Vibrio vulnificus metalloprotease VvpE is essentially required for swarming

Bacterial swarming constitutes a good in vitro model for surface adherence and colonization, and is accompanied by expressions of virulence factors related to invasiveness. In this study, it was determined that Vibrio vulnificus swarming was abolished by mutation of the vvpE gene encoding a metalloprotease VvpE and this swarming defect was recovered by complementation of the vvpE gene. Expression of the vvpE gene began simultaneously with the beginning of swarming and increased along with expression of the luxS gene encoding the synthase of the precursor of quorum-sensing signal molecule autoinducer 2, and this increased vvpE expression was decreased by mutation of the luxS gene. Moreover, VvpE destroyed IgA and lactoferrins, which are responsible for mucosal immunity. These results suggest that VvpE may play important roles in the surface adherence and colonization of V. vulnificus by facilitating swarming and in the mucosal invasion of V. vulnificus by destroying IgA and lactoferrin.     

The hemagglutinating action of Vibrio vulnificus metalloprotease

Vibrio vulnificus protease (VVP), a 45-kDa zinc metalloprotease, consists of two functional domains: an N-terminal 35-kDa polypeptide having endoproteinase activity, and a C-terminal 10-kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N-terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl2 or NiCl2, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution. Cu-treated 35-kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni-treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.     

Histamine-releasing reaction induced by the N-terminal domain of Vibrio vulnificus metalloprotease

A zinc metalloprotease secreted by Vibrio vulnificus, an opportunistic human pathogen causing septicemia and wound infection, stimulates exocytotic histamine release from rat mast cells. This protease consists of two functional domains: the N-terminal domain that catalyzes proteolytic reaction and the C-terminal domain that promotes the association with a protein substrate or cell membrane. Like the intact protease, the N-terminal domain alone also induced histamine release from rat peritoneal mast cells in a dose- and time-dependent manner. However, the reaction induced was apparently weak and went on more slowly. The nickel-substituted protease or its N-terminal domain, each of which has the reduced proteolytic activity due to decreased affinity to a substrate, showed much less histamine-releasing activity. When injected into the rat dorsal skin, the N-terminal domain also evoked enhancement of the hypodermic vascular permeability, while the activity was comparable to that of the protease. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane. The C-terminal domain may support the in vitro action of the N-terminal domain by coordination of the association of the protease with the membrane, but it may not modulate the in vivo action.     

Chemical modification of Vibrio vulnificus metalloprotease with activated polyethylene glycol

Abstract Vibrio vulnificus , an opportunistic human pathogen causing septicemia, produces a metalloprotease which is suspected to be a virulence determinant, but which is labile in vivo due to inactivation by α -macroglobulin. To obtain a derivative which is stable in vivo, the metalloprotease was modified with activated monomethoxy polyethylene glycol. The modified protease retained full activity to a peptide substrate and 10–20% activity to protein substrates, and was resistant to entrapment by α -macroglobulin because of the increased molecular size (approx. 90 kDa). These findings suggest that the modified protease is stable in vivo and may be used to investigate the pathological actions of the protease in the bloodstream.     

Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel β-strands with a unique fold that has a compact β-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe³⁺). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.     

Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.     

Vibrio vulnificus may produce a metalloprotease causing an edematous skin lesion in vivo

Abstract Oxalate decarboxylase was detected both intra- and extracellularly in liquid cultures of Coriolus versicolor . Induction of the enzyme by addition of oxalic acid to the medium on day 6 of growth resulted in a 50-fold increase in specific activity in the mycelia and a 30-fold increase in the extracellular specific activity in the media. The protein was isolated and purified from mycelia, and characterised by polyacrylamide gel electrophoresis and Western blotting against a polyclonal antibody raised to oxalate decarboxylase from Collybia velutipes (Basidiomycete). A major protein band of M _r 59000 cross-reacted with the antibody. Immunogold-cytochemical labelling of ultra-thin sections of beechwood infected with C. versicolor showed that the enzyme was localised close to the plasma membrane and in intracellular vesicles.     

Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus

A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the empV gene was expressed in minicells, a unique peptide of approx. 46 kDa was identified. Protease activity staining experiments also indicated a similar M_r for the protease. The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it. The crude enzyme prepared from the periplasmic fraction of recombinant E. coli was inhibited by a metalloprotease inhibitor and Zn²⁺ is essential for its protease activity. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long `pro' sequence consisting of 172 aa. The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V. vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference. The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V. vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining. The deduced aa sequence of EmpV showed high homology to V. anguillarum metalloprotease (EmpA), V. cholerae HA/protease (HprC), and V. proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.     

