rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth.

The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

Hemagglutinating and binding properties of botulinum C2 toxin.

To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition. Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II. Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones. On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes. Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose. Thyroglobulin and bovine salivary mucin were much stronger inhibitors. From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).

Involvement of phospholipids in the intoxication mechanism of botulinum neurotoxin.

Phospholipids were examined for their potential to interact with botulinum neurotoxin by an in vivo toxin-inactivation assay and a direct binding assay on a thin layer plate. Type E neurotoxin was inactivated by negatively charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). The toxicity of the neurotoxin was not affected by phosphatidylcholine (PC) without an electric charge or phosphatidylethanolamine (PE) with a positive electric charge. The neurotoxin bound directly to PS and PI but not to PC or PE. These results suggest that the negatively charged phospholipids in the cell membranes are involved in the intoxication mechanism of botulinum neurotoxin. The phospholipids PS and PI were tested for their potential to interact within three domains [L, H-1, and H-2] which compose the neurotoxin. All three domains bound to PS; whereas, PI specifically accepted the binding of the H-1 domain relative to the penetration of the neurotoxin into the lipid membrane. In this paper, we discuss the interaction between the neurotoxin and the lipid membrane in the intoxication mechanism.

Effects of Ca2+ and other cations on the action of Clostridium perfringens enterotoxin.

We investigated the role of extracellular Ca2+ in the Clostridium perfringens enterotoxin-induced alteration of the permeability of the plasma membrane. Enterotoxin released 86Rb and 51Cr from the Vero cells preloaded with the isotope. In the presence of EGTA, however, it released 86Rb but not 51Cr. The binding of enterotoxin to the cells was not influenced by Ca2+ or Mg2+. The effects of various cations on the enterotoxin-induced 51Cr release was also studied. The release depended on extracellular Ca2+ but not on Mg2+; it was inhibited by each of Zn2+, La3+ and Co2+. Zn2+ and Co2+ also inhibited 51Cr release caused by the enterotoxin previously bound to the cell membrane. In contrast, antibody against enterotoxin did not neutralize the toxin once it was bound to the Vero cells. When the cells were treated with enterotoxin, 45Ca influx occurred and reached the plateau in a few minutes, as did 86Rb release.

Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes.

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

A cold-adapted endo-arabinanase from Penicillium chrysogenum.

Previously, three arabinan-degrading enzymes were isolated from Penicillium chrysogenum 31B. Here we describe another arabinan-degrading enzyme, termed Abnc, from the culture filtrate of the same organism. Analysis of the reaction products of debranched arabinan by high-performance anion-exchange chromatography (HPAEC) revealed that Abnc cleaved the substrate in an endo manner and that the final major product was arabinotriose. The molecular mass of Abnc was estimated to be 35 kDa by SDS-PAGE. Enzyme activity of Abnc was highest at pH 6.0 to 7.0. The enzyme was stable up to 30 degrees C and showed optimum activity at 30 to 40 degrees C. Compared with a mesophilic counterpart from Aspergillus niger, Abnc exhibited a lower thermal stability and optimum enzyme activity at lower temperatures. Production of Abnc in P. chrysogenum was found to be strongly induced by arabinose-containing polymers and required a longer culture time than did other arabinanase isozymes in this strain.

Purification and characterization of the ganglioside-binding fragment of Clostridium botulinum type E neurotoxin.

A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin. The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin. In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride. Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41,000 Da. The recovery of the 41,000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12%. The 41,000-Da fragment bound to gangliosides GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound. The 41,000-Da fragment partially interfered with the binding of 125I-labeled neurotoxin to mouse brain synaptosomes. We have proposed a three-fragment structure (L.H-1.H-2) for botulinum type E neurotoxin. The characters of the 41,000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L.H-1.H-2, and that the ganglioside-binding fragment is H-2.

SNAP-25 is present on chromaffin granules and acts as a SNAP receptor.

SNAP-25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP-25 and syntaxin 1 via the interaction with VAMP-2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP-25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP-25 on chromaffin granules has essentially the same properties as does SNAP-25 on the plasma membrane.

TLC immunostaining characterization of Clostridium botulinum type A neurotoxin binding to gangliosides and free fatty acids.

The receptor structure of Clostridium botulinum neurotoxin type A was analysed by TLC immunostaining. GQ1b was found to be the most potent receptor, and the neurotoxin also bound to GT1b and GD1a, but not to GM3, GM2, GM1, GD3, GD1b and GT1a. Optimum binding of neurotoxin to the ganglioside appeared in 0.01 M phosphate buffer (pH 7.2) containing 0.2% NaCl. Higher and lower NaCl concentrations diminished neurotoxin binding to the ganglioside. In addition, the neurotoxin was able to bind to free fatty acids. Maximum binding was observed on stearic acid and neurotoxin binding to free fatty acids was not affected by NaCl concentration.

