Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of Aeromonas hydrophila OMVs is missing. In this study we analyzed the composition of purified OMVs from A. hydrophila ATCC® 7966TM by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90–170 nm. Moreover, 211 unique proteins were found in OMVs from A. hydrophila; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from A. hydrophila ATCC® 7966TM induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of A. hydrophila ATCC® 7966TM and their interaction with the host cell.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived roughmutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified