 300 nl/min. HPLC conditions and search parameters for tandem mass spectrometry (MS/MS) analysis were applied as described previously [20]. All MS and MS/MS spectra obtained using the LTQ-Velos ESI ion trap mass spectrometer were acquired in data-dependent mode (Thermo Fisher Scientific, USA).…”

“…Cells were plated onto 12-well culture plates, and OMVs were applied and incubated for 24 h. For apoptosis assays, cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V, propidium iodide (PI) and Hoechst reagent according to the manufacturer’s instructions. Stained cells were analysed using a NucleoCounter NC-3000 image cytometer (ChemoMetec, Denmark) [20]. …”

The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles

“…For example, in X. fastidiosa subsp fastidiosa strain Temecula, a proteomic approach to the secretome identified 17 proteins without signal peptides (Nascimento et al, 2016). Other studies have identified proteins related to glycolysis and transcription factors inside OMVs (Altindis et al, 2014; Chen et al, 2014; Choi et al, 2014). The pathways used by X. fastidiosa subsp pauca strain 9a5c to secrete wild-type XfYgiT protein remain unknown, necessitating further studies to unravel the underlying mechanism.…”

“…Many hypotheses have been proposed concerning the formation of OMVs and their function (i.e., modulation of the microbial environment to kill competing species, signaling likely sites of nodulation for biofilm formation and modulating the immune response system in host cells; Choi et al, 2014; Schwechheimer et al, 2014; Pérez-Cruz et al, 2015; Turner et al, 2015). This study is the first to report the presence of XfYgiT inside OMVs (Supplemental Figures 2A,C).…”