Erratum: Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L-cysteine, iodoacetic acid, N-ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl2 but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 μg protein g-1 fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.     

Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.     

Detection of a vascular permeability factor in the extracellular products of Renibacterium salmoninarum.

The presence of vascular permeability factors in the extracellular products (ECP) of 10 strains of Renibacterium salmoninarum with different geographical origin and serological characteristics are reported. All the ECP produced haemorrhagic and/or oedematous zones at the injection site with a diameter ranging from 10-30 mm. However, the ECP samples did not display toxic effect in fish at the same dose as inoculated in rabbit (180-400 micrograms protein/0.1 ml). No differences were observed in the production of this dermatotoxic factor between the two antigenic groups found in this microorganism. Whereas heating (80 and 100 degrees C/15 min) the ECP samples resulted in a complete loss of their proteolytic activity, only a decrease (but not total inactivation) of the dermatotoxic effects was detected. Therefore, although proteases could be implicated in the permeability factor, they are not totally responsible for this activity.     

Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila--part I

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.     

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.     

Affinity purification of staphylococcal toxic shock syndrome toxin 1 and its pathologic effects in rabbits.

Toxic shock syndrome toxin 1 (TSST-1) was purified to apparent homogeneity by chromatofocusing and affinity chromatography. The amino acid composition of the toxin was very similar to that reported for TSST-1 by other investigators. The amino-terminal amino acid was serine. A partial specific volume of 0.73 ml/g was calculated for the toxin from the amino acid data, and a molecular weight of 19,200 +/- 1,300 was determined by hydrodynamic methods. New Zealand white rabbits of both sexes were equally susceptible to the lethal effects of the toxin; however, older rabbits (greater than 12 months) were far more susceptible than young adults or weanlings. The 50% lethal dose of TSST-1 in older rabbits was 50 to 60 micrograms/kg when injected subcutaneously and 20 to 30 micrograms/kg when injected intravenously. Enhancement of lethal endotoxin shock by TSST-1 could not be demonstrated when both toxins were injected subcutaneously; however, lethal shock did occur when endotoxin (10 micrograms/kg) was injected intravenously after TSST-1 had been injected by either the subcutaneous (50 to 60 micrograms/kg) or the intravenous (20 to 30 micrograms/kg) route. Endotoxin alone was not lethal at a dose of 500 micrograms/kg of body weight when injected subcutaneously. When injected intravenously, endotoxin at a dose of 500 micrograms/kg was not lethal in weanling males or in females in any age group; however, young (6 to 7 months) and adult (greater than 12 months) males were killed by endotoxin doses as low as 45 to 50 micrograms/kg. Histopathologic studies of rabbits by both sexes which died as a result of TSST-1 alone or in combination with endotoxin showed extensive damage to organs rich in lymphoid and mononuclear phagocytic cells such as the thymus, mesenteric lymph nodes, liver, and spleen. Severe congestion of these organs as well as erythrophagocytosis and lymphoid depletion in the spleen and mesenteric lymph nodes were noted. Congestion and hemorrhage were also found in the heart, lungs, trachea, and thymus. The systemic pathology produced by TSST-1 was strikingly similar to that seen in humans who had died of toxic shock syndrome and in rabbits with subcutaneous chamber inoculated with toxic shock case strains of Staphylococcus aureus. Rabbits that were not killed by the toxin suffered a very rapid and severe leukopenia followed by leukocytosis with a left shift. Lymphopenia was also noted as was a mild but persistent anemia. With the exception of the early leukopenia, very similar hematologic findings have been noted in humans with toxic shock syndrome.     

DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila

Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam(AhSSU)) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N(6)-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P(BAD) promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.     

Immunological cross-reactivity of enterotoxins of Aeromonas hydrophila and cholera toxin.

Pre-incubation with anticholera toxin (ACT) significantly reduced intestinal secretion induced by cell-free broth preparations of heat-labile toxins (LT) of Escherichia coli and Aeromonas hydrophila in jejunal perfusion experiments in rats in vivo. Pre-incubation with ACT also prevented cytotoxicity by E. coli LT in the Y1 cell culture system. Pre-incubation had no effect on cytotoxicity in Y1 and L132 cell lines or on haemolytic activity with cell-free preparations of A. hydrophila. In another series of experiments rats were immunized with cholera toxin given as an intraperitoneal priming dose followed 12 days later by intraduodenal boosting. Immunization significantly protected against net intestinal fluid secretion induced by enterotoxigenic E. coli and A. hydrophila and by cholera toxin.     

Purification of dermonecrotic toxin from a sonic extract of Pasteurella multocida SP-72 serotype D

A procedure was developed to purify dermonecrotic toxin (DNT) from a sonic extract of a serotype D strain of Pasteurella multocida. Sonic extract containing DNT was applied to a DEAE-Sephacel column and eluted by a linear gradient of NaCl. Upon rechromatographing, fractions with dermonecrotic activity for guinea pigs were applied on a second Sephacel column, and a pooled fraction with the toxic activity was filtered through a Sephadex G-200 column. Pooled fractions with the toxic activity were subjected to polyacrylamide disc gel electrophoresis (PAGE), and the toxic substance was eluted from each sliced gel. Eluted fractions with the toxic activity were rechromatographed on a second Sephadex G-200 column, and a pooled fraction with high dermonecrotic activity was referred to as a purified DNT. The activity of purified DNT was increased by 1,000 times, and the average yield was about 1.8%. The purified DNT was homogeneous as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and thin-layer isoelectric focusing in polyacrylamide gels and gave a single band on PAGE and sodium dodecyl sulfate-PAGE. The molecular weight of the toxin was ca. 160,000 as determined by sodium dodecyl sulfate-PAGE. The isoelectric point of the toxin was ca. 4.7 to 4.8. Amino acid analysis of the purified DNT revealed that the toxin was composed of characteristically high proportions of glutamic acid, aspartic acid, glycine, proline, alanine, and leucine. The minimal necrotizing dose of the toxin was about 1 ng of protein, and the 50% lethal dose per mouse was 0.2 micrograms. The purified DNT was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde.     

Interactions Between Aerolysin, Erythrocytes, and Erythrocyte Membranes

Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids. Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods. Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action. There is some correlation between sensitivity and phosphatidyl choline content. Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis. Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases. Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent. Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin. Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant.     

Effect of toxic shock syndrome toxin 1 on chicken embryos.

Staphylococcus aureus strains associated with toxic shock syndrome produce toxic shock syndrome toxin 1 (TSST1). This toxin has a variety of biological effects, including enhanced lethality in rabbits in the presence of sublethal amounts of lipopolysaccharide (LPS). Because chicken embryos are highly susceptible to LPS, the synergistic effect of TSST1 and LPS was examined in this system. Although TSST1 per se had no effect on chicken embryos, it potentiated the lethal effect of LPS. The 50% lethal dose of LPS was greatly reduced in the presence of up to 10 micrograms of TSST1 per ml. However, at high doses of TSST1 (greater than 100 micrograms/ml), no enhanced lethality was observed. The lowest dose of TSST1 tested which potentiated lethality was 10 ng/ml.     

Purification and characterisation of an extracellular metalloprotease, serine protease and haemolysin of Aeromonas hydrophila strain B32: all are lethal for fish.

Three different lethal (for rainbow trout, Salmo gairdneri) extracellular toxins were purified by HPLC from the culture supernatants of Aeromonas hydrophila strain B32 which had been isolated from rainbow trout. A metalloprotease, MW 38 kDa, was stable at 56 degrees C for 10 min, had no cytotoxic activity and and LD50 of 150 ng/g fish. In narrow range isoelectric-focusing (IEF) the enzyme had 11 isomers with (pls) between 4.12 and 4.8. A serine protease (22 kDa) was stable at 56 degrees C for 10 min, possessed cytotoxic activity and had an LD50 of 150 ng/g fish. In IEF, multiple isomers possessed pls between 4.5-5.2. The haemolysin had alpha-haemolytic activity (68 kDa) multiple isomers in IEF with pl range 4.5-5.1 and an LD50 of 2 micrograms/g fish. It was stable after heating to 56 degrees C for 20 min, 60 degrees C for 10 min and possessed esterase activity on beta-naphthyl acetate. These latter properties suggest it may be a novel haemolysin distinct from alpha- and beta-haemolysin.     

Identification of lethal toxin with the thermostable direct hemolysin produced by Vibrio parahaemolyticus, and some physicochemical properties of the purified toxin.

Lethal toxin was purified extensively from the culture filtrate of a Kanagawa phenomenon-positive strain of Vibrio parahaemolyticus. The purified toxin was a protein, and its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. It was demonstrated that the thermostable direct hemolysin was identical to the lethal toxin and that it was the main, if not only, lethal toxin in the culture filtrate. The purified toxin had a lethal effect when injected into mice either intravenously or intraperitoneally. Its lethal effect was very rapid, a dose of 5 mug of toxin per mouse killing the animals within 1 min. The lethal activity was inhibited by a ganglioside mixture. Some physicochemical properties of the purified toxin are reported.     

Detection of extracellular toxin(s) produced by Vibrio vulnificus.

