Haemolysins in Vibrio species

Many Vibrio species are pathogenic to humans, and/or marine vertebrates and invertebrates. The pathogenic species produce various virulence factors including enterotoxin, haemolysin, cytotoxin, protease, lipase, phospholipase, siderophore, adhesive factor and/or haemagglutinins. Haemolysin, which is an exotoxin that lyses erythrocyte membranes with the liberation of haemoglobin, is arguably the most widely distributed toxin among pathogenic vibrios and exerts various roles in the infection process. Haemolysins act on erythrocytes membranes thus lysing the cells which leads to the freeing up of the iron-binding proteins namely haemoglobin, transferrin and lactoferrin. This iron can then be picked up by various siderophores, and is subsequently taken up through receptors in the cell membrane. In many cases, the pore-forming activity of haemolysin is not restricted to erythrocytes, but extends to a wide range of other cell types including mast cells, neutrophils, and polymorphonuclear cells, and enhances virulence by causing tissue damage. There are four representative haemolysin families in Vibrio spp., including the TDH (thermostable direct haemolysin) family, the HlyA (E1 Tor haemolysin) family, the TLH (thermolabile haemolysin) family and the δ-VPH (thermostable haemolysin) family. Some haemolysins, for example, TDH of Vibrio parahaemolyticus and HlyA of Vibrio cholerae have been studied extensively, and are closely associated with virulence. However, the role of some haemolysins, e.g. TLH and δ-VPH are unclear, and await the outcome of further research. Bacteria of the genus Vibrio are Gram-negative, straight or curved rods, motile by one or more polar flagella, that give a positive oxidase test, grow on thiosulfate citrate bile salt sucrose agar and are facultative anaerobes. Most species are sensitive to the vibriostatic agent O/129, and have both a respiratory and a fermentative type of metabolism. Sodium ions stimulate the growth of all species and are an absolute requirement for most species (Holt et al. 1994). Vibrios are normal inhabitants in aquatic environments, being very common in marine and estuarine habitats and on the surface and in the intestinal contents of marine animals (Colwell 1984; Fouz et al. 1990). Bergey's Manual of Systematic Bacteriology (Holt et al. 1994) described 35 Vibrio species, but there has been constant changes in the taxonomy of the group, which is reflected in the number of studies leading to improvements in taxonomy and the description of new species (e.g. Alsina and Blanch 1994; Pedersen et al. 1998; Hayashi et al. 2003; Shieh et al. 2003; Thompson et al. 2003; Gomez-Gil et al. 2004; Sawabe et al. 2004). Many vibrios are pathogenic for humans and/or marine vertebrates and invertebrates (Table 1), with the virulence mechanisms reflecting the presence of enterotoxin, haemolysin, cytotoxin, protease, lipase, phospholipase, siderophore, adhesive factor and/or haemagglutinins (Iida and Honda 1997; Austin and Austin 1999; Shinoda 1999). Haemolysin is an exotoxin that attacks blood cell membranes and causes cell rupture. Haemolysis, which results from the lysis of erythrocyte membranes with the liberation of haemoglobin, consists of β-haemolysis, i.e. the complete degradation of haemoglobin, and α-haemolysis, i.e. the incomplete degradation of haemoglobin. Haemolysins are produced by many different species of bacteria including Escherichia coli, Pseudomonas aeruginosa and vibrios. In most cases, epidemiological and experimental evidence suggests that haemolysins are involved in disease pathogenesis (Iida and Honda 1997; Ludwig and Goebel 1997; Shinoda 1999). Haemolysis may result from the enzymic activities demonstrated by some species of bacteria, including phospholipase C of Ps. aeruginosa and phospholipase D in Photobacterium damselae ssp. damselae (formally known as V. damsela). However, most other protein haemolysins act by forming pores in the cytoplasmic membrane of erythrocytes (Kreger et al. 1987; Iida and Honda 1997; Ludwig and Goebel 1997). Haemolysin is arguably the most widely distributed toxin