Rapid detection of Salmonella sp. by means of a combination of selective enrichment broth and MALDI-TOF MS.

The identification of Salmonella sp. in stool samples usually takes 2 days when employing routine procedures. Fast approaches are necessary in order to shorten the analysis time. The aim of this work was the development of a rapid procedure for the detection of Salmonella sp. from clinical stool samples. Spiked stool samples were cultured in selective selenite enrichment broth. Identifications were directly performed from the liquid broth by the MALDI Biotyper. After the evaluation of this method, the same procedure was applied to clinical samples. Coevally, the samples were streaked on Hektoen agar and single colonies were analyzed by the MALDI Biotyper. For comparison, the liquid broth was plated according to the standard laboratory procedure. A total of 4,847 samples were analyzed for Salmonella sp. In total, 108 Salmonella sp.-positive samples were identified; 66 of these were identified after the streaking of stool samples on Hektoen agar and subsequent MALDI Biotyper analysis of Salmonella sp. suspicious colonies. These and a further 34 samples were detected as Salmonella sp.-positive directly from the selenite enrichment broth on day one. Eight Salmonella sp.-positive samples were not detected before plating of the selenite broth and subsequent MALDI Biotyper analysis on day two. The combination of MALDI Biotyper analysis and selective selenite enrichment broth identification delivers positive results for the majority of the samples already after one day.

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

Characterization of SNARE protein expression in beta cell lines and pancreatic islets.

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin.

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.

Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells.

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells.

Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain.

The ability of the two-chain form of tetanus toxin (TeTx), its constituent light (LC) or heavy (HC) chains, and papain fragment to block evoked acetylcholine (ACh) release in the buccal ganglia of Aplysia californica was studied electrophysiologically. Extracellularly applied, TeTx or its B fragment (consisting of LC and beta 2, the amino-terminal portion of HC) blocked ACh release, whereas LC, HC, or the beta 2 fragment did not affect it. Toxicity was restored when LC was bath applied together with HC or the beta 2 fragment. When injected into the presynaptic neuron, TeTx, the B fragment or LC, but not HC, induced inhibition of ACh release. These results indicate that the blockade of ACh release by TeTx is mimicked by intracellular action of LC, the internalization of which is mediated by the HC via its amino-terminal moiety.

The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells.

The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.

Production of biologically active light chain of tetanus toxin in Escherichia coli. Evidence for the importance of the C-terminal 16 amino acids for full biological activity.

The activity of the light (L) chain of tetanus toxin, and of mutants constructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E. coli. Wild-type recombinant L chain (pTet87) was active in the inhibition of exocytosis from cultured bovine adrenal chromaffin cells, although at a level 5-15% of that of L chain purified from tetanus toxin. L chain mutants which terminated at Leu-438 (pTet89), or which contained a Cys-to-Ser mutation at residue 439 (pTet88) were equally as active as the full-length recombinant protein. The reduced activity of pTet87 L chain correlated with C-terminal proteolysis of the protein upon purification. A tryptic fragment derived from native light chain and which terminated at Leu-434 also showed reduced activity in the exocytosis assay, consistent with a requirement of the C-terminal region of the L chain for maximal activity. pTet87 L chain, but neither of the mutants, could be associated with purified H (heavy) chain to form a covalent dimer which induced the symptoms of tetanus in mice. The ability to form biologically active toxin using recombinant L chain will be of great value in structure-function studies of tetanus toxin.

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin.

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by microM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.

The tetanus toxin light chain inhibits exocytosis.

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two-chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half-maximal effect at about 5 nmol/l. Heavy chain (up to 11 nmol/l) and unprocessed single-chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single-chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.

Comparison of the intracellular effects of clostridial neurotoxins on exocytosis from streptolysin O-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells.

The inhibitory effects of tetanus toxin, botulinum toxin A, their constituent light chains, and botulinum toxin B were compared using streptolysin O-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells in primary culture. In both types of chromaffin cells exocytosis can be triggered by micromolar amounts of free Ca2+, bovine adrenal chromaffin cells in addition require ATP. In PC 12 cells the isolated tetanus toxin light chain alone blocks exocytosis without any additive. The time-course of the inhibitory action of tetanus toxin light chain in permeabilized PC 12 cells in the absence of ATP is similar to the one obtained with permeabilized bovine adrenal chromaffin cells, in the presence of ATP. Thus, ATP does not seem to be crucial for tetanus toxin (two-chain form) poisoning. Botulinum toxin B (two-chain form), if preactivated by dithiothreitol, also inhibits exocytosis from permeabilized PC 12 cells up to 90% in the absence of ATP. By contrast, botulinum toxin A (two-chain form) or its isolated light chain, which are highly potent in permeabilized bovine adrenal chromaffin cells, causes only a weak inhibition in PC 12 cells. In streptolysin O-permeabilized bovine adrenal chromaffin cells omission of ATP during the incubation with the toxin increases the potency of botulinum toxin A light chain. Under the same conditions the effect of tetanus toxin light chain remains unchanged. Tetanus toxin and botulinum toxin B (two-chain forms) probably block a step which occurs during exocytosis from both PC 12 cells and adrenal chromaffin cells and which could be closely related to the final fusion event.(ABSTRACT TRUNCATED AT 250 WORDS)

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings.

The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin. The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain.

