Bacteria in the genus Vibrio are normal habitants of aquatic environments and play important roles in maintaining the aquatic ecosystem. Although more than 100 species are currently in this genus, at least, 11 species listed in Table 1 are human pathogens (Janda et al., 1988; Chakraborty et al., 1997). Amongst, Vibrio hollisae is recommended to place into a novel genus Grimontia (Thompson et al., 2003). Human pathogenic species can be classified into two groups according to types of infectious diseases: the group causing gastrointestinal diseases and that causing extra-intestinal diseases. Representative species in the former group are V. cholerae and V. parahaemolyticus, whereas V. vulnificus is the most important species in the latter group. Two species, V. damsela and V. vulnificus, are also fish pathogens.

Of human pathogenic vibrios, V. cholerae is the most extensively studied species because it is a causative agent of severe watery diarrheal disease, cholera. Cholera is characterized by uncontrolled purging of copious rice water stools leading to serious electrolyte depletion, dehydration, acidosis, shock, and, if left untreated, to death. Although V. cholerae is divided serologically into more than 200 groups, only two serogroups, O1 and O139, are etiologic agents of epidemic cholera. The non-O1/non-O139 serogroups are etiologic agents causing sporadic diarrheal cases and occasionally extra-intestinal infections, including skin, ear, sputum, urine, and cerebrospinal fluid infections. Cholera toxin (CT) produced by the O1 and O139 serogroups is a major toxic factor evoking severe watery diarrhea, whereas, hemolysin (Shinoda and Miyoshi, 2006), Zonula occludens toxin (Fasano et al., 1991), and accessory CT (Trucksis et al., 1993) have also been demonstrated to be additional enterotoxic factors.

Vibrio parahaemolyticus inhabits commonly coastal and estuary areas in the tropical and temperate regions, but this species has been recognized to cause gastroenteritis following consumption of seafood. The outstanding features of gastroenteritis are severe abdominal pain, diarrhea (frequently bloody stools), nausea, vomiting, mild fever, and headache. The mean incubation period is 6 to 12 h and diarrhea or soft stools persist for 4–7 days. Although the food poisoning caused by V. parahaemolyticus had been frequent in Japan, the number of outbreaks has been decreased drastically in the last 10 years. Production of the hemolysin designated as thermostable direct hemolysin is closely related to the bacterial pathogenicity (Shinoda and Miyoshi, 2006). V. parahaemolyticus also causes wound-infection through exposure of a new wound to contaminated seawater or estuarine water, whereas this type of diseases is independent of production of the hemolysin.

The first clinical isolation of V. vulnificus was from a human leg ulcer (Roland, 1970). However, because of similar bacteriological characteristics of the bacterium isolated, this case was reported as an extra-intestinal infectious disease by V. parahaemolyticus (Hollis et al., 1976). V. vulnificus causes two types of infections, the primary septicemia and the wound-infection, to human (Blake et al., 1980; Klontz et al., 1988). The primary septicemia is associated with consumption of contaminated raw seafood, especially shellfish such as oysters. However, this type is a typical opportunistic infection. Namely, most patients have an underlying disease(s) of liver dysfunction, alcoholic cirrhosis or hemochromatosis, which leads to increase in the plasma ferric ion level and to decrease in the activity of the innate immunity (Strom and Paranjpye, 2000; Inoue et al., 2008). In two-thirds of the patients, the edematous and/or hemorrhagic secondary skin lesions appear on the extremities and the trunk (Blake et al., 1980; Inoue et al., 2008). The wound-infection is characterized by the development of edema, erythema or necrosis around a new wound exposed to seawater contaminated with V. vulnificus (Blake et al., 1980; Klontz et al., 1988). This type of infection can occur in healthy persons, as well as in the immuno-compromised hosts, and may occasionally progress to septicemia.

Human pathogenic vibrios produce various extracellular factors including enterotoxin, hemolysin, cytotoxin, protease, collagenase, phospholipase, siderophore, and hemagglutinin (Janda et al., 1988). Of these factors, enterotoxin, hemolysin, and cytotoxin are directly related to the clinical symptoms; however, siderophore and hemagglutinin may play roles in the establishment of the infection.

Proteolytic enzymes hydrolyzing a peptide bond in proteins and peptides are essential for the homeostatic control in both eukaryotes and prokaryotes, and thus, the bacterial enzymes also have various physiological roles in the life cycle of the microorganisms. However, the enzymes produced by pathogenic species occasionally act as toxic factors to the infected host (Hase and Finkelstein, 1993; Harrington, 1996). Proteolytic enzymes are classified into several groups, such as aspartic, cysteine, and serine protease, and metalloprotease; however, many of the bacterial toxic proteases are in the metalloprotease group, which often contains a zinc (II) ion in the catalytic center (Hase and Finkelstein, 1993; Hooper, 1994). As shown in Table 2, human pathogenic Vibrio species also produce and secrete proteolytic enzymes, and several enzymes have been extensively characterized as direct toxic factors causing skin damage or indirect virulence factors processing other protein toxins. The enzymes produced by vibrios are in two metalloprotease groups (vibriolysin and collagenases) or one serine protease group (chymotrypsin-like proteases).

