Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion

TLDR

Pseudomonas aeruginosa releases membrane vesicles during normal growth that can transport DNA and virulence factors, potentially contributing to genetic transformation, disease, and septic shock. The study isolated natural and gentamicin‑induced membrane vesicles by filtration and differential centrifugation and characterized their biological activities. Gentamicin exposure tripled vesicle release, producing spherical 50–150 nm bilayer vesicles enriched in B‑band lipopolysaccharide, containing virulence enzymes, and carrying more DNA than natural vesicles.

Abstract

Pseudomonas aeruginosa blebs-off membrane vesicles (MVs) into culture medium during normal growth. Release of these vesicles increased approximately threefold after exposure of the organism to four times the MIC of gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs and g-MVs, respectively) were isolated by filtration and differential centrifugation, and several of their biological activities were characterized. Electron microscopy of both n-MVs and g-MVs revealed that they were spherical bilayer MVs with a diameter of 50 to 150 nm. Immunoelectron microscopy and Western blot (immunoblot) analysis of the vesicles demonstrated the presence of B-band lipopolysaccharide (LPS), with a slightly higher proportion of B-band LPS in g-MVs than in n-MVs. A-band LPS was occasionally detected in g-MVs but not in n-MVs. In addition to LPS, several enzymes, such as phospholipase C, protease, hemolysin, and alkaline phosphatase, which are known to contribute to the pathogenicity of Pseudomonas infections were found to be present in both vesicle types. Both types of vesicles contained DNA, with a significantly higher content in g-MVs. These vesicles could thus play an important role in genetic transformation and disease by serving as a transport vehicle for DNA and virulence factors and are presumably involved in septic shock.