Differences in the multiple step process of inhibition of neurotransmitter release induced by tetanus toxin and botulinum neurotoxins type A and B at Aplysia synapses.

In order to gain insights into the steps (binding, uptake, intracellular effect) which differ in the inhibitory actions of tetanus toxin and botulinum neurotoxins types A or B, their temperature dependencies were investigated at identified cholinergic and non-cholinergic synapses in Aplysia. Upon lowering the temperature from 22 degrees C to 10 degrees C, extracellularly applied botulinum neurotoxin type A and B appeared unable to inhibit transmitter release whilst tetanus toxin exhibited a residual activity. Binding of each toxin to the neuronal membrane appeared virtually unaltered following this temperature change. By contrast, the intracellular effects of botulinum neurotoxin type B and tetanus toxin were strongly attenuated by temperature reduction whereas the inhibitory action of botulinum neurotoxin type A was only moderately reduced. Importantly, this discrepancy relates to the known proteolytic cleavage of different synaptic proteins by these two toxin groups. Since both the binding and intracellular activity of botulinum neurotoxin type A are minimally affected at 10 degrees C, its inability to inhibit neurotransmission at this low temperature when applied extracellularly indicated attenuation of its uptake. Due to the strict temperature dependence of the intracellular action of tetanus toxin and botulinum neurotoxin type B, but not A, an examination of the effects of changes in temperature on the internalization step was facilitated by the use of heterologous mixtures of the toxins' heavy and light chains. At 10 degrees C, heavy chain from tetanus toxin but not from botulinum neurotoxin type B mediated uptake of botulinum neurotoxin type A light chain. Collectively, these results provide evidence that, at least in Aplysia, the uptake mechanism for botulinum neurotoxin types A and B differs from that of tetanus toxin.

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin.

The intracellular action on exocytosis of botulinum A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 microM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain.

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals.

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules.

Anti-B-50 (GAP-43) antibodies decrease exocytosis of glutamate in permeated synaptosomes.

The involvement of the protein kinase C substrate, B-50 (GAP-43), in the release of glutamate from small clear-cored vesicles in streptolysin-O-permeated synaptosomes was studied by using anti-B-50 antibodies. Glutamate release was induced from endogenous as well as 3H-labelled pools in a [Ca(2+)]-dependent manner. This Ca(2+)-induced release was partially ATP dependent and blocked by the light-chain fragment of tetanus toxin, demonstrating its vesicular nature. Comparison of the effects of anti-B-50 antibodies on glutamate and noradrenaline release from permeated synaptosomes revealed two major differences. Firstly, Ca(2+)-induced glutamate release was decreased only partially by anti-B-50 antibodies, whereas Ca(2+)-induced noradrenaline release was inhibited almost completely. Secondly, anti-B-50 antibodies significantly reduced basal glutamate release, but did not affect basal noradrenaline release. In view of the differences in exocytotic mechanisms of small clear-cored vesicles and large dense-cored vesicles, these data indicate that B-50 is important in the regulation of exocytosis of both types of neurotransmitters, probably at stages of vesicle recycling and/or vesicle recruitment, rather than in the Ca(2+)-induced fusion step.

Cooperative action of the light chain of tetanus toxin and the heavy chain of botulinum toxin type A on the transmitter release of mammalian motor endplates.

Purified heavy chain of botulinum toxin type A and light chain of tetanus toxin were combined to form a chimeric toxin. It was active on the mouse phrenic nerve-hemidiaphragm with a potency 6 times higher than that of native tetanus toxin. Electrophysiological data from poisoned neuromuscular junctions revealed that the pattern of nerve-evoked and spontaneous transmitter release was equivalent to that seen with tetanus toxin i.e. asynchronous release, and did not resemble that after botulinum toxin type A poisoning. We conclude that the light chain of tetanus toxin alone is responsible for the characteristic effects on spontaneous and nerve-evoked transmitter release of the native toxin and that these properties can be introduced into a new, more potent complex with the heavy chain of botulinum toxin A.

Differences in the temperature dependencies of uptake of botulinum and tetanus toxins in Aplysia neurons.

The respective neuroselective actions of botulinum type A (BoNT) and tetanus (TeTx) neurotoxins on cholinergic and non-cholinergic synapses of Aplysia are mainly due to differences in their extracellular neuronal targetting. Further information was gained on this neuroselectivity by examining the temperature dependencies of binding, internalization and intracellular action of both toxins. After reduction of temperature from 22 degrees C to 10 degrees C, the binding of neither BoNT nor TeTx was significantly altered whereas the neuronal uptake of BoNT, but not of TeTx, was prevented. Although TeTx internalization could be detected at the low temperature, its intracellular activity was greatly attenuated compared to that of BoNT. It is inferred that the uptake mechanisms are different for these two related but distinct toxins.

Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis.

Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BE1) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT greater than BE greater than S) in hydrophobic interaction HPLC, and anionic behaviour (BC greater than BE greater than BT greater than S) in DEAE ion exchanger HPLC. The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD50, mouse s.c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i.e. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([3H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([3H]noradrenaline, with toxin BA, BC, BE and BT). Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal to the spinal cord.

Single-chain toxin was investigated in vitro and in vivo for limited proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [3H]noradrenaline release in vitro. Cultured mouse brain generated fragments from 125I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i.v. into rats, 125I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to limited proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.