Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom.

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 micrograms/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.

Immunological properties of the cell surface haemagglutinins (sHAs) of Helicobacter pylori strain NCTC 11637.

Rabbits were immunised with stage 1 and stage 2 soluble haemagglutinins (sHA) of Helicobacter pylori strain NCTC 11637 and with rabbit erythrocytes coated with stage 1 sHA. After adsorption of stage 1 sHA on erythrocytes, SDS-PAGE analysis showed that 4 major protein bands were removed from the preparation. The anti-sHA coated erythrocyte serum had the highest HA inhibition titre of 16. Crossed immunoelectrophoresis of the stage 1 sHA, against stage 1 and 2 antisera showed multiple precipitin arcs; however, the anti-sHA coated erythrocyte serum produced only two arcs. One arc produced by the anti-stage 2 serum was absent with the anti-stage 1 serum. This arc could have been produced against a 20 kDa polypeptide which was absent in the stage 1 sHA. The other arc was stronger when compared with that produced by anti-stage 1 serum. These two arcs corresponded to the two arcs produced by the anti-sHA coated erythrocyte serum, which had the highest inhibition titre. The two arcs were markedly reduced in crossed immunoelectrophoresis with an adsorbed stage 1 sHA preparation, which indicates that these arcs were produced against the sHAs.

Inhibition of Staphylococcus adherence to biomaterials by extracellular slime of S. epidermidis RP12.

Adherence of selected strains of coagulase-negative staphylococci to various biomaterials, and the inhibition of their adherence by extracellular slime obtained from the RP12 strain of Staphylococcus epidermidis were studied in vitro. S. epidermidis RP12 adhered considerably more to polymethylmethacrylate (PMMA) discs than did the SP2 strain of S. hominis and the SE-360 strain of S. hyicus. Strain RP12 was less adherent to titanium alloy, ultrahigh molecular weight polyethylene (UHMWPE), and Teflon discs than to PMMA discs. Exposure of PMMA discs to extracellular slime extracted from strain RP12 greatly reduced adherence of strain RP12, SP2, SE-360, and S. epidermidis RP-62A. The active component(s) was present in the > 10 kD mol wt fraction obtained by Amicon YM10 ultrafiltration of crude slime; heat treatment of the fraction did not affect its inhibitory activity. When the bacteria and RP12 slime fractions were added simultaneously to the PMMA discs, the > 10 kD mol wt fraction of slime competitively inhibited adherence of strain RP12 to PMMA discs; in contrast, the < 10 kD mol wt fraction enhanced adherence of strain RP12 to PMMA discs.

Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637.

A deionized water extract of Helicobacter pylori NCTC 11637 contained haemagglutinin activity that was (i) soluble (i.e., not associated with particulate material sedimented by centrifugation at 100,000 x g for 1 h), (ii) stable to lyophilization, (iii) heat-labile, (iv) chymotrypsin-sensitive, (v) inhibited by fetuin, orosomucoid, and NANLac, but not by asialofetuin and (vi) inactive against guinea pig erythrocytes incubated with Clostridium perfringens neuraminidase, but active against untreated guinea pig erythrocytes. The data support the idea that the haemagglutinin is a protein which recognizes the alpha-(2-3) structure of sialylated glycoconjugates. Fractionation of the extract by isoelectric focusing and by gel filtration with Sephacryl S-400 indicated that the haemagglutinin has a pI of 3.7 and consist of high molecular-weight-protein aggregates. SDS-PAGE analysis of the preparation purified by gel filtration showed 3 protein bands at ca. 64 kD, 56 kD and 20 kD. Electron microscopy of H. pylori incubated with gold-labelled fetuin indicated that the haemagglutinin was associated with loosely adherent material on the bacterial surface, and that the purified haemagglutinin did not reveal a fimbrial structure. The ability to bind to sialoglycoconjugates on the erythrocyte membrane suggests that the haemagglutinin may be an important colonization factor enabling H. pylori to bind to similar saccharide structures on epithelial cells.