A recombinant metalloprotease antigen of Vibrio vulnificus elicits protective antibodies in a murine model

Aims: To investigate whether Vibrio vulnificus metalloprotease (VvpE) can induce the production of specific anti‐VvpE antibody to confer effective protection against Vibrio vulnificus infection and to evaluate the possibility of VvpE as a potential vaccine candidate against disease caused by V. vulnificus. Methods and Results: The gene encoding the 65‐kDa VvpE of V. vulnificus was amplified by PCR and cloned into the expression vector pET21(b). The recombinant VvpE of V. vulnificus was expressed in Escherichia coli BL21(DE3). This His₆‐tagged VvpE was purified and injected intramuscularly into mice to evaluate its ability to stimulate immune response. Specific antibody levels were measured by ELISA. The 75% protective efficacy of recombinant VvpE was evaluated by active immunization and intraperitoneal challenge with V. vulnificus in mice. Conclusions: The recombinant His₆‐tagged VvpE of V. vulnificus is capable of inducing high antibody response in mice to confer effective protection against lethal challenge with V. vulnificus. VvpE might be a potential vaccine candidate to against V. vulnificus infection. Significance and Impact of the Study: This study uses His₆‐tagged VvpE to act as vaccine that successfully induces effective and specific anti‐VvpE antibody and offers an option for the potential vaccine candidate against V. vulnificus infection.     

Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.     

The N-terminal propeptide of Vibrio vulnificus extracellular metalloprotease is both an inhibitor of and a substrate for the enzyme

Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP-vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease-were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.     

Role of the metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar E in surface colonization and fish virulence

The virulence for eels of Vibrio vulnificus biotype 2 serovar E (VSE) is conferred by a plasmid that codifies ability to survive in eel serum and cause septicaemia. To find out whether the plasmid and the selected chromosomal gene vvp plays a role in the initial steps of infection, the VSE strain CECT4999, the cured strain CT218 and the Vvp-deficient mutant CT201 (obtained in this work by allelic exchange) were used in colonization and virulence experiments. The eel avirulent biotype 1 (BT1) strain YJ016, whose genome has been sequenced, was used for comparative purposes. The global results demonstrate that the plasmid does not play a significant role in surface colonization because (i) CECT4999 and CT218 were equally chemoattracted towards and adherent to eel mucus and gills, and (ii) CT218 persisted in gills from bath-infected eels 2 weeks post infection. In contrast, mutation in vvp gene reduced significantly chemoattraction and attachment to eel mucus and gills, as well as virulence degree by immersion challenge. Co-infection experiments by bath with CECT4999 and CT201 confirmed that Vvp was involved in eel colonization and persistence in gills, because CECT4999 was recovered at higher numbers compared with CT201 from both internal organs of moribund fish (ratio 4:1) and gills from survivors (ratio 50:1). Interestingly, YJ016 also showed chemoattraction and attachment to mucus, and complementation of CT201 with BT1- vvp gene restored both activities together with virulence degree by immersion challenge. Additional experiments with algae mucus and purified mucin gave similar results. In conclusion, the protease Vvp of V. vulnificus seems to play an essential role in colonization of mucosal surfaces present in aquatic environments. Among the V. vulnificus strains colonizing fish mucus, only those harbouring the plasmid could survive in blood and cause septicaemia.     

Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity

Vibrio vulnificus biotype 2 serovar E (VSE) is a bacterial pathogen that produces a haemorrhagic septicaemia called vibriosis in eels. Its ability to grow in blood is conferred by a recently described virulence plasmid [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.]. In this study, we analyzed the role of this plasmid together with the role played by the metalloprotease (Vvp) in the interaction between bacteria and eel innate immunity. To this end, we compared and statistically analyzed the differences in resistance to serum and mucus factors (complement, selected antimicrobial peptides, transferrin and lysozyme) and also to phagocytosis/opsonophagocytosis between one VSE strain and its derivatives: a plasmid-cured strain and a vvp-deficient mutant. The wild-type and the metalloprotease-deficient strains were resistant to both the bactericidal action of fresh serum and the phagocytosis and opsonophagocytosis by eel phagocytes, confirming that Vvp is not involved in resistance to eel innate immunity. In contrast, the cured strain was sensitive to both the bactericidal action of eel serum activated by the alternative pathway and phagocytosis/opsonophagocytosis. Since no plasmid-encoded ORF, with homology to known genes, is related to the resistance to innate immunity [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.], this function could be codified by one or more new genes. Further studies are underway to characterize the plasmid-encoded system responsible for V. vulnificus resistance to the innate immune system of eels.     

High-frequency phase variation of Vibrio vulnificus 1003: isolation and characterization of a rugose phenotypic variant

The marine bacterium Vibrio vulnificus is a human pathogen that can spontaneously switch between virulent opaque and avirulent translucent phenotypes. Here, we document an additional form, the rugose variant, which produces copious biofilms and which may contribute both to pathogenicity of V. vulnificus and to its survival under adverse environmental conditions.     

Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca2(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca2(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca2(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca2(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca2(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.