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 as a molecular that it exerts a role in the of the mature HlyA haemolysin et al. 1997). It is considered that HlyA haemolysin as a pore-forming toxin through formation et al. with the having molecular of kDa on et al. 1996). In the has a molecular mass of kDa et al. 1999). it was estimated that the pores were of nm in size et al. 1996). in the target cell membrane was considered to have an important role in the of the toxin for formation et al. et al. 1996). The is that HlyA with and at the of and et al. The data that disruption of the of HlyA by and of the to an Furthermore, the HlyA when in and on the which was and to the in the target lipid and 2003). Thus, it was suggested that the HlyA polypeptide a to the as the in to the and 2003). Moreover, of human erythrocyte membranes was reported to be a for HlyA haemolysin or at least an associated of the for HlyA haemolysin in human erythrocytes et al. 1999). studies have reported that bacteria other than V. cholerae (e.g. V. V. V. and V. produce haemolysins, which common with HlyA haemolysin (e.g. et al. 1987; Yamamoto et al. et al. et al. et al. 1997; et al. 1997; et al. 2002; et al. 2002; et al. 2003). example, the structural gene of the haemolysin from V. mimicus was and the nucleotide sequence was determined et al. 1997; et al. 1997). This gene was found to an of which for a protein of amino with a molecular mass of kDa et al. 1997; et al. 1997). The amino acid sequence revealed identity with V. cholerae HlyA was that the mature haemolysin in V. mimicus was the amino and the molecular mass is as the two-step processing of V. cholerae haemolysin (Yamamoto et al. 1990a; et al. 1997; et al. 1997). Most of the clinical and V. mimicus isolates examined were to the gene et al. Shinoda et al. 2004). of the structural gene of V. haemolysin the gene of HlyA haemolysin, both in of sequence and (Yamamoto et al. et al. of which the a molecular mass of erythrocytes from a of species by forming pores in the cytoplasmic membrane (Yamamoto et al. of in V. was by the addition of to the culture expression being by which suggested that haemolysin is regulated by et al. 1999). Haemolysin activity and the level of a in and were when cells et al. 1999). and revealed that was with a gene, of the gene is which was located of et al. It was that the expression of the V. haemolysin gene by binding to the et al. Moreover, the expression of the V. of haemolysin et al. 2003). However, the transmembrane stimulated expression of the haemolysin gene et al. extracellular cytolysin from V. a which causes in and et al. was et al. of the N-terminal amino acid residues are identical to of the V. (Yamamoto et al. et al. Moreover, this which is sensitive to heat and and is by has a molecular weight of kDa et al. In addition to lysing various erythrocytes including of and the toxin is cytolytic and/or to cells, cells and cells in tissue culture et al. The V. haemolysin gene is base in and to a protein of amino acid residues et al. 1996). The amino acid sequence of the gene, and the reported V. cholerae HlyA haemolysin, V. haemolysin, haemolysin and haemolysin, a significant of sequence and the amino acid were and respectively et al. 1996). DNA as a demonstrated that with 25 of strains of V. including but not with other Vibrio species et al. 1996). The haemolysin gene of V. which is in a protein of amino with a molecular weight of kDa et al. The molecular weight of the was estimated to be kDa by and N-terminal amino acid sequence revealed that the is synthesized in the and then secreted the extracellular as the mature haemolysin after of 25 N-terminal amino et al. This is unlike the two-step processing found in V. cholerae (Yamamoto et al. 1990a). of amino from the a haemolytic whereas of C-terminal amino haemolytic activity (Yamamoto et al. 1990a). The of the amino acid sequence to V. cholerae HlyA haemolysin, V. mimicus haemolysin and V. haemolysin and homologies respectively et al. was erythrocytes from and et al. 2003). However, erythrocytes from and were the most and least sensitive respectively et al. 2003). In addition to lysing erythrocytes, was cells in tissue culture et al. 2003). In addition to Vp-TDH and Vp-TRH, a thermolabile haemolysin also coined the haemolysin was in V. parahaemolyticus (Taniguchi et al. 1985, Shinoda et al. 1991). TLH was heat labile for 10 Taniguchi et al. and phospholipase activity et al. 1991). The TLH haemolysin gene contained an of et al. 1991). the nucleotide sequence of the it was revealed that the and mature protein consisted of and amino with molecular to and kDa respectively et al. 1991). The + C content of the gene is which is the as that of the V. parahaemolyticus (Taniguchi et al. Two were located near the (Taniguchi et al. but it was which was the was not found between the nucleotide sequences of the tdh gene and the gene of V. parahaemolyticus (Taniguchi et al. the gene has been found in the of all V. parahaemolyticus isolates of they have been from clinical or (Taniguchi et al. 1985; et al. 1999; et al. 1999). However, the role of this haemolysin in the of V. parahaemolyticus is et al. 1991). Zhang and Austin the and virulence factors of 21 V. isolates obtained from various and found that the most pathogenic produced extracellular with the of haemolytic activity to carried two haemolysin genes (designated and which were cloned and the majority of of the other cultures possessed only single genes, or at all et al. The of the and genes were both in which is the size as the of the gene of V. parahaemolyticus (Taniguchi et al. Both and encoded composed of amino acid and the amino acid sequences encoded by and were identical et al. the nucleotide sequences of and were not but were that the of the gene was a revealed that the nucleotide sequence of and to were and respectively et al. the amino acid sequence of V. protein of identity to V. parahaemolyticus TLH haemolysin et al. the phospholipase of V. the of V. mimicus et al. and the of V. cholerae et al. 1997). In addition to and TLH haemolysins, another thermostable haemolysin has been and found to be in all cultures of V. parahaemolyticus examined (Taniguchi et al. 1990). The gene a polypeptide of amino acid the nucleotide and amino acid sequence from of the trh and genes, or other haemolysins of V. parahaemolyticus (Taniguchi et al. 1990). a haemolysin, which was from the V. cholerae O1 HlyA haemolysin, was cloned from the O1 et al. This haemolysin possessed similarity to the δ-VPH haemolysin of V. parahaemolyticus, and was coined as the V. haemolysin encoded by the et al. The complete of a protein of kDa in to the TDH of V. parahaemolyticus, signal peptide could be from the sequence et al. However, this is in agreement with the V. which does not typical of a signal sequence at (Taniguchi et al. 1990). The + C content of and the was which is with that of V. cholerae (Taniguchi et al. 1990). It was determined that the gene is as a single copy on the of the two V. cholerae in The gene in V. cholerae O1 strains and and but in which is a clinical from et al. In as determined by DNA δ-VPH to be in all V. parahaemolyticus strains of their of (Taniguchi et al. 1990). from an in Escherichia coli, was found to be a protein on However, a direct role could not be for in V. cholerae O1 pathogenesis et al. haemolysin gene which a polypeptide of amino acid residues and haemolytic activity when in coli, has been cloned from V. cholerae O1 et al. The gene was in and El Tor V. cholerae V. cholerae non-O1 and V. but not in V. parahaemolyticus et al. et al. However, the of the gene to the of the organisms has not been In addition to the haemolysin gene (Yamamoto et al. et al. another haemolysin gene from V. has been et al. 1997). sequence an of encoding a amino acid polypeptide with an estimated of et al. 1997). The amino acid sequence of similarity to the sequence of which is responsible for and in et al. 1994). and of V. strains revealed that all of the V. clinical isolates contained genes et al. 1997). a new haemolysin gene from V. has been cloned and sequenced et al. 2004). sequence an of encoding a amino acid that sequence identity to the haemolysin of and and sequence identity to a haemolysin from V. cholerae of V. was in coli, haemolytic activity to that of haemolysin from was and an virulence with the when this was to and It is apparent that Vibrio spp. a range of haemolysins, some of which are very similar, although not 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 await the outcome of further research. more work is to the origin of the haemolysin This work was by of