Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection.
Keywords: Caco-2 cells; Cytotoxicity; Endocytosis; Host-pathogen interactions; Listeria monocytogenes; Membrane vesicles.
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