among pathogenic vibrios, and exerts various roles in the infection process (Iida and Honda 1997; Shinoda 1999). Iron is essential for bacterial growth and replication, and plays an important role in the pathogenesis of Vibrio spp. (Sigel and Payne 1982; Dai et al. 1992). Haemolysins act on erythrocyte membranes leading to cell lysis, which in turn frees up the iron-binding proteins, such as haemoglobin, transferrin and lactoferrin. The iron may then be picked up by a high-affinity iron acquisition system capable of competing with the host iron-binding proteins. The main system centres on siderophores, which are low molecular weight chelators that specifically bind Fe3+ outside the cell, and are subsequently taken up through receptors in the cell membrane (Sigel and Payne 1982; Stoebner and Payne 1988; Dai et al. 1992). Haemolysin expression in marine vibrios is regulated in iron-limited conditions, which occur in the host during infection (Stoebner and Payne 1988). In many cases, the pore-forming activity of haemolysin is not restricted to erythrocytes, but extends to a wide range of other cell types including mast cells, neutrophils and polymorphonuclear cells, and enhances virulence by causing tissue damage (Iida and Honda 1997; Ludwig and Goebel 1997; Shinoda 1999). Often, the haemolysin is also appropriately designated as a cytolysin. The various haemolysins produced by Vibrio spp. are considered to be similar, although not identical. Overall there are four representative haemolysin families, including the thermostable direct haemolysin (TDH) of V. parahaemolyticus, the El Tor haemolysin of V. cholerae (HlyA), the thermolabile haemolysin (TLH) of V. parahaemolyticus and another thermostable haemolysin, that is, the δ-VPH of V. parahaemolyticus (e.g. Taniguchi et al. 1985, 1986, 1990; Yamamoto et al. 1990a; Zhang et al. 2001; Fallarino et al. 2002; Table 2). The majority of V. parahaemolyticus strains isolated from cases of gastroenteritis in humans produced a β-haemolysin on Wagatsuma agar, which is a type of blood agar (Wagatsuma 1968). The ability to cause haemolysis on this medium has been termed the Kanagawa phenomenon (KP), and is because of a thermostable TDH (Vp-TDH) encoded by the tdh gene, which has been cloned and sequenced (Kaper et al. 1984; Nishibuchi and Kaper 1985). Vp-TDH has been considered a major virulence factor in cases of gastroenteritis (Miyamoto et al. 1969; Takeda 1988). The molecular weight was determined to be c. 42 kDa by gel filtration, and 21 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Takeda et al. 1978; Iida and Honda 1997). This proteinaceous toxin, which does not have a lipid or carbohydrate moiety (Iida and Honda 1997), is a dimer which is composed of two identical subunits each having a molecular mass of c. 21 kDa (Takeda et al. 1978). Purified Vp-TDH is heat-stable, even at 100°C for 10 min (Taniguchi et al. 1985; Iida and Honda 1997), and has an amino acid sequence of 165 amino acid residues, with one disulfide bond near the carboxyl terminus (Tsunasawa et al. 1987). These data are in good agreement with that obtained from the nucleotide sequence of the tdh gene (Nishibuchi and Kaper 1985). Purified Vp-TDH has haemolytic, cytotoxic, enterotoxic, mouse lethality and cardiotoxic activities (Iida and Honda 1997). It is believed that Vp-TDH damages the erythrocyte membrane by acting as a pore-forming toxin, with the pores estimated at 2 nm in diameter (also Vp-TDH has the ability to lyse target eucaryotic cells by punching holes in the plasma membrane) (Honda et al. 1992). Evidence suggests that Vp-TDH causes haemolysis by three sequential steps, namely binding to the erythrocyte membrane, followed by formation of a transmembrane pore, and then disruption of the cell membrane (Honda et al. 1992). It was reported that the N-terminal region is involved in the binding process, whereas the region near the C-terminal region has been implicated with postbinding (Tang et al. 1997). It is clear that phosphorylation of a 25-kDa host protein induced by Vp-TDH is essential for haemolysis after binding to the erythrocyte membrane (Yoh