Effects of stonefish (Synanceia trachynis) venom on murine and frog neuromuscular junctions.

The neuromuscular toxicity of stonefish (Synanceia trachynis) venom was characterized by electrophysiological and electron microscopic examination of isolated murine and frog nerve-skeletal muscle preparations exposed to various concentrations of venom. Low concentrations of venom (2.5-10 micrograms/ml) acted presynaptically by causing release and depletion of neurotransmitter from the nerve terminal. The response was Na+ channel-independent (resistant to tetrodotoxin), required the presence of either Ca2+ or Mg2+, and was observed with botulinum neurotoxin-paralyzed nerve-muscle preparations. Higher concentrations of venom (100-300 micrograms/ml) acted postsynaptically and presynaptically. They caused irreversible depolarization of muscle cells and microscopically observable muscle and nerve damage. We conclude that the previously observed neuromuscular toxicity of stonefish venom is a consequence of the venom's dose-dependent, presynaptic and postsynaptic actions at the myoneural junction.

Detection of a cytolytic toxin in the venom of the stonefish (Synanceia trachynis).

The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an Mr of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.

Cloning and expression of the damselysin gene from Vibrio damsela.

The gene encoding damselysin, an extracellular cytolysin produced by virulent Vibrio damsela strains, was cloned and expressed in Escherichia coli. DNA sequences homologous to that of the cloned gene were detected in hemolytic strains of V. damsela but not in other hemolytic Vibrio species.

Detection of Vibrio vulnificus cytolysin in V. vulnificus-infected mice.

Enzyme-linked immunosorbent assays using polyclonal and monoclonal antibodies specific for V. vulnificus cytolysin detected the toxin in an extract of skin lesions and in serum from mice showing local and systemic V. vulnificus disease after subcutaneous injection of the bacterium. The cytolysin also was detected in skin lesions by an indirect immunofluorescence procedure using polyclonal and monoclonal antibodies. Our findings provide direct evidence that the cytolysin is produced in vivo during the development of the disease process, and this observation is consistent with the hypothesis that the toxin is involved in the pathogenesis of V. vulnificus disease.

Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes.

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.

Purification and characterization of an elastolytic protease of Vibrio vulnificus.

Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an Mr of about 50,500 (estimated by SDS-PAGE), a pI of 5.7, and a temperature optimum range of 55 to 60 degrees C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu- Phe-Gly . The purified protease was toxic for mice (about 1.5 mg kg-1 and 4.5 mg kg-1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.

Mouse skin damage caused by cytolysin from Vibrio vulnificus and by V. vulnificus infection.

Light and electron microscopy of mouse skin damage caused by intradermal infection with a virulent strain of Vibrio vulnificus and by a single intradermal injection of the cytolytic toxin produced by the bacterium revealed similar structural alterations. The epidermis was intact; however, the infection and toxin produced acute cellulitis characterized by extensive extracellular edema; disorganization of collagen bundles; large accumulations of cell debris and plasma proteins; damaged or necrotic fat cells, capillary endothelial cells, and muscle cells; and mild inflammatory cell infiltration. The virulent strain of V. vulnificus produced a capsule and was resistant to phagocytosis in vivo, whereas a weakly virulent strain of the bacterium did not produce a capsule and was readily phagocytized and digested. Factors that may be important in the pathogenesis of V. vulnificus wound infections include a capsule that inhibits phagocytosis and an extracellular cytolytic toxin that is responsible, at least in part, for the severe tissue damage characteristic of such wound infections.

Immunization against experimental Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with lipopolysaccharide endotoxins and proteases.

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseudomonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis.

Detection of anti-Vibrio vulnificus cytolysin antibodies in sera from mice and a human surviving V. vulnificus disease.

An enzyme-linked immunosorbent assay and a cytolysin neutralization assay were used detect anti-Vibrio vulnificus cytolysin antibodies in sera from mice and a human that survived V. vulnificus disease. The detection of antibodies against the cytolysin indicated that the cytolysin is produced in vivo, and this observation is consistent with the hypothesis that the cytolysin is involved in the pathogenesis of V. vulnificus disease.

