Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.

Salmonella enterica serovar Enteritidis is the most prevalent serotype that causes human infections worldwide. Consumption of S. Enteritidis-contaminated animal foods is a major source of human infections; however, eradicating bacteria from animals remains difficult. Therefore, it is necessary to develop new measures to prevent and control salmonellosis. Here, we used the outer-membrane vesicles (OMVs) of S. Enteritidis and assessed their protective efficacy and immune response in mice. Deletion of tolR in S. Enteritidis increased the production and size of OMVs compared to those in the wild type (WT) and ΔrfaQ strains. Intramuscular immunization with OMVs conferred greater protection than intraperitoneal and intranasal immunization. Moreover, OMVs extracted from both WT and ΔtolR strains provided an 83.3% protective rate in mice challenged with S. Enteritidis, which was higher than that provided by OMVs extracted from the ΔrfaQ strain. However, compared with OMVs from the ΔtolR strain, OMVs from WT and ΔrfaQ strains rapidly eradicated S. Enteritidis colonizing the liver, spleen, ileum, and cecum of BALB/c mice after immunization. Immunization with OMVs from each of the three strains induced humoral immune responses and showed no side effects on the growth of mice. Our study revealed that OMVs from various S. Enteritidis strains could be developed for use as subunit vaccine candidates against nontyphoidal Salmonella infections in mammals.

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation-driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.

Outer membrane and membrane vesicles (OMV/MV) are released from bacteria and participate in cell communication, biofilm formation and host-pathogen interactions. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that catalyze post-translational deimination/citrullination of proteins, causing structural and functional changes in target proteins. PADs also play major roles in the regulation of eukaryotic extracellular vesicle release. Here we show phylogenetically conserved pathways of PAD-mediated OMV/MV release in bacteria and describe deiminated/citrullinated proteins in E. coli and their derived OMV/MVs. Furthermore, we show that PAD inhibitors can be used to effectively reduce OMV/MV release, both in Gram-negative and Gram-positive bacteria. Importantly, this resulted in enhanced antibiotic sensitivity of both E. coli and S. aureus to a range of antibiotics tested. Our findings reveal novel strategies for applying pharmacological OMV/MV-inhibition to reduce antibiotic resistance.