Purification and some properties of Aeromonas hydrophila hemolysin

A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.     

Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila.

The extracellular hemolysin (AHH1) gene of Aeromonas hydrophila ATCC7966 was cloned into Charomid9-28 in Escherichia coli DH1, and its complete nucleotide sequence determined. Escherichia coli carrying this gene expressed an extracellular heat-labile hemolysin for rabbit red blood cells. The minimum size of the coding region of the 2.6 kilobase-pair BamHI-SphI fragment was subcloned into pUC118 and pUC119, selecting for hemolytic activity. The nucleotide sequence of this region contained a single open reading frame of 1734 base pairs, corresponding to a protein of 577 amino acid residues (63,658 daltons). A consensus promoter sequence was present upstream of the AHH1 open reading frame. Maxicell analysis of [35S]methionine-labelled proteins in E. coli CSR603 carrying the AHH1 plasmid suggested that AHH1 gene codes for an approximately 60,000 dalton polypeptide. By colony DNA-DNA hybridization analysis, the AHH1 gene was detected in 43 of 62 hemolysin-producing strains of A. hydrophila (isolated from various sources and areas) and in all 43 hemolysin-producing strains of A. salmonicida (isolated from fish). Three hemolysin-negative strains of A. hydrophila did not react with the AHH1 probe, whereas three non-hemolytic A. salmonicida strains hybridized with the probe.     

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.     

Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.

Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000- and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.     

Medium for the accumulation of extracellular hemolysin and protease by Aeromonas hydrophila.

A medium for the accumulation of extracellular hemolysin (300 to 1,600 hemolytic units per ml) and protease (2 to 3 proteolytic units per ml) was developed for an anaerogenic strain of Aeromonas hydrophila. In this medium, growth yields were less but levels of accumulated toxin were greater or equivalent when compared with the same responses in brain heart infusion and nutrient broths. The medium was considered to be partially defined since the conditions for maximum observed hemolysin accumulation (1,600 hemolysin units per ml) were not identified. The results showed that iron and zinc contributed to the control of the extracellular accumulation of both toxins. Whereas iron exerted an inhibitory effect, zinc stimulated the accumulation of both toxins.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria.

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58-68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.     

抗嗜水气单胞菌单克隆抗体的制备及初步鉴定

嗜水气单胞菌是养殖及野生鱼、虾、蛙、甲鱼等生物常见的细菌性病原体[1],可通过受伤的皮肤及伤口感染,并可通过食物链感染人,造成腹泻、败血症等疾病而严重威胁人类健康,是我国沿海地区人群中最常见的致病性弧菌之一[2].     

Purification and chemical characterization of a cholera toxin-cross-reactive cytolytic enterotoxin produced by a human isolate of Aeromonas hydrophila.

A bacterial protein toxin possessing hemolytic, enterotoxic, and cytotoxic activities as well as cross-reactivity to cholera toxin was purified from culture filtrates of a human diarrheal isolate of Aeromonas hydrophila (SSU). This cytolytic enterotoxin was purified by ammonium sulfate precipitation, hydrophobic chromatography using phenyl-Sepharose, anion-exchange chromatography on DEAE-Bio-Gel A, and size-exclusion high-performance liquid chromatography. The factor was a single polypeptide with an apparent molecular weight of 52,000 as determined by polyacrylamide gel electrophoresis. Automated amino acid sequence analysis confirmed that the toxin was a single chain and established a 25-residue N-terminal segment which was identical to that of aerolysin purified from culture supernatants of A. hydrophila isolate Ah65 originally obtained from rainbow trout as reported by Howard et al. (S. P. Howard, W. J. Garland, M. J. Green, and J. T. Buckley, J. Bacteriol. 169:2869-2871, 1987). However, the amino acid compositional analysis of the toxin produced by our human isolate (SSU) differed significantly from that of the Ah65 isolate. Taken together, these results strongly indicated that several toxic phenomena associated with A. hydrophila (SSU) culture filtrates, including hemolysis, cytotoxicity, and enterotoxicity as well as cross-reactivity to cholera toxin, all can occur on a single polypeptide. In addition, these results underline the fact that although aerolysin-related toxins isolated from culture filtrates of A. hydrophila are biologically similar, significant chemical and immunological differences may exist between toxins produced by individual isolates.     

Cloning and characterization of three hemolysin genes from Aeromonas salmonicida

Two hemolysin genes (ASH3 and ASH4) of Aeromonas salmonicida strain 17-2 and one (ASH1) of A. salmonicida ATCC14174 were cloned into the cosmid vector charomid 9-36 in Escherichia coli DH1. The overall amino acid sequence of the ASH3 was similar to that of the aerolysins of Aeromonas hydrophila and Aeromonas sobria, and hemolysins AHH3, AHH4, and AHH5 of A. hydrophila, and hemolysin ASA1 of A. sobria. The sequence of ASH4 was similar to that of the AHH1 hemolysin of A. hydrophila. The ASH4 hemolysin contains some homologous sequence regions of the Vibrio vulnificus and Vibrio cholerae cytolysin-hemolysin. Both ASH3 and ASH4 DNA probes reacted with all 104 strains of A. salmonicida, whereas the ASH1 DNA probe did not hybridize with any of the 104 strains studied except strain ATCC14174. ASH1 and ASH3 were broad spectrum hemolysins with most activity against rabbit and horse erythrocytes, respectively, whereas ASH4 hemolysin did not lyse bovine and horse erythrocytes. ASH3 and ASH4, but not ASH1, were activated by trypsin.     