The progress in the molecular biology has provided much information on the DNA-derived amino acid sequences of metalloproteases and has shown the presence of the consensus sequence HEXXH as the zinc-binding motif. This motif was also found in some bacterial protein toxins including clostridial neurotoxins, Bacteroides fragilis enterotoxin, and Bacillus anthracis lethal factor. Indeed, these bacterial toxins were verified to show the remarkably specific proteolytic action toward a target host protein (Miyoshi and Shinoda, 2000). For instance, clostridial neurotoxins can cleave the restricted protein components of the neuroexocytosis machinery, which leads to the blockade of neurotransmitter release and consequent muscle paralysis (Schiavo et al., 1993). In addition, a novel cytotoxin that consists of one A subunit and five B subunits was isolated from some enterohemorrhagic Escherichia coli strains, and the A subunit was indicated to be a subtilase-like serine protease (Paton and Paton, 2010). The RTX (repeated-in-toxin) toxins are large multifunctional cytotoxins and are possible to modulate the virulence of a number of gram-negative bacterial pathogens including V. cholerae and V. vulnificus (Satchell, 2007; Prochazkova et al., 2009; Roig et al., 2011). In V. cholerae RTX toxin, the cysteine protease domain was reported to mediate autoprocessing of the toxin (Sheahan et al., 2007; Shen et al., 2009).

Yu and Lee (1999) and Kim et al. (2002) carried out cloning of the V. parahaemolyticus gene encoding a collagenase, which was designated as PrtV/PrtVp (562 aa, 63,156 Da) and VppC (814 aa, 89,833 Da), respectively. These enzymes were revealed to be metalloproteases in the zincins superfamily having the consensus zinc-binding HEXXH motif, but neither of the collagenases was in the thermolysin family. Miyoshi et al. (2008) showed that, only when V. parahaemolyticus was cultivated at 26°C, the vppC gene was sufficiently expressed, and VppC was secreted from the bacterial cell after removal of the N-terminal 72 amino acid residues. In contrast, expression of the prtV/prtVp gene was negligible in the wild type strain. The gene was significantly expressed by disruption of the vppC gene; however, the product PrtV/PrtVp was not secreted into the cultivation broth (Miyoshi et al., 2008), suggesting PrtV/PrtVp is a cell-associated enzyme. VppC purified showed the steady activity to hydrolyze Z-Gly-Pro-Gly-Gly-Pro-Ala, the specific substrate for bacterial collagenases, and to digest gelatin. This indicates that VppC may contribute to the wound-infection by V. parahaemolyticus because putative digestion of the components of the extracellular matrix by VppC may accelerate the bacterial dissemination and may form cellulitic skin damage. V. alginolyticus, another species causing the wound-infection, is known to produce a VppC homolog (Takeuchi et al., 1992).

In 2002, two research groups reported individually purification of a serine protease, which was termed protease A and VPP1 respectively, from the culture supernatant of V. parahaemolyticus (Ishihara et al., 2002; Lee et al., 2002). These proteases were identical and corresponded to the VPA0227 protein (677 aa, 71,038 Da) of strain RIMD 2210633 (GenBank accession number: BA000032). However, the amino acid sequencing of the purified enzyme indicated that the N-terminal 121 amino acid residues were removed during the maturating process. This serine protease designated herein as protease A/VPP1 showed the immunological cross-reactivity with serine proteases from V. metschnikovii and V. alginolyticus (Ishihara et al., 2002). Indeed, protease A/VPP1 revealed highly similarity of the amino acid sequence to the enzyme from V. metschnikovii (Kwon et al., 1995). Production of protease A/VPP1 was much higher at 25 than 37°C and was induced by the addition of gelatin to the cultivation broth. Miyoshi et al. (2008) reported that production of this serine protease was remarkably increased by disruption of the vppC gene, suggesting that protease A/VPP1 is a substitutive enzyme of VppC. The purified protease was found to be sensitive to chymostatin, the well-known competitive inhibitor for chymotrypsin, and to hydrolyze the specific peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates for chymotripsin, such as Glutaryl-Ala-Ala-Phe-MCA and Succinyl-Ala-Ala-Pro-Phe-MCA. Therefore, it may be concluded that protease A/VPP1 is a chymotrypsin-like serine protease. The cytotoxicity against CHO, HeLa, or Vero cells and the mouse lethal toxicity of purified protease A/VPP1 was demonstrated by Lee et al. (2002). Our preliminary study showed the proteolytic activity of the purified enzyme toward extracellular matrix components, laminin and type I collagen. These results suggest that protease A/VPP1 also modulate the bacterial pathogenicity.