et al. 1996). In addition, it was determined that Vp-TDH induces cation permeability and activates endogenous Gardos potassium (K+) channels (Lang et al. 2004). The consequences of the activity include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+ (Lang et al. 2004). Honda et al. (1988) reported that KP-negative isolates of clinical origin produced a Vp-TDH related haemolysin, coined Vp-TRH, which is regarded also as an important virulence factor. In particular, Vp-TRH stimulated fluid secretion in the rabbit ileal loop test, which suggests a possible role for the toxin in inducing diarrhoea (Honda et al. 1988). Furthermore, it was determined that the amino acid sequences of Vp-TRH is c. 67% homologous with Vp-TDH (Honda et al. 1988). However, unlike the tdh genes, significant nucleotide differences exist within the trh family, with two subgroups, trh1 and trh2, sharing 84% sequence identity (Nishibuchi et al. 1989). Vp-TRH was found to be immunologically related, but not identical to TDH, and was heat labile to 60°C for 10 min (Honda et al. 1988). Both Vp-TDH and Vp-TRH induce chloride secretion in human colonic epithelial cells (Takahashi et al. 2000a,b). Nishibuchi and Kaper (1990) examined the relationship between phenotypic variation and nucleotide sequence variation of the tdh gene encoding Vp-TDH. These workers noted that cultures showing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2), whereas tdh-gene-positive isolates showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Moreover, it was revealed that tdh2 was primarily responsible for the haemolytic phenotype (Nishibuchi and Kaper 1990). Two other tdh gene copies were cloned from a phenotypically haemolysin-negative strain, which is unusual in that it contained only a single gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3) (Nishibuchi and Kaper 1990). It was revealed that all the four gene copies encoded polypeptides, which are composed of 189 amino acid residues (Nishibuchi and Kaper 1990). Furthermore, the nucleotide sequences in this region were very similar: homologies of tdh2, tdh3 and tdh4 with tdh1 are 97·2, 96·75 and 96·7% respectively (Nishibuchi and Kaper 1990). The tdh5 gene was cloned from a KP-negative strain, that also carried the trh gene and produced TDH at a very low level, and has a 98·9% nucleotide similarity to tdh2 (Baba et al. 1991a). It was considered that differences in the transcriptional control were primarily responsible for the differences seen in the haemolytic phenotype (Nishibuchi and Kaper 1990; Okuda and Nishibuchi 1998). In particular, it was realized that the base substitution of the tdh promoters of KP-negative strains only at position −34 were sufficient to increase the expression of these genes to the KP-positive level (Okuda and Nishibuchi 1998). Therefore, the tdh genes of KP-negative strains were considered to be potentially important because they could generate a KP-positive subclone by a point mutation in their promoters (Okuda and Nishibuchi 1998). As the initial report on the cloning of a tdh gene, >10 variants of the tdh/trh genes have been found in V. parahaemolyticus (Nishibuchi and Kaper 1990) and V. cholerae non-O1, V. mimicus and V. hollisae (Nishibuchi et al. 1990; Terai et al. 1990; Baba et al. 1991b; Yamasaki et al. 1991; Shinoda et al. 2004). Although most of the tdh genes were located on chromosomal DNA, there was evidence that some organisms possessed the gene on plasmid DNA (Nishibuchi and Kaper 1990). Further work revealed that the guanine plus cytosine (G + C) content of the tdh gene (35·6%) was lower than that of the total genomic DNA in the case of V. parahaemolyticus (46–47%) (Baumann et al. 1984). In many cases, the tdh genes were determined to be flanked by elements that appeared to be insertion sequences, which suggested that the tdh gene may well be a transposable unit that sometime in the past has undergone transposition between different replicons (e.g. between chromosome and plasmid) (Terai et al. 1991). Vibrio cholerae E1 Tor O1 and non-O1 strains are capable of producing a water-soluble cytolytic toxin that has