Production and partial characterization of an elastolytic protease of Vibrio vulnificus.

Conditions are described for the production of large amounts of an extracellular elastolytic protease by Vibrio vulnificus. The yield of enzyme was maximal during the late exponential growth phase and was stable during the stationary growth phase in a medium composed of 2% Proteose Peptone and 1.5% NaCl. The protease has a molecular weight of ca. 50,500 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of ca. 5.8, and a pH optimum range against azocasein and elastin of pH 7 to 8. The caseinolytic and elastase activities in protease preparations partially purified by ammonium sulfate precipitation were inseparable by gel filtration, hydrophobic interaction chromatography, and isoelectric focusing. Both activities were deleteriously affected by heat, low pH, heavy-metal ions, chelating agents, reducing agents, sodium cyanide, N-bromosuccinimide, alpha-2-macroglobulin, and phosphoramidon, but were unaffected by various trypsin inhibitors, chymostatin, aprotinin, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, and N-ethylmaleimide.

Identification of Vibrio vulnificus by indirect immunofluorescence.

A rapid, sensitive, and specific indirect immunofluorescence (IIF) procedure is described for identifying Vibrio vulnificus. Reference antisera were prepared by vaccinating rabbits with surface antigen preparations of V. vulnificus, and the antisera were examined for the ability to react with and serologically group 85 isolates of V. vulnificus grown in heart infusion broth, and to detect V. vulnificus in tissue specimens from mice experimentally infected with a virulent isolate of the bacterium. The antisera detected 100% of the V. vulnificus isolates examined and gave false-positive results in approximately 0.9% of 445 IIF tests performed with non-V. vulnificus clinical isolates. V. vulnificus also was detected in frozen tissue sections from infected mice; however, the most easily observed positive results were obtained by examining V. vulnificus from lesion specimens and blood cultured briefly in heart infusion broth. The bacteria in 2-hr-old cultures of local lesions fluoresced brilliantly and were easily detectable. The IIF procedure could be of value in rapidly diagnosing fulminating and potentially fatal human disease caused by V. vulnificus.

Purification and characterization of an extracellular cytolysin produced by Vibrio damsela.

Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium Vibrio damsela were obtained free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-100, and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The cytolysin is heat labile and protease sensitive and has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of ca. 69,000 and an isoelectric point of ca. 5.6. The first 10 amino-terminal amino acid residues of the cytolysin are Phe-Thr-Gln-Trp-Gly-Gly-Ser-Gly-Leu-Thr. The cytolysin was very active against erythrocytes from 4 of the 18 animal species examined (mice, rats, rabbits, damselfish) and against Chinese hamster ovary cells and was lethal for mice (ca. 1 microgram/kg, intraperitoneal median lethal dose). Lysis of mouse erythrocytes by the cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step followed by a temperature-dependent, membrane-perturbation step(s).

Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus.

An extracellular cytolytic toxin produced by the halophilic bacterium Vibrio vulnificus was isolated free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-75, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and isoelectric focusing in an ethylene glycol density gradient. The cytolysin is a heat-labile, hydrophobic protein that is inhibited by large amounts of cholesterol, is partially inactivated by proteases and trypan blue, has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid analysis) of ca. 56,000, and has an isoelectric point of ca. 7.1. The first 10 amino-terminal amino acid residues of the cytolysin are Gln-Glu-Tyr-Val-Pro-Ile-Val-Glu-Lys-Pro. Lysis of mouse erythrocytes by the purified cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step, followed by a temperature-dependent, membrane-perturbation step(s). In addition to possessing cytolytic activity against erythrocytes from 17 animal species and against Chinese hamster ovary cells in tissue culture, the purified cytolysin preparation was lethal for mice (ca. 3 micrograms/kg, intravenous 50% lethal dose) and had vascular permeability factor activity in guinea pig skin.