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BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.

RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.

CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.

Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. siRNA-mediated gene knock-down revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain, leucine rich repeat containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum sensing regulator HapR which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs in infective conditions. This work thus identified a new facet of how Vibrio affects host immune responses and defines a role for the quorum sensing machinery in this process.

Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similar to several other Gram-negative species this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G(2) cell cycle arrest and/or apoptosis in many mammalian cell types. In this study we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a) in contrast to OMVs from a D7SS cdtABC mutant induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates, belonging to serotypes b, and c, respectively, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although, the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

Background: Gram-negative bacteria release large amounts of outer membrane vesicles (OMVs) during normal growth. OMVs from pathogenic bacteria are known to carry different biologically active toxins and enzymes into the surrounding environment. We hypothesized that OMVs may therefore be able to mediate the transport of bacterial products into host cells. We present here an analysis of the V. cholerae OMV-associated virulence factor PrtV. 

Methodology/Principal Findings: We observed that PrtV, a M6 family, zinc-binding metalloprotease, is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs. The association of PrtV with OMVs was determined by immunoblotting and electron microscopy using immunogold labeling. In addition, we observed that the PKD-domain(s) of PrtV has a role in the protein’s association with OMVs. We also demonstrated that OMV-associated PrtV was biologically active because HCT8 cells treated with OMVs from the wild type V. cholerae strain C6706 exhibited altered morphology, whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Moreover, our data suggest that OMV-associated PrtV might be transported into target eukaryotic cells by a vesicle fusion mechanism in association with lipid raft microdomains in the plasma membrane.

Conclusion/Significance: Our findings suggest that OMVs can deliver biologically active PrtV into target host cells.