Vibrio vulnificus sometime causes severe hemorrhagic septicemia called vibriosis in eels of the culture farms (Tison et al., 1982; Biosca et al., 1991). A few strains isolated from diseases eels were recently found to have lost the 80 kb genomic region including the gene encoding vibriolysin, but instead of vibriolysin, these strains secrete a serine protease termed VvsA, which is an ortholog of protease A/VPP1 from V. parahaemolyticus (Miyoshi et al., 2012b). As protease A/VPP1, production of VvsA was extremely increased in the absence of the functional gene encoding vibriolysin (Wang et al., 2008). The vvsA gene constitutes an operon with a downstream gene vvsB, of which product VvsB may act as the chaperon supporting the maturation process of VvsA. The database analysis showed that several Vbrio species including V. parahaemolyticus have the homologus genes to vvsAB, indicating widely distribution of the chymotrypsin-like serine protease in Vibrio species.

Vibrio species are ubiquitous microorganisms in aquatic environments, but 11 species cause intestinal or extra-intestinal infections to humans. During the infection process, the bacterial cells must sense the change of environmental factors, such as temperature, pH, salinity, and osmolarity, and then, the bacterium must transmit the signals into the cells through the specific signal-transduction systems, which result in the change of expression of the genes. Especially, the genes encoding the toxic or virulence factors, which may be required for in vivo survival and growth, must be expressed at an appropriate place and time in a tightly regulated fashion (Heithoff et al., 1997; Lee et al., 1999). Amongst the environmental factors, temperature is thought to be the most important. The vibriolysin genes of the bacterium causing intestinal infections are expressed sufficiently at 37°C, while the genes of the bacterium causing extra-intestinal infections are expressed more effectively around the body surface temperature (Watanabe et al., 2004; Miyoshi et al., 2006). The gene expression is also often affected by the salinity. For instance, the expression level of the vppC gene in V. parahaemolyticus is higher at 3% NaCl than 0.9% NaCl (Miyoshi et al., 2008). Although the intracellular second messengers, such as cyclic AMP and cyclic di-GMP, and the global general regulators including RpoS and histone-like nucleotide structuring protein are also involved in the gene regulation (Benitez et al., 2001; Wang et al., 2011, 2012), the molecular mechanisms how the bacterium senses the environmental signals and transmits the signals into the cells are not clarified.

Production of proteolytic enzymes is tightly dependent on the growth phase and reaches to the maximum level at the early stationary phase. Many pathogenic bacteria coordinate expression of the virulence genes in response to the bacterial cell density. This regulation system is termed the quorum sensing (QS) and is controlled by the small diffusible signal molecule called autoinducer (AI; Whitehead et al., 2001; Henke and Bassler, 2004). At the low cell density, the QS system cannot modulate the gene expression because the concentration of AI is too low. However, at the high cell density, the AI concentration around the bacterial cell reaches the threshold level, the AI molecule is sensed by the sensor protein, the signals are transmitted into the cell, and finally, the expression of a set of genes is started or stopped.

In Vibrio species including V. cholerae, V. mimicus, and V. vulnificus (Miller et al., 2002; Milton, 2006; Sultan et al., 2006), the AI molecule is detected by the specific membrane-bound sensor protein, which causes conversion of the sensor protein from kinase to phosphatase. Subsequently, the sensor protein/phosphatase mediates dephosphorylation of LuxU-LuxO, the response regulator proteins. The dephosphorylated LuxO has no activity to inhibit LuxR or its homolog, the master transcriptional regulator for the genes under the control of the QS system. Therefore, at the high cell density, the transcriptional status of the target genes is changed by the function of LuxR or its homologue. Production of the proteolytic enzymes by pathogenic vibrios is closely related with the extracellular AI level (Kim et al., 2003; Kawase et al., 2004; Raychaudhuri et al., 2006). For instance, the mutant of the AI synthetase showed apparently reduced production of vibriolysin. Therefore, the QS system markedly controls the expression of the proteolytic genes in Vibrio species. However, it should be noted that the QS system of V. cholerae or V. mimicus is operated sufficiently at 37°C (Sultan et al., 2006), whereas, the system of V. vulnificus is operated effectively at 26°C but not at 37°C (Miyoshi et al., 2006).

The proteolytic enzymes produced by human pathogenic Vibrio species may play a variety of pathological roles: direct roles by digesting many kinds of host proteins or indirect roles by processing other toxic protein factors. Especially, vibriolysin from V. vulnificus is thought to be a major virulence factor. However, some contradictions of the pathogenic roles were also reported (Shao and Hor, 2000; Sun et al., 2006). It must be mentioned that the purified enzymes from V. vulnificus and V. proteolyticus, a non-pathogenic species, are difficult to distinguish in the in vivo actions, because both enzymes are members of vibriolysin and have comparative biochemical and toxic activities. However, it has been demonstrated that the high growing ability of V. vulnificus in the mammal host is important for the pathogenicity of the bacterium (Watanabe et al., 2004). In addition, production of the toxic or virulence factors including proteolytic enzymes is tightly regulated by environmental factors, the bacterial cell density and so on. Therefore, the overall experiments from various approaches are necessary for evaluation of the extracellular proteolytic enzymes as the virulence factors.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.