| Vibrios are normal inhabitants in aquatic environments, being very common in marine and estuarine habitats. Many studies have reported that Vibrio species are pathogenic to marine shrimps and fish. Haemolysin, which is an exotoxin that attacks blood cell membranes and causes cell rupture, is arguably the most widely distributed toxin among pathogenic vibrios. Haemolysis may result from the enzymatic activities demonstrated by some species of bacteria, including phospholipase C of Pseudomonas aeruginosa and phospholipase D in Photobacterium damselae ssp. damselae. However, many other haemolysins act by forming pores in the cytoplasmic membrane of erythrocytes. 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. Currently, five representative haemolysin families have been found in Vibrio sp., including the TDH (thermostable direct haemolysin) family of V. parahaemolyticus, the HlyA (E1 Tor haemolysin) family of V. cholerae, the TLH (thermolabile haemolysin) family of V. parahaemolyticus, theδ-VPH (thermostable haemolysin) family of V. parahaemolyticus and the HLX family of V. cholerae O1. Each type of haemolysin gene was aligned using ClustalW (http://www.ebi.ac.uk/clustalW/), and five pairs of PCR primers were designed from the conserved region of the haemolysin genes and the TLH, TDH, HlyA,δ-VPH and HLX haemolysin gene fractions were amplified. Five DIG-labelled haemolysin gene probes were synthesized by PCR amplification from total DNA of different Vibrio strains, and were used to detect the distributions of five haemolysin genes among 57 Vibrio strains by Southern Blot. The result showed that 2 Vibrio strains had TDH haemolysin gene, 2 strains had HlyA haemolysin gene, 49 strains had TLH haemolysin gene, 3 strains hadδ-VPH haemolysin gene and 30 strains had HLX haemolysin gene. From these results, it seems that the TLH haemolysin gene was widely distributed in different Vibrio species, and the positive ratio was as high as 85.96%. HLX haemolysin gene was widely distributed among V. harveyi, V. parahaemolyticus and was also detected in V. alginolyticus, V. cincinatiensis, V. diazotrophicus, Grimontia hollisae and the positive ratio is 52.63%. However, the positive ratios of TDH,HlyA andδ-VPH haemolysin genes were less than 6%. Moreover, three strains of V. harveyi had duplicated tlh genes, one strain of Grimontia hollisae and one strain of V. parahaemolyticus had duplicated tdh genes. It seems that duplication of haemolysin genes in vibrios is not rare.To support this conclusion, the vhh/tlh homologue genes in V. anguillarum VIB 72, V. aestuarianus VIB 281, V. fischeri VIB 291, V. fluvialis VIB 292, V. campbellii VIB 285, V. natriegens VIB 299 and V. harveyi VIB 647 were cloned and sequenced, and the deduced amino acid sequences showed high degree of identities to VHH (67～99%) and TLH haemolysin (69～91%).57 isolates of vibrios were inoculated on the fish blood agar plate and egg yolk plates respectively, and haemolytic and phospholipase activities were determined by the appearance of a lytic zone and opalescence around the bacterial growth. It was found that all the vibrios strains with TLH haemolysin gene had haemolytic activities. Except V. fischeri, V. gazogenes, V. logei and V. harveyi VIB 647, all the other vibrio strains that had TLH haemolysin gene also had phospholipase activities. Most vibrio strains that had strong haemolytic and phospholipase activities also had strong hybridization signals for TLH haemolysin gene. However, there are also some exceptions, e.g. Photobacterium damselae subsp. damselae, V. furnissii, V. tubiashii and V. harveyi VIB 649, which have strong haemolytic or phospholipase activities, have weak or no hybridization signals. Almost all the vibrio strains which had no hybridization signals for TLH haemolysin gene had no haemolytic or phospholipase activities. The haemolytic or phospholipase activities in the 3 V. harveyi isolates which had duplicated TLH haemolysin genes were stronger than those in others strains. This suggests that the haemolytic or phospholipase activities of V. harveyi were also correlated with the duplication of TLH haemolysin genes. From these results, it was concluded that haemolytic and phospholipase activities of Vibrio species were closely correlated with the distribution of TLH haemolysin gene, but not with other 4 types of haemolysin genes.To further study the vhh/tlh haemolysin gene, the chromosomal DNA of V. campbellii VIB 285 was used as template to amplify the whole vhh/tlh haemolysin gene. The PCR product was cloned into pET-24d(+) vector following the manufacture's instruction. The recombinant plasmid containing the insert of expected size was transfered into E. coli express strain BL21(DE3). When induced by IPTG (1 mM), the recombinant strain had haemolytic activity.
|
PDF Full Text Request