Isolation and activity of Listeria monocytogenes-derived membrane vesicles

Abstract:

[Background] Extracellular vesicles are lipid bound vesicles harboring specific subsets of bioactive proteins, lipids, nucleic acids, and metabolites, secreted by all living cells into the extracellular space. They have been named as membrane vesicles (MVs) in Gram-positive bacteria. Recently, vesicle production is described in Listeria monocytogenes, a Gram-positive intracellular foodborne pathogen that causes listeriosis in human as well as in animals. However, the knowledge about the impact of L. monocytogenes-derived MVs on host health and disease is still limited. [Objective] To establish and improve the isolation method to extract MVs from L. monocytogenes, and to explore its roles of biological activities. [Methods] Two methods, the ultrafiltration concentration method and Optiprep density gradient centrifugation method, were used to extract MVs from the wild strain EGDe and its virulence mutants (EGDe?prfA, EGDe?prfA+pERL3-prfA*) and compared. The impact of different virulent L. monocytogenes-derived membrane vesicles on the bacterial biofilm formation and pathogenesis to the insect infection model was investigated. [Results] Comparing with the ultrafiltration concentration method, the MVs isolated by the Optiprep density gradient centrifugation have got higher yield and presented more uniform and clear image under transmission electron microscope, but it’s relatively complicated and took a long time; All different virulent L. monocytogenes strains can secrete membrane vesicles with 20?200 nm in diameter, and no obvious differences in their shapes and sizes. However, these vesicles inhibited biofilm formation of L. monocytogenes, harbored a certain hemolytic activity, reduced the pupation and survival rate of the Helicoverpa armigera larvae, and even caused them death. The toxicity of MVs to the larvae showed a clear link with the virulence of strains they originated. [Conclusion] L. monocytogenes-derived membrane vesicles have got versatile roles, and likely participate in bacteria-bacteria and bacteria-host interactions. These results may be of great significance for the further study of the formation and function of Gram-positive bacteria-derived MVs and the pathogenic mechanism of L. monocytogenes.