been designated HlyA or E1 Tor haemolysin (Ichinose et al. 1987) and is also known as V. cholerae cytolysin (VCC; Olson and Gouaux 2003). HlyA haemolysin lyses erythrocytes and other mammalian cells, and exhibits enterotoxicity in experimental diarrhoea models (Ichinose et al. 1987). Thus, the haemolysin may well play a role in the pathogenesis of gastroenteritis caused by V. cholerae strains (Ichinose et al. 1987). Although the apparent molecular size of the HlyA haemolysin is 65 kDa when secreted and detected in the culture supernatant of V. cholerae cultures, the structural gene (hlyA) has been recognized to encode an 82-kDa polypeptide (Yamamoto et al. 1990a). Moreover, these workers demonstrated that the HlyA haemolysin was synthesized as an 82-kDa precursor form (prepro-HlyA), which consisted of a signal peptide of 25 amino acid residues at the N-terminus, a pro-region of 132 amino acid residues, and a mature region (of 584 residues) at the C-terminus. Furthermore, the mature form of the haemolysin was determined to be produced by a two-step process. Thus, during passage through the inner membrane the signal peptide was cleaved off, followed immediately by extracellular secretion in which the pro-region was lost. This two-step processing was regarded as necessary to activate the haemolysin (Yamamoto et al. 1990a). The pro-region of HlyA was reported to function as a molecular chaperone, suggesting that it exerts a role in the correct folding of the mature HlyA haemolysin (Nagamune et al. 1997). It is considered likely that HlyA haemolysin acts as a pore-forming toxin through oligomer formation (Ikigai et al. 1996), with the oligomers having molecular masses of 170–350 kDa on SDS-PAGE (Ikigai et al. 1996). In comparison, the monomer has a molecular mass of 60 kDa (Ikigai et al. 1996, 1999). Overall, it was estimated that the pores were of 1·2–1·6 nm in size (Ikigai et al. 1996). Cholesterol in the target cell membrane was considered to have an important role in the assembly of the toxin oligomers needed for pore formation (Ikigai et al. 1996; Menzl et al. 1996). The conclusion is that HlyA interacts with cholesterol and forms oligomers at the interface of cholesterol microcrystals and water (Harris et al. 2002). The data indicated that mild disruption of the native tertiary structure of HlyA by 1·75 m urea triggered rapid and quantitative conversion of the monomer to an oligomer. Furthermore, the HlyA monomer when unfolded in 8 m urea refolded and reconstituted on renaturation into the oligomer, which was biochemically and functionally similar to the heptamer formed in the target lipid bilayer (Chattopadhyay and Banerjee 2003). Thus, it was suggested that the HlyA polypeptide had a strong propensity to adopt the oligomer as the stable native state in preference to the monomer (Chattopadhyay and Banerjee 2003). Moreover, glycophorin B of human erythrocyte membranes was reported to be a receptor for HlyA haemolysin or at least an associated molecule of the receptor for HlyA haemolysin in human erythrocytes (Zhang et al. 1999). Several studies have reported that bacteria other than V. cholerae (e.g. V. mimicus, V. vulnificus, V. anguillarum and V. fluvialis) produce haemolysins, which share common features with HlyA haemolysin (e.g. Ichinose et al. 1987; Yamamoto et al. 1990b; Hirono et al. 1996; Ikigai et al. 1996; Kim et al. 1997; Rahman et al. 1997; Choi et al. 2002; Han et al. 2002; Kothary et al. 2003). For example, the structural gene (vmhA) of the haemolysin from V. mimicus (VMH) was cloned, and the nucleotide sequence was determined (Kim et al. 1997; Rahman et al. 1997). This gene was found to contain an open reading frame (ORF) consisting of 2232 nucleotides, which coded for a protein of 744 amino acids with a predicted molecular mass of 83 kDa (Kim et al. 1997; Rahman et al. 1997). The amino acid sequence revealed 81·6% identity with V. cholerae HlyA haemolysin. A suggestion was made that the mature haemolysin in V. mimicus was processed upon deleting the first 151 amino acids, and the molecular mass is 66 kDa, as the two-step processing of V. cholerae haemolysin (Yamamoto et al. 1990a; Kim et al. 1997; Rahman et al. 1997). Most of the clinical and environmental V. mimicus isolates examined were shown to possess the vmh gene (Shi et al. 2000; Shinoda et al. 2004). Similarly, regions of the structural gene (vvhA) of V. vulnificus haemolysin (VVH) resembled the corresponding gene of HlyA haemolysin, both in terms of sequence and organization (Yamamoto et al. 1990b; Choi et al. 2002). VVH, of which the purified molecule had a molecular mass of 51 kDa, lysed erythrocytes from a variety of animal species by forming small pores in the cytoplasmic membrane (Yamamoto et al. 1990b). Interestingly, production of VVH in V. vulnificus was repressed by the addition of glucose to the culture media; expression being depressed by cAMP, which suggested that haemolysin synthesis is regulated by cAMP-CRP (cAMP receptor protein) controlled catabolite repression (Bang et al. 1999). Haemolysin activity and the level of vvh transcript reached a maximum in late exponential phase, and were repressed when cells entered stationary phase (Bang et al. 1999). Northern blot and primer extension analyses revealed that vvhA was co-transcribed with a second gene, vvhB (the function of the vhhB gene product is unknown), which was located upstream of vvhA (Choi et al. 2002). It was concluded that CRP activated the expression of the V. vulnificus haemolysin gene by binding directly to the vvhBA promoter (Choi et al. 2002). Moreover, the temporal alternative expression of the V. vulnificus elastase prevented proteolytic inactivation of haemolysin (Eun et al. 2003). However, the transmembrane transcription activator ToxRS stimulated expression of the haemolysin gene vvhA (Lee et al. 2000). An extracellular cytolysin from V. tubiashii, a pathogen which causes bacillary necrosis in larval and juvenile bivalve (Tubiash et al. 1970), was purified (Kothary et al. 2001). Twelve of the first 17 N-terminal amino acid residues are identical to those of the V. vulnificus VVH (Yamamoto et al. 1990b; Kothary et al. 2001). Moreover, this cytolysin, which is sensitive to heat and proteases and is inhibited by cholesterol, has a molecular weight of 59 kDa (Kothary et al. 2001). In addition to lysing various erythrocytes including those of sheep, goat and rabbits, the toxin is cytolytic and/or cytotoxic to Chinese hamster ovary cells, carco-2 cells and Atlantic menhaden liver cells in tissue culture (Kothary et al. 2001). The V. anguillarum haemolysin gene (vah1) is 2253 base pairs (bp) in size, and corresponds to a protein of 751 amino acid residues (Hirono et al. 1996). The deduced amino acid sequence of the vah1 gene, and the previously reported V. cholerae HlyA haemolysin, V. vulnificus VVH haemolysin, Aeromonas hydrophila AHH1 haemolysin and A. salmonicida ASH4 haemolysin, showed a significant degree of sequence homology, and the overall amino acid identities were 57·3, 25·8, 46·2 and 43·7% respectively (Hirono et al. 1996). DNA hybridization analysis, under highly stringent conditions using vah1 as a probe, demonstrated that vah1 hybridized with 25 of 28 strains of V. anguillarum including serotypes A–I, but did not hybridize with other Vibrio species (Hirono et al. 1996). The haemolysin gene of V. fluvialis (VFH), which is 2200 bp in size, encodes a protein of 740 amino acids with a molecular weight of 82 kDa (Han et al. 2002). The molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE, and N-terminal amino acid sequence analysis revealed that the 82–kDa prehaemolysin is synthesized in the cytoplasm and then secreted into the extracellular environment as the 79-kDa mature haemolysin after cleavage of 25 N-terminal amino acids (Han et al. 2002). This is unlike the two-step processing found in V. cholerae (Yamamoto et al. 1990a). Deletion of 70 amino acids from the C-terminus exhibited a smaller haemolytic activity, whereas deletion of 148 C-terminal amino acids prevented haemolytic activity (Yamamoto et al. 1990a). The alignment of the deduced VFH amino acid sequence to V. cholerae HlyA haemolysin, V. mimicus VMH haemolysin and V. anguillarum VAH1 haemolysin showed 71, 70 