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.     

Cytotoxic activity of Aeromonas hydrophila.

Most strains of Aeromonas hydrophila tested demonstrated cytotoxic activity on several tissue-cultured cell lines. The cytotoxin is heat-labile, non-dialyzable, and immunologically distinct from that of Shigella dysenteriae and Clostridium perfringens. None of the aeromonas isolates was found to be enterotoxigenic by either tissue culture or rabbit ileal loop assays.     

Aeromonas hydrophila bacteremia

This patient with septicemia caused by Aeromonas hydrophila demonstrates clinical features similar to those of patients in previous reports. Particularly interesting were the skin lesions that appeared at the time of onset of septicemia. It has been shown that these bacteria have particular affinity for muscle tissue, causing necrosis and intense inflammation (3, 5). Presumptive identification of this species may be accomplished if the oxidase test is routinely performed along with additional physiologic and biochemical tests (3, 7). However, further taxonomic clarification is needed for members of this genus.     

Meningitis due to Aeromonas hydrophila.

A case of meningitis caused by Aeromonas hydrophila is reported. The infection complicated an otherwise successful frontotemporal craniotomy. Survey of the literature revealed that human infections due to this organism are relatively uncommon, and its causal relationship in meningitis has not been previously reported. A. hydrophila is known to cause bacteremia in patients under treatment with immunosuppresive agents. We report meningitis with bacteremia, caused by this organism, in an immunologically competent host.     

Bioactivity and immunological characterization of a cholera toxin-cross-reactive cytolytic enterotoxin from Aeromonas hydrophila.

A cytolytic enterotoxin of molecular weight 52,000 was isolated and purified from culture supernatants of a human diarrheal isolate (SSU) of Aeromonas hydrophila. The toxin reacted with cholera antitoxin when tested in an enzyme-linked immunosorbent assay and by Western blot (immunoblot) analysis. The appearance of cytotoxic and hemolytic activities in culture supernatant occurred simultaneously 8 h after the initial inoculation of the culture. Loss of hemolytic activity and cholera toxin cross-reactivity was correlated with heat and pH inactivation. Homologous antibodies neutralized the cytotoxic and hemolytic activities associated with the toxin, but cholera antitoxin did not neutralize these activities. The toxin also possessed enterotoxic activity as demonstrated by fluid accumulation in rabbit ligated intestinal loops. When purified cytolytic enterotoxin was injected intravenously into mice, death occurred within 2 min, whereas mice injected with whole cells or sonicated cell fragments died after several hours or days. Results from 51Cr release experiments demonstrated that the cytolytic enterotoxin had significant membrane-damaging capability. These results indicated that the cytolytic and enterotoxic activities expressed by the described A. hydrophila toxin may contribute significantly to the pathogenesis of disease associated with A. hydrophila.     

Identification and characterization of the Aeromonas sobria hemolysin glycoprotein receptor on intestine 407 cells.

Aeromonas sobria hemolysin is important in the pathogenesis of diarrhoea caused by this enteropathogenic bacterium. By immunoprecipitation analysis using hemolysin and anti-hemolysin antibody, a 66 kDa protein (p66) was identified as a receptor for A. sobria hemolysin on Intestine 407 cells. Treatment of p66 with N-glycosidase F reduced the apparent sized of p66 to 60 kDa on SDS-polyacrylamide gels. p66, released from Intestine 407 cells following incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, bound A. sobria hemolysin. Thus treatment of Intestine 407 cells with PI-PLC resulted in the remarkable decrease of the sensitivity to A. sobria hemolysin. These results are consistent with the hypothesis that p66, the binding protein for A. sobria hemolysin, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the surface of Intestine 407 cells and probably plays a role as a receptor for A. sobria hemolysin on the intestinal cells.     

Unusual case of Aeromonas hydrophila endocarditis.

We describe a case of Aeromonas hydrophila endocarditis in a 66-year-old man with myelodysplastic syndrome and non-A, non-B hepatitis, The infection resolved with antibiotic therapy, but the patient succumbed to complications of his underlying illness. This is the second case of Aeromonas endocarditis reported in the world literature.     

Faecal carriage rate of Aeromonas hydrophila.

Over a four and a half month period, 1004 unselected routine faecal specimens from 815 patients were cultured for Aeromonas hydrophila. Forty-two specimens (4.2%) representing 38 patients were culture-positive. The study specimens also yielded Salmonella on 116 occasions, Shigella on seven, Campylobacter species on six and other bacterial pathogens on 17 occasions, respectively. Seven specimens had A hydrophila together with another bacterial pathogen. In only 19 of 38 patients (50%) was A hydrophila possibly associated with gastrointestinal symptoms. All the Aeromonas isolates were resistant to ampicillin but sensitive to gentamicin, piperacillin, mecillinam, chloramphenicol, ceftazidime and colistin.