Yukako Fujinaga

Structure and Function of Clostridium botulinum Toxins

Clostridium botulinum strains produce seven immunologically distinct neurotoxins, types A to G. The neurotoxins inhibit the release of acetylcholine (Ach) at the neuromuscular junctions and synapses, and cause botulism in humans and animals. The molecular mass (Mr) of all types of neurotoxins are approximately 150 kDa. The neurotoxins associate with nontoxic components in culture, and become large complexes varying from 300 kDa (12S) to 900 kDa (19S), which are designated as progenitor toxins. Recently, the genes coding for type A to G neurotoxins have been cloned, and their whole nucleotide sequences have been determined. Furthermore, it has become clear that the neurotoxins are Zn2+ -binding proteins and possess protease activities. Structures of the nontoxic components of the progenitor toxins have also been investigated genetically in types C, E and F. In this paper, the gene organization of the neurotoxins and the nontoxic components is summarized, and their structure and function are discussed.

The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin

Binding of the purified type C 7S (neurotoxin), 12S and 16S botulinum toxins to epithelial cells of ligated small intestine or colon of the guinea pig (in vivo test) and to pre-fixed gastrointestinal tissue sections (in vitro test) was analysed. The 16S toxin bound intensely to the microvilli of epithelial cells of the small intestine in both in vivo and in vitro tests, but did not bind to cells of the stomach or colon. The neurotoxin and 12S toxin did not bind to epithelial cells of the small intestine or to cells of the stomach or colon. Absorption of the toxins was assessed by determining the toxin titre in the sera of guinea pigs 6-8 h after the intra-intestinal administration of the toxins. When the 16S toxin [1 x 10(5) minimum lethal dose (MLD)] was injected, 200-660 MLD ml-1 was detected in the sera, whereas when the 12S toxin (2 x 10(5) MLD) or 7S toxin (2 x 10(5) MLD) was injected, little toxin activity was detected in the sera. Therefore, the haemagglutinin of type C 16S toxin is apparently very important in the binding and absorption of botulinum toxin in the small intestine.

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.

Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms

We isolated the gene encoding a botulinum neurotoxin (BoNT) of 1285 amino acids with a molecular weight of 147,364 from the toxigenic bacteriophage d-sA of Clostridium botulinum type D strain South African (Dsa). The BoNT of Dsa (BoNT/Dsa) is composed of three regions on the basis of the homology to BoNT types C1 (BoNT/C1) and D (BoNT/D). The N-terminal (Met-1 to Val-522) and the C-terminal regions (Trp-945 to Glu-1285) have high identity to corresponding regions of BoNT/D (96% identity) and BoNT/C1 (74% identity), respectively. The core region (Pro-523 to Lys-944) is common to three toxins (83% to 92% identity). These results suggest that neurotoxins produced from Clostridium botulinum types C and D are composed in a mosaic-like fashion.

The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.

The type B botulinum neurotoxin (BoNT) elicits flaccid paralysis and death in humans by intoxicating peripheral nerves after oral absorption. Here, we examine the function of the haemagglutinin (HA), a non‐toxic component of the large 16S BoNT complex. We find that the HA acts in the intestine to disrupt epithelial barrier function by opening intercellular tight and adherens junctions. This allows transport of BoNT and other large solutes into the systemic circulation and explains how the type B BoNT complexes are efficiently absorbed. In vitro, HA appears to act on the epithelial cell via the basolateral membrane only, suggesting the possibility of another step in the absorptive process. These studies show that the 16S BoNT complex is a multifunctional protein assembly equipped with the machinery to efficiently breach the intestinal barrier and act systemically on peripheral nerves.

Structure and Function of Clostridium Botulinum Progenitor Toxin

Clostridium botulinum strains produce seven immunologically distinct neurotoxins (NTX), type A to G. the NTXs associate with nontoxic components in cultures, and become large complexes with three forms (12S, 16S, and 19S) designated progenitor toxins. the 12S toxin consists of a NTX and a nontoxic component having no hemagglutinin (HA) activity (described here as non-toxic non-HA, NTNH), and the 16S and 19S toxins are formed by conjugation of the 12S toxin with HA. Based on the genetic-and protein chemical-analyses of the progenitor toxins it became clear that 1) the HA consists of four subcomponents namely HA1 (Mr. 33–35 kDa), HA2 (15–17 kDa), HA3a (19–23 kDa), and HA3b (52–53 kDa), 2) the genes coding for NTX (ntx), NTNH (ntnh), and HA (ha) occur as a cluster; ha lies just upstream of ntnh, and ntx lies just downstream of ntnh, 3) ha is in the opposite orientation from that of ntnh and ntx, 4) ha consists of three ORFs (ha1, ha2, and ha3), 5) the gene product (70 kDa) of ha3 is split into HA3a and HA3b ...

Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction

A polymerase chain reaction (PCR)‐based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin‐producing C. butyricum strain, and a type F toxin‐producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR‐based detection method can be used for the rapid diagnosis of botulism.

botR/A is a positive regulator of botulinum neurotoxin and associated non‐toxin protein genes in Clostridium botulinum A

The genes of the botulinum neurotoxin A (BoNT) complex are clustered in a locus consisting of two divergent polycistronic operons, one containing the non‐toxic, non‐haemagglutinin (NTNH) component and bontA genes, the other containing the haemagglutinin (HA) component genes. The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein. A recombinant Clostridium botulinum A strain that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing botR/A under the control of its own promoter. The transformed strain produced more BoNT/A and associated non‐toxic proteins (ANTPs) and the corresponding mRNAs than the non‐transformed strain. Partial inhibition of botR/A by antisense mRNA resulted in lower levels of BoNT/A, NTNH and HA70 and the levels of the corresponding mRNAs. Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons. These results show that botR/A activated transcription of the genes encoding BoNT/A and ANTPs in C. botulinum A by interacting directly with the region promoter, and that the homologous genes in C. botulinum B, C and D presumably have the same function.

Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins - differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C.

Orally ingested botulinum neurotoxin (BoNT) causes food-borne botulism, but BoNT must pass through the gut lining and enter the bloodstream. We have previously found that type B haemagglutinin (HA) proteins in the toxin complex play an important role in the intestinal absorption of BoNT by disrupting the paracellular barrier of the intestinal epithelium, and therefore facilitating the transepithelial delivery of BoNT. Here, we show that type A HA proteins in the toxin complex have a similar disruptive activity and a greater potency than type B HA proteins in the human intestinal epithelial cell lines Caco-2 and T84 and in the canine kidney epithelial cell line MDCK I. In contrast, type C HA proteins in the toxin complex (up to 300 nM) have no detectable effect on the paracellular barrier in these human cell lines, but do show a barrier-disrupting activity and potent cytotoxicity in MDCK I. These findings may indicate that type A and B HA proteins contribute to the development of food-borne botulism, at least in humans, by facilitating the intestinal transepithelial delivery of BoNTs, and that the relative inability of type C HA proteins to disrupt the paracellular barrier of the human intestinal epithelium is one of the reasons for the relative absence of food-borne human botulism caused by type C BoNT.

Characterization of haemagglutinin activity of Clostridium botulinum type C and D 16S toxins, and one subcomponent of haemagglutinin (HA1)

The 16S toxin and one subcomponent of haemagglutinin (HA), designated HA1, were purified from a type D culture of Clostridium botulinum by a newly established procedure, and their HA activities as well as that of purified type C 16S toxin were characterized. SDS-PAGE analysis indicated that the free HA1 forms a polymer with a molecular mass of approximately 200 kDa. Type C and D 16S toxins agglutinated human erythrocytes in the same manner. Their HA titres were dramatically reduced by employing erythrocytes that had been previously treated with neuraminidase, papain or proteinase K, and were inhibited by the addition of N-acetylneuraminic acid to the reaction mixtures. In a direct-binding test to glycolipids such as SPG (NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc beta1-Cer) and GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-Cer), and glycoproteins such as glycophorin A and/or B prepared from the erythrocytes, both toxins bound to sialylglycolipids and sialoglycoproteins, but bound to neither neutral glycolipids nor asialoglycoproteins. On the basis of these results, it was concluded that type C and D 165 toxins bind to erythrocytes through N-acetylneuraminic acid. HA1 showed no haemagglutination activity, although it did bind to sialylglycolipids. We therefore speculate that binding to glycoproteins rather than to glycolipids may be important in causing haemagglutination by type C and D 16S toxins.