and 59% homologies respectively (Han et al. 2002). VFH was active against erythrocytes from sheep, guinea pig, rabbit, calf, goat, horse and goose (Kothary et al. 2003). However, erythrocytes from guinea pig and chicken were the most and least sensitive respectively (Kothary et al. 2003). In addition to lysing erythrocytes, VFH was cytotoxic towards Chinese hamster ovary cells in tissue culture (Kothary et al. 2003). In addition to Vp-TDH and Vp-TRH, a thermolabile haemolysin (TLH), also coined the lecithin-dependent haemolysin (LDH), was discovered in V. parahaemolyticus (Taniguchi et al. 1985, 1986; Shinoda et al. 1991). TLH was heat labile (60°C for 10 min; Taniguchi et al. 1985), and had phospholipase A2/lysophospholipase activity (Shinoda et al. 1991). The TLH haemolysin gene (tlh) contained an ORF of 1254 bp (Shinoda et al. 1991). From the nucleotide sequence of the tlh, it was revealed that the pre- and mature protein consisted of 418 and 398 amino acids with molecular weights corresponding to 47·5 and 45·3 kDa respectively (Shinoda et al. 1991). The G + C content of the tlh gene is 47·6%, which is almost the same as that of the V. parahaemolyticus genome (Taniguchi et al. 1986). Two methionine codons (ATG) were located near the 5′-end (Taniguchi et al. 1986), but it was unclear which was the initiation codon. Using computer analysis, homology was not found between the nucleotide sequences of the tdh gene and the tlh gene of V. parahaemolyticus (Taniguchi et al. 1986). Yet, the tlh gene has been found in the genomes of all V. parahaemolyticus isolates examined, regardless of whether they have been derived from clinical or environmental sources (Taniguchi et al. 1985; Bej et al. 1999; McCarthy et al. 1999). However, the role of this haemolysin in the enteropathogenicity of V. parahaemolyticus is unknown (Shinoda et al. 1991). Zhang and Austin (2000) compared the pathogenicity and putative virulence factors of 21 V. harveyi isolates obtained from various sources, and found that the most pathogenic isolate, VIB 645, produced extracellular products with the highest titre of haemolytic activity to fish erythrocytes. VIB 645 carried two haemolysin genes (designated vhhA and vhhB), which were cloned and sequenced. Conversely, the majority (19 of 20) of the other cultures possessed only single genes, or none at all (Zhang et al. 2001). The ORF of the vhhA and vhhB genes were both 1254 nucleotides in length, which is the same size as the ORF of the tlh gene of V. parahaemolyticus (Taniguchi et al. 1986). Both vhhA and vhhB encoded polypeptides composed of 418 amino acid residues; and the predicted amino acid sequences encoded by vhhA and vhhB were identical (Zhang et al. 2001). Yet, the nucleotide sequences of vhhA and vhhB were not identical, but were nevertheless extremely similar (98·8%), suggesting that the duplication of the vhh gene was a relatively recent event. Data revealed that the nucleotide sequence identities of vhhA and vhhB to tlh were 77·5 and 77·2% respectively (Zhang et al. 2001). Interestingly, the amino acid sequence of V. harveyi VHH protein showed high degrees of identity to V. parahaemolyticus TLH haemolysin (85·6%) (Shinoda et al. 1991), the phospholipase VPL (Genbank AF291424) of V. vulnificus (75·6%), the lecithinase PHL of V. mimicus (65·3%) (Kang et al. 1998) and the lecithinase LEC of V. cholerae (64·3%) (Fiore et al. 1997). In addition to TDH/TRH and TLH haemolysins, another thermostable haemolysin (δ-VPH; Genbank AB012597) has been identified, and found to be present in all cultures of V. parahaemolyticus examined (Taniguchi et al. 1990). The gene encodes a polypeptide of 203 amino acid residues. Interestingly, the nucleotide and predicted amino acid sequence differ from those of the tdh, trh and tlh genes, or any other haemolysins of V. parahaemolyticus (Taniguchi et al. 1990). Recently, a previously undescribed haemolysin, which was distinct from the V. cholerae O1 HlyA haemolysin, was cloned from the O1 classical biotype strain Z17561 (Genbank AJ007495) (Fallarino et al. 2002). This novel haemolysin possessed 71·5% overall similarity to