Characterization of a novel monoclonal antibody that senses nitric oxide-dependent activation of soluble guanylate cyclase.

Two monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the alpha- and beta-subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the beta-subunit and its affinity did not change with NO. Neither mAb cross-reacted with particulate GC. Cultured Purkinje cells from rats were treated with S-nitroso-N-acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased.

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes.

Clostridium botulinum type A hemagglutinin-positive progenitor toxin consists of three distinct components: neurotoxin (NTX), hemagglutinin (HA), and non-toxic non-HA (NTNH). The HA consists of four subcomponents designated HA1, 2, 3a and 3b. By employing purified toxin and GST-fusion proteins of each HA subcomponent, we found that the HA-positive progenitor toxin, GST-HA1 and GST-HA3b bind to human erythrocytes and microvilli of guinea pig upper small intestinal sections. The HA-positive progenitor toxin and GST-HA1 bind via galactose moieties, GST-HA3b binds via sialic acid moieties. GST-2 and GST-3a showed no detectable binding.

Distribution of synaptosomal-associated protein 25 in nerve growth cones and reduction of neurite outgrowth by botulinum neurotoxin A without altering growth cone morphology in dorsal root ganglion neurons and PC-12 cells.

Synaptosomal-associated protein 25 has been regarded as one of the target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptors essential for exocytosis of vesicles in synapses. We have previously reported that cleavage of syntaxin, which is another target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, with botulinum neurotoxin C1 resulted in inhibition of neurite extension and morphological changes including growth cone collapse and large vacuole formation. As an attempt to explore the mechanism of growth cone extension, we examined the ultrastructural localization of synaptosomal-associated protein 25 in growth cones with or without treatment of botulinum neurotoxin A, which cleaves synaptosomal-associated protein 25. In dorsal root ganglion neurons, light microscopy demonstrated synaptosomal-associated protein 25 immunoreactivity throughout the neurons, including the cell bodies, neurites and growth cones. Using electron microscopy, gold signals immunoreactive for synaptosomal-associated protein 25 were identified diffusely in the cytoplasm of the growth cones. In contrast, in PC-12 cells, a large number of gold signals were localized on the plasma membranes. High levels of signal were also found in the cytoplasm in the central region of the growth cones. We also confirmed that botulinum neurotoxin A treatment reduced neurite extension by about 50%. However, both in dorsal root ganglion neurons and in PC-12 cells we found no differences in the ultrastructure nor in the localization of synaptosomal-associated protein 25 between growth cones with and without toxin treatment. These results indicate that cleavage of synaptosomal-associated protein 25 inhibits growth cone extension in a manner different than that of syntaxin cleavage. The results of this study suggest the possibility that synaptosomal-associated protein 25 is involved in growth cone extension through a process independent of vesicle fusion.

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule.

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.

Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties.

Two strains of Clostridium botulinum type A associated with food-borne botulism and six strains associated with infant botulism in Japan were compared in intraintestinal toxin production in infant mice, in vitro toxin and hemagglutinin production, molecular sizes of the toxins, and some other properties. The infant botulism-associated strains, producing M toxin (Mr 300 kDa) but no hemagglutinin, showed significantly lower 50% infective doses in infant mouse intestines. The antigenicities of the toxin differed between the two groups, while the biochemical properties of the cultures did not. Besides infant botulism-associated strains, this set of properties were found only in a strain isolated from honey of South American origin.

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin.

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.

Production of monoclonal antibody against Aeromonas hydrophila haemolysin.

Two hybridoma cell lines that produce monoclonal antibodies against Aeromonas hydrophila haemolysin were established by fusion of myeloma and spleen cells obtained from a mouse immunised with haemolysin detoxified with tetranitromethane. Enzyme-linked immunosorbent assay (ELISA) showed that the two purified monoclonal antibodies, B7 and B11, recognised the same epitope on the haemolysin molecule. Antibody B7 neutralised the haemolytic and enterotoxic activities of the haemolysin. It is concluded that the same site on the haemolysin molecule is responsible for both haemolytic and enterotoxic activities.

Dissociation of SNAP-25 and VAMP-2 by MgATP in permeabilized adrenal chromaffin cells.

In digitonin-permeabilized adrenal chromaffin cells, Ca(2+)-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca(2+)-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.