Conditions are described for the production, in high titers, a heat-labile, antigenic, extracellular toxin(s) by Vibrio vulnificus, a recently recognized human pathogen. Bacteriologically sterile culture filtrate preparations obtained from mid-logarithmic-phase cultures of the bacterium possessed cytolytic activity against mammalian erythrocytes, cytotoxic activity for Chinese hamster ovary cells, vascular permeability factor activity in guinea pig skin, and lethal activity for mice. The specific activity of toxin preparations from cultures of a virulent strain of the bacterium was ca. 25-fold more than that of toxin preparations obtained from cultures of a weakly virulent strain. The four toxic activities were inseparable by gel filtration with Sephadex G-100; however, two components, which had markedly different elution behavior but which possessed the four activities mentioned above, were obtained. The major (ca. 88% of the recovered activity) and minor components had apparent molecular weights of ca. 38,500 and greater than 150,000, respectively.     

Purification and Properties of a Toxin Isolated from Mortierella wolfii

A substance toxic for mice has been extracted from the washed mycelia of Mortierella wolfii. This toxin could also be demonstrated in culture fluids after spontaneous autolysis of the fungal hyphae. Purification of the toxin was carried out using Sephadex chromatography (G-200, G-50) and resulted in a single band on sodium dodecyl sulfate polyacrylamide gels. Properties of the toxin included heat lability at 60 C, trypsin sensitivity, activity over a wide pH range, and a molecular weight of 23,600. The mean lethal dose for 16-week-old male mice was 3.3 μg of protein.     

Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections.

We report here on the isolation of isogenic strains of Aeromonas hydrophila AB3 deleted for a segment of the aerolysin gene. All aer mutants obtained lacked the 49-kilodalton aerolysin gene product and were neither hemolytic for blood erythrocytes nor cytotoxic for Chinese hamster ovary tissue culture cells. One such mutant, AB3-5, was used in a mouse toxicity model to evaluate the role of aerolysin in the pathogenesis of A. hydrophila infections. The strain had a 50% lethal dose (LD50) of greater than 10(9) as compared with the parental strain which had an LD50 of 5 X 10(7). Reintegration of the deleted segment into AB3-5 resulted in an LD50 of 6 X 10(7) cells for this revertant. Furthermore, all mice injected with a sublethal dose of the parental strains developed necrotic lesions; this was never obtained with the aerolysin-deficient strain AB3-5. More importantly, specific neutralizing antibody to aerolysin was detected in mice surviving A. hydrophila infection, demonstrating that aerolysin is produced during the course of systemic A. hydrophila infections.     

Partial Characterization of Aerolysin, a Lytic Exotoxin from Aeromonas hydrophila

Conditions are defined for the production, in high titers, of an extracellular hemolytic toxin of Aeromonas hydrophila, here termed "aerolysin." Substantial purification of the toxin was accomplished by means of salt fractionation, dialysis, and gel filtration, with a yield of 24% of the starting activity. Analysis of the product by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single, heavy protein band and a number of faint protein bands. The estimated molecular weight of the heavy band (50,000) was in close agreement with that (53,000) of the substance responsible for hemolytic activity as determined by gel filtration. Purified aerolysin is a labile substance, apparently protein. It is not inactivated by any of several proteases under the conditions employed nor is it inhibited by any of several lipids tested. About 0.1 mug administered to mice intravenously is lethal. The physical properties of aerolysin show considerable resemblance to those described for the exotoxin of Pseudomonas aeruginosa.     

Characterization of macrophage sensitivity and resistance to anthrax lethal toxin.

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.     

Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish.

Protease-deficient strains of Aeromonas hydrophila TF7 were induced by transposon Tn5 mutagenesis, with Escherichia coli 1830(pJB4JI) as the Tn5 donor. The parent strain has the cell surface characteristics associated with virulence for fish, and as it produces a single metalloprotease, mutants could be distinguished by direct plating on brain-heart infusion skim milk agar. Mutants Pd-7 and Pd-10 still produced metalloprotease, but at reduced levels and only after prolonged incubation. The activities of other exoenzymes and hemolysin were unaffected, and the mutants autoagglutinated in broth, indicating that the cell surface characteristics of A. hydrophila TF7 had been retained. Unlike the parent strain, the mutants did not produce lesions or mortalities in rainbow trout (Salmo gairneri) when 5 X 10(6) CFU were injected intramuscularly. The bacterial cells were completely cleared from the site of the injection and the organs within 7 days. For 60-g rainbow trout held at 10 degrees C, the 50% lethal dose of Pd-10 was greater than 10(7) CFU, compared with 8.1 X 10(5) CFU for the parent strain. The mutants were significantly more susceptible than the parent strain to the bactericidal effect of fresh normal trout serum in vitro. Mutants Pd-7 and Pd-10 grew as well as the parent on M9 salts-glucose medium but more slowly on heat-inactivated fish serum. Thus, protease appears to be able to contribute to the establishment of A. hydrophila infection in fish both by overcoming initial host defenses and by providing nutrients for cell proliferation.