Four novel hemolysin genes of Vibrio anguillarum and their virulence to rainbow trout

Four nucleotide sequences showing homology to known hemolysin genes were cloned and sequenced from V. anguillarum strain H775-3. The four genes, vah2, vah3, vah4 and vah5, have open reading frames encoding polypeptides of 291, 690, 200 and 585 amino acid residues, respectively, with predicted molecular masses of 33, 75, 22 and 66KDa, respectively. VAH2 is most closely related to a putative hemolysin of Vibrio vulnificus YJ016 (89% identity). VAH3 is most closely related to a hemolysin-related protein in Vibrio cholerae O1 (68% identity). VAH4 is most closely related to a thermostable hemolysin in V. cholerae O1 (72% identity). VAH5 is most closely related to a putative hemolysin in V. cholerae O1 (73% identity). The purified hemolysin proteins showed hemolytic activities against erythrocyte of fish, sheep and rabbit. Four strains of V. anguillarum mutants were constructed, each deficient in one of the hemolysin genes. Each mutant was less virulent than V. anguillarum H775-3 to juvenile rainbow trout (Oncorhynchus mykiss), indicating that each hemolysin gene contributes to the virulence of V. anguillarum H775-3.     

Purification and characterization of a hemolysin produced by Vibrio mimicus.

Vibrio mimicus is a causative agent of human gastroenteritis. This pathogen secretes a pore-forming toxin, V. mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the cell membrane. VMH with a molecular mass of 63 kDa was purified by ammonium sulfate precipitation and column chromatography with phenyl Sepharose HP and Superose 6 HR. The hemolytic reaction induced by VMH continued up to disruption of all erythrocytes in the assay system. Moreover, VMH that bound preliminarily to erythrocyte ghosts showed a sufficient ability to attack intact erythrocytes. These results suggest reversible binding of the toxin molecule to the membrane. The final cell-disrupting stage was effectively inhibited by various divalent cations. Additionally, some cations, such as Zn2+ and Cu2+, blocked the pore-forming stage at high concentrations. Although VMH could disrupt all kinds of mammalian erythrocytes tested, those from horses were most sensitive to the hemolysin. Horse erythrocytes were found to have the most toxin-binding sites and to be hemolyzed by the least amount of membrane-bound toxin molecules, suggesting that toxin binding to and pore formation on erythrocytes are more effective in horses than in other mammals. Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner. In addition, the antibody against the hemolysin obviously reduced enteropathogenicity of living V. mimicus cells. These findings clearly demonstrate that VMH is probably involved in the virulence of this human pathogen.     

Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum.

An invasiveness-defective mutant of the fish-pathogenic bacterium Vibrio anguillarum was isolated. Compared with the wild type, this mutant had a 1,000-fold higher 50% lethal dose after immersion infection of rainbow trout, Oncorhynchus mykiss, while after intraperitoneal infection, the mutant had only a 10-fold higher 50% lethal dose. In addition, the mutant showed a lower level of protease activity. Two forms of the protease (Pa and Pb) were found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonheated samples. Pa was found predominantly in protease preparations of the wild type, while Pb was the predominant form in the mutant. Conversion of Pb to Pa was observed in protease preparations after incubation at 4 degrees C. Characterization of the protease showed that it was an elastolytic enzyme which required Zn2+ for activity and Ca2+ for stability. The molecular mass of the protease was 36 kilodaltons. N-terminal amino acid sequence analysis of the protease of V. anguillarum revealed homology to the elastase of Pseudomonas aeruginosa and the protease of Legionella pneumophila.     

Purification and partial characterization of a non-O1 Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus.

A newly identified hemolysin (NAG-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus produced by non-O1 Vibrio cholerae, was studied. NAG-rTDH was purified by successive column chromatographies on DEAE-cellulose and an immunoaffinity column coupled with anti-Vp-TDH immunoglobulin. The molecular weight of NAG-rTDH was estimated as 18,500, similar to that of Vp-TDH, as judged by sodium dodecyl sulfate slab gel electrophoresis, but its charge or molecular shape was different, judging from its electrophoretic mobility. The lytic activities of NAG-rTDH on erythrocytes of most animals were essentially similar to those of Vp-TDH, but that on sheep erythrocytes was different. The hemolytic activity of NAG-rTDH was stable on heating at 100 degrees C for 10 min, as was that of Vp-TDH. Immunological cross-reactivity between NAG-rTDH and Vp-TDH was demonstrated by both the Ouchterlony test and the neutralization test. Thus, we conclude that non-O1 V. cholerae produce a new type of hemolysin that is similar but not identical to the thermostable direct hemolysin of V. parahaemolyticus.     

Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis

The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus. This homology was further confirmed by PCR analysis using a 5' primer derived from the amino-terminal sequence of the hemolysin and a 3' primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V. cholerae.     

Mutation of a novel virulence-related gene mltD in Vibrio anguillarum enhances lethality in zebra fish

Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis, which is a major problem for the aquaculture industry worldwide. Previously, a virulence-related gene fragment of V. anguillarum was obtained from a suppression subtractive hybridization (SSH) library. In this study, the complete gene sequence was obtained by long and accurate PCR (LA-PCR). After sequence analysis and homologous comparison, this new virulence-related gene was revealed to encode a putative membrane-bound lytic murein transglycosylase D (MltD), which consisted of 547 amino acids, and showed 34% identity to the MltD in Escherichia coli. An mltD mutant of pathogenic V. anguillarum CW-1 was constructed by homologous recombination. Production of extracellular gelatinase and protease of the mltD mutant decreased markedly compared with those of the wild-type strain, and the hemolytic activity was totally lost. Sodium chloride challenge and antibiotic sensitivity assay showed that the resistance of the mltD mutant to high concentrations of sodium chloride, and rocephin, fortun, cefobid, gentamicin, kanamycin and carbenicillin was enhanced. Most importantly, virulence of the mltD mutant was enhanced compared with that of the wild type when it was inoculated intraperitoneally into zebra fish; the LD₅₀ of the wild type and the mutant was 3.92 × 10³ CFU and 1.01 × 10² CFU fish⁻¹, respectively. The mltD was cloned and overexpressed in E. coli, and the recombinant MltD protein showed hemolytic, phospholipase, gelatinase and diastase activities. This is the first report that MltD possibly has a virulence-related function.     

Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin.

A clinical isolate (strain 4037) of Kanagawa phenomenon-negative Vibrio parahaemolyticus was studied. Although the strain was judged to be Kanagawa phenomenon-negative by various conventional tests, it produced a new hemolysin (named Vp-TRH, for thermostable direct hemolysin [Vp-TDH]-related hemolysin) that was related to the Vp-TDH produced by ordinary Kanagawa phenomenon-positive V. parahaemolyticus. Vp-TRH was purified by ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, and Mono Q. The molecular weight of Vp-TRH was estimated as 48,000 by Sephadex G-100 gel filtration, and the molecular weight of the subunit was estimated to be 23,000 by sodium dodecyl sulfate-slab gel electrophoresis. Thus, like Vp-TDH, Vp-TRH seems to be composed of two subunits. The isoelectric point of Vp-TRH was determined to be 4.6. Vp-TRH showed lytic activities different from those of Vp-TDH on erythrocytes from various animals, especially those from calves, chickens, and sheep. The hemolytic activity of Vp-TRH was labile on heat treatment at 60 degrees C for 10 min, unlike that of Vp-TDH. The immunological similarities, but not the identities of Vp-TRH and Vp-TDH, were demonstrated by Ouchterlony, neutralization, and latex agglutination tests. Thus, we conclude that this clinical isolate of Kanagawa phenomenon-negative V. parahaemolyticus produces a new type of hemolysin that is similar, but not identical, to Vp-TDH, which is usually produced by Kanagawa phenomenon-positive V. parahaemolyticus.     

Overexpression, purification, characterization, and pathogenicity of Vibrio harveyi hemolysin VHH

Vibrio harveyi VHH hemolysin is a putative pathogenicity factor in fish. In this study, the hemolysin gene vhhA was overexpressed in Escherichia coli, and the purified VHH was characterized with regard to pH and temperature profiles, phospholipase activity, cytotoxicity, pathogenicity to flounder, and the signal peptide.     

Identification of a lacZ gene in Vibrio cholerae.

Plasmids that express an enzymatically active beta-galactosidase in an Escherichia coli delta lac strain were isolated from libraries of recombinant plasmids containing Vibrio cholerae chromosomal DNA. Deletion analysis localized the gene responsible for beta-galactosidase activity on a 3.1-kb DNA fragment, and the gene product was identified as a protein of approximately 110 kDa. Primary sequence comparisons indicated that this V. cholerae gene is homologous to the E. coli lacZ gene. In contrast to the lac loci of other bacteria, no gene that could specify a lactose transport system was detected in the vicinity of the V. cholerae lacZ gene, which may account for the inability of this species to use lactose. In V. cholerae, portions of open reading frames encoding proteins homologous to the Alcaligenes eutrophus chrA and E. coli galR gene products were detected upstream and downstream from the lacZ gene, respectively.     

