Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System.

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of ß-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

Bacterial ghost technology for pesticide delivery.

Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis. Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery. We show, using fluorescence-labeled P. cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm). Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole. In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested. The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics. The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens. The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion. This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.