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Background: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV., Methodology/principal Findings: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37., Conclusion/significance: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

Background: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL)., Methodology/principal Findings: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure., Conclusion: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

The Vibrio metalloprotease PrtV was purified from the culture supernatant of a Vibrio cholerae derivative that is deficient in several other secreted peptidases, including the otherwise abundant hemagglutinin/protease HapA. The PrtV is synthesized as a 102 kDa protein, but undergoes several N- and C-terminal processing steps during V. cholerae envelope translocation and prolonged incubation. Purified V. cholerae PrtV protease forms of 81 or 73 kDa were stabilized by calcium ions. Removal of calcium resulted in further rapid autoproteolysis. The two major products of autoproteolysis of the PrtV protease were approximately 37 and 18 kDa and could not be separated under non-denaturing conditions, indicating they are interacting domains. In an assay using cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell death. Using human blood plasma as a source of potential substrates of mammalian origin for the PrtV protease, we found that the extracellular matrix components fibronectin and fibrinogen were degraded by the enzyme. Additional tests with individual protein substrates revealed that plasminogen was also a possible target for the PrtV protease.