The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene.

A cytolysin of ca. 56 kilodaltons has been suggested as a possible virulence factor in Vibrio vulnificus infections. We sequenced the DNA encoding cytolytic activity and found that the sequence contained two open reading frames, vvhA and vvhB. vvhA encoded the structural gene for the cytolysin and contained the N-terminal amino acid sequence previously reported for the protein. Regions of the vvhA gene showed homology to the structural gene for the Vibrio cholerae E1 Tor hemolysin.     

Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin.

Hemolysin of a non-O1 Vibrio cholerae strain was purified and characterized. The purified hemolysin gave a single protein band on conventional and sodium dodecyl sulfate-gel electrophoresis. Its molecular weight was estimated as 60,000 by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. It had a pI of 5.7. The purified hemolysin caused increased vascular permeability of rabbit skin and rapid death of mice on intravenous injection and also lysed erythrocytes of various animal species. An Ouchterlony double gel diffusion test using antiserum against the purified hemolysin indicated that the hemolysin from non-O1 V. cholerae was immunologically related, but not identical, to the hemolysin from El Tor V. cholerae. Antiserum against the purified hemolysin neutralized the hemolytic activity of the hemolysins from not only non-O1 but also El Tor V. cholerae.     

Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae

The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors.     

Cloning and nucleotide sequence of the gene (trh) encoding the hemolysin related to the thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates derived from an outbreak of gastroenteritis in the Republic of Maldives did not have the genetic potential to produce the thermostable direct hemolysin, but one such isolate produced a hemolysin immunologically related to the thermostable direct hemolysin (T. Honda, Y. Ni, and T. Miwatani, Infect. Immun. 56:61-965, 1988). The Maldives isolates hybridized with the DNA probe for the gene encoding the thermostable direct hemolysin (the tdh gene) under reduced stringencies. A DNA fragment containing the probe-reactive nucleotide sequence was isolated from a selected strain and cloned into pBR322 in Escherichia coli. A clone producing the thermostable direct hemolysin-related hemolysin was obtained by screening with hemolysis assays and by an immunological assay. Nucleotide sequence analysis of the cloned DNA fragment revealed that the gene encoding the thermostable direct hemolysin-related hemolysin (the trh gene), like the tdh gene, encoded the hemolysin subunit composed of 189 amino acid residues. The trh gene had significant nucleotide sequence homology with the tdh gene (68.4% with the tdh1 gene copy and 68.6% with the tdh2 gene copy). The amino acid sequences of the hemolysin subunits deduced from the nucleotide sequences of the trh gene and tdh gene were homologous (61.9% homology with the tdh1-encoded subunit and 63.0% homology with the tdh2-encoded subunit) and contained the two cysteine residues to form an intrachain bond at the same positions, and their possible conformations appeared to be similar as determined by hydrophobicity-hydrophilicity analysis and a secondary structure prediction. The trh and tdh genes may have had a common ancestor and may have evolved by single-base changes so that they maintained the fundamental architecture of the molecules.     

Molecular cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae.

This article describes the molecular cloning and expression of a hemolysin gene from a serotype 1 strain of Actinobacillus pleuropneumoniae. The hemolysin was a thermolabile protein with an apparent molecular weight of 29,500 (29.5K hemolysin). Unlike expression of the recently described 105K hemolysin of A. pleuropneumoniae (J. Frey and J. Nicolet, FEMS Microbiol. Lett. 55:41-46, 1988), expression of this hemolysin was not regulated by Ca2+. Antiserum prepared against the 105K hemolysin did not neutralize the activity of the 29.5K hemolysin; conversely, antiserum prepared against the 29.5K hemolysin did not neutralize the activity of the 105K hemolysin. The hemolytic activity was not neutralized with antisera against hemolytic Escherichia coli, Streptococcus agalactiae, or purified streptolysin O, but antisera prepared against recombinants containing the 29.5K gene and convalescent pig sera abrogated hemolytic activity. Although hemolytic activity could be detected in several strains of E. coli K-12 and in minicells expressing several different constructs encoding the 29.5K hemolysin, we could not rigorously exclude the possibility that the gene which we have isolated encodes a regulator of hemolytic activity rather than a hemolysin per se.