Inhibition of Clostridium botulinum 52A toxicity and protease activity by sodium acid pyrophosphate in media systems

The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP.     

Response of type B and E Botulinum toxins to purified sulfhydryl-dependent protease produced by Clostridium botulinum type F.

A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C. botulinum types A and F in culture. The toxicity of type E progenitor toxin potentiated by the treatment with SHP persisted, whereas that of derivative toxin decreased rapidly by further incubation with SHP. This may indicate that only the progenitor toxin, the complex of the toxic and nontoxic components, activated by SHP withstands the subsequent exposure to the enzyme in cultures of proteolytic C. botulinum.     

The role of hydrogen peroxide in the in vitro cytotoxicity of 3-hydroxykynurenine

Previous studies have indicated that the generation of H₂O₂ may be a key step in the mechanism mediating the in vitro cytotoxicity of 3-hydroxykynurenine (3HK). An exposure protocol resulting in a delayed toxicity was utilized in order to further examine the role of H₂O₂ in the in vitro toxicity of 3HK in a neural hybrid cell line. 3HK-induced cell lysis was significantly attenuated by administration of catalase after termination of 3HK exposure and was abolished when intracellular peroxidase activity was elevated by pretreatment of cultures with horseradish peroxidase. In addition, a dose-dependent attenuation of 3HK toxicity was observed when cultures were exposed to 3HK in the presence of the iron chelator, desferrioxamine (DFO). Pretreatment with DFO also resulted in a significant attenuation of 3HK toxicity. These data suggest a direct role for H₂O₂ and metal ions in the cytotoxic action of 3HK and indicate that cell lysis results from the intracellular accumulation of toxic levels of H₂O₂.     

Dependence of Clostridium botulinum gas and protease production on culture conditions

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine.

Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine

Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum.     

Grazing activity of Pfiesteria piscicida (Dinophyceae) and susceptibility to ciliate predation vary with toxicity status

Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in <40% of actively toxic cells for ≤16 h (high light) and ≤23 h (low light). These results suggest a relatively high reliance on phagotrophic carbon assimilation and more rapid response to algal prey availability in Pfiesteria cells with lower toxicity. Grazing by two euplotid benthic ciliates (Euplotes vannus and E. woodruffi) on P. piscicida also varied among functional types. Grazing on actively toxic P. piscicida cells did not occur, whereas net positive ingestion rates were calculated for the other prey cultures. These results support concurrent experimental findings that a natural assemblage of microzooplankton displayed lower grazing potential on actively toxic P. piscicida than on cultures with reduced toxicity. In summary, pronounced differences in trophic interactions were found between actively toxic cultures and those with reduced or undetectable toxicity, providing additional evidence of the importance of cellular toxicity in the trophic ecology of Pfiesteria.     

The Ability of 2-Deoxyglucose to Promote the Lysis of Streptococcus bovis JB1 via a Mechanism Involving Cell Wall Stability

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture superntant. Received: 11 April 1997 / Accepted: 10 June 1997     

Expression of protease and protease-inhibitory activity in human mononuclear leukocyte cultures : Effect of conA stimulation

Trypsin-activated protease activity was observed in the supernatants of conA-treated unfractionated or monocyte-depleted cultures at 1 h of incubation and trypsin-inhibitory activity was detected at 24 h. In contrast, supernatants from wheat germ agglutinin (WGA)-treated cultures or untreated cultures exhibited trypsin-inhibitory activity at 1 h and trypsinactivated protease activity after 24 h. The expression of proteases and protease inhibitors may be early events that occur during the activation of quiescent lymphocytes to a proliferative state.     

Subcellular distribution of [14C]5-hydroxytryptamine in the blood platelets of rabbits: effects of imipramine and and haloperidol

Long-term (10 days) administration of imipramine [20 mg/(kg X d)] to rabbits significantly increases the Km value (4.0 micron) of 5-hydroxytryptamine uptake in their platelets compared to those of saline- (0.7 micron) or haloperidol- (0.4 micron) treated rabbits. Administration of haloperidol inhibits the 5-hydroxytryptamine uptake non-competitively, and in vitro it had an ID50 value of 22 micron. Intravenous injections of [14C]5-hydroxytryptamine were given to the animals 1 h before blood collection. After isolation of platelets, their sonicates were subjected to 30-60% continuous sucrose gradient centrifugation. The subcellular distribution of [14C]5-hydroxytryptamine indicates that imipramine treatment, in contrast to the control and haloperidol treatment, led to a shift in the exogenous 5-hydroxytryptamine peak from within the granular zone (d 1.18) to the extragranular cytoplasm (d 1.15). Compared to control values, the imipramine treatment caused 63% inhibition in the platelet Na-K-ATPase activity.     

Absence of trichothecenes in toxigenic isolates of Fusarium moniliforme

Thirty-four isolates of Fusarium moniliforme were obtained from cereal grains collected in various parts of the world. The isolates were grown on rice and tested as a diet for toxicity to rats. Of these isolates, 53% caused death, 12% caused congestion and hemorrhage of the stomach and intestine as well as hematuria, 21% caused diarrhea, 38% caused weight loss, and 9% were nontoxic. The cultures were tested to T-2, HT-2, neosolaniol, acetyl-T-2, T-2-tetraol, iso-T-2, diacetoxyscirpenol, monoacetoxyscirpenol, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, moniliformin, fusarochromanone, fusarin-C, and wortmannin; all were negative. In addition, F. moniliforme NRRL A25820 was grown on corn and banana fruit as solid substrates as well as on a defined liquid medium; none of the above toxins were found. When F. moniliforme NRRL A25820 was incorporated into a rat diet, no toxicity was noted. Twenty-eight additional isolates of F. moniliforme, isolated from feed associated with equine leukoencephalomalacia, were grown on cracked corn for 2 weeks. The cultures were negative when tested for deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, monoacetoxyscirpenol, nivalenol, and fusarenone X. Seventy-five percent of the isolates were toxic to ducklings, indicating the presence of a toxin other than trichothecenes. Our results support the conclusion that F. moniliforme does not produce trichothecenes.     

Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type?A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25?kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type?E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.     

Assessment of the efficacy of Japanese Bacillus thuringiensis isolates against the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

A total of 2,652 Japanese isolates of Bacillus thuringiensis, belonging to at least 54 H serogroups, were examined for assessment of the toxicity against the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae). When tested with spore/parasporal inclusion mixtures, strong larvicidal activities were associated with 28 isolates (1.1%). Serologically, these toxic isolates fell into 4 known H serovars: thuringiensis (9 isolates), kurstaki (2), kenyae (2), and darmstadiensis (15). Purified parasporal inclusions of the 10 selected isolates exhibited no larvicidal activity, while the supernatants of liquid cultures showed larvicidal and/or growth inhibitory effects. The activities were fully retained after heat treatment at 100 degrees C for 10 min. Overall results suggest that beta-exotoxin (or thuringiensin)-related substances are responsible for the toxicity of the present B. thuringiensis isolates against the cigarette beetle.