Duplication of Hemolysin Genes in a Virulent Isolate of Vibrio harveyi

Abstract

Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated vhhA and vhhB), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene. Both genes from VIB 645 were cloned and sequenced. The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues. The VHH protein shows homology to the lecithinase of V. mimicus and V. cholerae. Transformants of Escherichia coli containing the ORF of either vhhA or vhhB displayed weak hemolytic activity in rainbow trout blood agar. The hemolytic activity was very high when the ORF of vhhB was cloned in E. coli together with the native promoter. Surprisingly, the level of vhh-specific RNA transcript produced by VIB 645 was found to be very low. We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of the vhh gene.Vibrio harveyi is a gram-negative, luminous bacterium which is widely distributed in the marine environment (28,29). Recently, this organism has emerged as a serious pathogen of marine animals. For example, the organism has been reported as a primary pathogen of cultured penaeid shrimp, especially in South America and Asia (1,10,18,26,33,36). Additionally, V. harveyi has been associated with diseases in finfish (1,8,9,15,30) and pearl oysters (24). However, little is known about the pathogenicity mechanisms of V. harveyi. Liu et al. (19) considered that proteases, phospholipases, or hemolysins might be important for pathogenicity; cysteine protease has been reported as the major exotoxin to penaeid shrimp (16,17,20). Montero and Austin (21) suggested that the lipopolysaccharide might constitute the lethal toxin of V. harveyi E2 to penaeid shrimp. We have previously examined a large number of well-characterized V. harveyi cultures and found that the hemolysin activity in the extracellular product was involved in pathogenesis in salmonids. V. harveyi VIB 645, which was the most pathogenic isolate, produced extracellular product with the highest titer of hemolytic activity towards Atlantic salmon (1:256) and rainbow trout erythrocytes (1:32) (40).In general, bacterial hemolysins have been suggested to be important factors of pathogenic vibrios by causing hemorrhagic septicemia and diarrhea in the host. Many of these hemolysins are well characterized, and genes encoding them have been cloned from V. parahaemolyticus (22, 35), V. cholerae (3, 27), V. hollisae (38), V. mimicus (12), V. vulnificus (38), and V. anguillarum (7).In this study, we describe the cloning and characterization of the hemolysin gene of V. harveyi as a first step towards assessing the role of V. harveyi hemolysin in fish disease. MATERIALS AND METHODSBacterial strains and plasmids. Thirteen V. harveyi isolates from a diverse range of h...

PCR detection of hemolysin (vhh) gene in Vibrio harveyi

“…2B) but did not amplify genes of the isolates from Thailand (VIB 391) or Ecuador . When the F2 forward primer designed in this study was used in combination with the reverse primer R1 from a previous study (Zhang et al, 2001), the expected 1.2-kb amplicon was observed in nine (9) V. harveyi strains, but still no PCR product was produced in the Thailand or Ecuador isolates (Fig. 2D).…”

“…The primers reported in a previous study (Zhang et al, 2001) were utilized in the PCR amplification of the 1.3 kb hemolysin gene from Vibrio harveyi strains obtained from different geographic locations (Table 1). Amplification using the reported primer set successfully generated 1.3 kb putative hemolysin gene from only nine out of eleven strains including the reference V. harveyi strain IFO 15634 ( Fig.…”

“…Subsequently, they isolated the duplicated hemolysin genes (vhhA and vhhB) of a strain of V. harveyi pathogenic to salmons (Zhang et al, 2001). Sequences of the two copies of the V. harveyi (VIB 645) hemolysin genes (vhhA and vhhB), are almost identical and primers that could amplify the 1.3 kb open reading frame of both copies are already available but have not been used for detection of the species.…”

“…One such gene implicated in the virulence of Vibrio species encodes for hemolysins. Hemolysins are responsible for the disruption of the erythrocyte membrane or hemolysis and genes encoding for hemolysins have been reported to be present in several members of the genus including V. harveyi (Hirono et al, 1996;Nishibuchi and Kaper, 1995;Nishibuchi et al, 1990;Zhang et al, 2001). Hemolysin genes have been targeted in PCR for the detection of pathogenic strains of Vibrio species including hemolysin genes (tl, tdh and trh) of V. parahaemolyticus (Bej et al, 1999) and V. vulnificus cytolysin gene (Lee et al, 1998).…”

Diversity and functional analysis of luxS genes in Vibrios from marine sponges Mycale laxissima and Ircinia strobilina

“…Associations of vibrios with other marine organisms range from pathogenic to mutualistic. Close relatives of the Vibrio isolates described in this study include V. harveyi, a major pathogen of marine animals (Zhang et al, 2001) and V. shiloli (AF007115), well established as a causative agent of coral bleaching (Kushmaro et al, 1996(Kushmaro et al, , 1997. Pathogenicity is often strain-specific rather than species-specific so the close relationship between the sponge-associated Vibrio species and known pathogens does not necessarily indicate that our Vibrio isolates are potential pathogens of their host sponges.…”