the δ-VPH haemolysin of V. parahaemolyticus, and was coined as the V. choleraeδ-thermostable haemolysin (Vc-δ TH; encoded by the dth gene) (Fallarino et al. 2002). The complete ORF of dth encodes a predicted protein of 22·8 kDa but, in contrast to the TDH of V. parahaemolyticus, no signal peptide could be deduced from the predicted sequence (Fallarino et al. 2002). However, this is in agreement with the V. parahaemolyticusδ-VPH, which does not contain any features typical of a signal sequence at its amino-terminus (Taniguchi et al. 1990). The overall G + C content of dth and the downstream ORF was 50·46 mol%, which is comparable with that of V. cholerae (Taniguchi et al. 1990). It was determined that the dth gene (VCA1111) is present as a single copy on the smaller of the two V. cholerae chromosomes (chromosome 2) in strain Z17561. The dth gene occurred in V. cholerae O1 strains Z17561, O17, 569B and H1, and O139 strain AI-1837, but absent in O139 strain Arg3, which is a recent clinical isolate from Argentina (Fallarino et al. 2002). In contrast, as determined by DNA hybridization studies, δ-VPH appears to be present in all V. parahaemolyticus strains regardless of their source of isolation (Taniguchi et al. 1990). When expressed from an inducible promoter in Escherichia coli, Vc-δ TH was found to be a 22·8-kDa protein active on sheep erythrocytes. However, a direct role could not be shown for Vc-δ TH in V. cholerae O1 pathogenesis (Fallarino et al. 2002). Another haemolysin gene (hlx), which encodes a polypeptide of 92 amino acid residues and shows haemolytic activity when expressed in E. coli, has been cloned from V. cholerae O1 (Nagamune et al. 1995). The hlx gene was observed in classical and El Tor biotype V. cholerae O1, V. cholerae non-O1 and V. mimicus, but not in V. parahaemolyticus (Nagamune et al. 1995; Shi et al. 2000). However, the contribution of the hlx gene to the pathogenicity of the organisms has not been resolved. In addition to the vvhA haemolysin gene (Yamamoto et al. 1990b; Choi et al. 2002), another haemolysin gene (vllY) from V. vulnificus has been identified (Chang et al. 1997). Nucleotide sequence analysis predicted an ORF of 1071 bp, encoding a 357 amino acid polypeptide with an estimated pI of 5·02 (Chang et al. 1997). The deduced amino acid sequence of vllY showed high similarity to the sequence of legiolysin, which is responsible for haemolysis, pigment production, and fluorescence in Legionella pneumophila (Wintermeyer et al. 1994). PCR screening and Southern blotting of V. vulnificus strains revealed that all of the 41 V. vulnificus clinical isolates contained vllY-like genes (Chang et al. 1997). Recently, a new haemolysin gene (hlyIII) from V. vulnificus has been cloned and sequenced (Chen et al. 2004). Nucleotide sequence analysis predicted an ORF of 642 bp, encoding a 214 amino acid polypeptide, that showed 48% sequence identity to the haemolysin III of Bacillus cereus (Baida and Kumin 1995), and 80% sequence identity to a putative haemolysin from V. cholerae (Genbank NP_229669). When HlyIII of V. vulnificus was expressed in E. coli, crude extracts exhibited haemolytic activity similar to that of haemolysin III from B. cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally to mice (Baida and Kumin 1995). It is apparent that Vibrio spp. express a range of haemolysins, some of which are very similar, although not necessarily identical. There are four representative haemolysin families, including the TDH (thermostable direct haemolysin) family, the HlyA (E1 Tor haemolysin) family, the TLH (thermolabile haemolysin) family and the δ-VPH (thermostable haemolysin) family. Some haemolysins, for example, TDH of V. parahaemolyticus and HlyA haemolysin of V. cholerae have been studied extensively, and are closely associated with virulence. However, the role of some haemolysins, that is, TLH and δ-VPH is unclear, and must await the outcome of further research. Also, more work is needed to understand the evolutionary origin of the haemolysin genes. This work was supported by National Natural Science Foundation of China (NSFC) project no. 30371119.