A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Abstract

The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa nontoxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with "three arms" attached. The "body" section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a "delivery vehicle" for BoNT.Different strains of Clostridium botulinum produce seven distinct serotypes of neurotoxins (BoNTs), 2 classified A through G. BoNT has attracted much interest in recent years due to extensive research on its biochemistry, determination of its crystal structure, and investigations into the pharmacology and applications of BoNTs as therapeutic agents for the treatment of human disease (1-3). After ingestion of BoNT, the BoNT is absorbed from intestinal epithelial cells into the bloodstream, after which it consequently reaches the neuromuscular junctions. BoNT enters nerve cells via receptor-mediated endocytosis, where it cleaves specific sites on target proteins, inhibiting release of neurotransmitters from peripheral cholinergic synapses through its zinc protease activity (4 -6). This process causes muscular paralysis in humans and animals, leading to the disease botulism.Toxins with serotypes A-D and G are encoded by two gene clusters in close proximity to each other; cluster 1 contains the bont and ntnha genes, and cluster 2 contains three genes : ha-70, ha-33, and ha-17 (7, 8). Therefore, botulinum TC consists of five components: BoNT, non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, HA-33, and HA-17). All serotypes of BoNT associate non-covalently with auxiliary non-toxic proteins, thereby forming large toxin complexes (TCs). Serotype A-D strains produce the M-TC (BoNT⅐NTNHA complex) and L-TC (BoNT⅐NTNHA⅐HAs complex) in the culture medium, while serotype E and F strains produce only M-TC. The major biological function of t...

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

“…The β-trefoil domains of HA-33 from BoNT/A, C, and D are highly similar. However, the domains are capable of twisting against each other by as much as 10°, via the helical linker, suggesting significant inter-domain conformational plasticity (Arndt et al 2005;Hasegawa et al 2007;Inoue et al 2003). HA-17 seems to tightly associate with HA-33, forming a complex that remains bound even in the presence of high concentrations of guanidine hydrochloride .…”

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

“…The concentrations of the toxin complexes in molarity were calculated from the molecular masses of 16S toxin (745 kDa) and 12S toxin (287 kDa) for the type A HApositive and HA-negative toxin complexes; the molecular masses of 16S toxin (744 kDa) and 12S toxin (289 kDa) for the type B HApositive and HA-negative toxin complexes; and the molecular masses of 16S toxin (749 kDa) and 12S toxin (287 kDa) for the type C HApositive and HA-negative toxin complexes, respectively. The molecular mass of each toxin was calculated based on the stoichiometry of subunits contained in the type D toxin complexes (Hasegawa et al, 2007). In the case of the type A HA-positive toxin complex (a mixture of 16S and 19S toxins), one molecule of 19S toxin was regarded as two molecules of 16S toxins, and concentrations of the toxin complex in molarity were calculated as those of 16S toxins.…”

“…In addition, in a human intestinal epithelial cell line (HT-29), the cell surface sialic acid-containing Olinked glycoproteins that are recognized by the type C 16S toxin have been shown to induce the internalization of the toxin into the cells (Nishikawa et al, 2004;Uotsu et al, 2006). Recently, it has been reported that type D HA proteins in the toxin complex enhance permeation through a monolayer of human intestinal epithelial cells (Caco-2) (Niwa et al, 2007). More recently, we have reported that type B HA proteins in the toxin complex have a novel activity, that of disrupting the paracellular barrier of the intestinal epithelium, which facilitates the transepithelial delivery of macromolecules including BoNT (Matsumura et al, 2008).…”

Molecular assembly of botulinum neurotoxin progenitor complexes

“…Despite these observations, there is limited information regarding the architecture of the BoNT 16S and 19S PCs. A 2D electron crystallography study of the BoNT/A1 19S PC suggested features with trigonal symmetry (22), and individual electron microscopy (EM) micrographs of the BoNT/D 16S PC suggested that the complex has three extended "arms" (23). Here, we have generated 3D reconstructions of the BoNT/A1, /B, and /E PCs, building on a series of 2D class averages obtained by single particle EM and using the random conical tilt (RCT) approach (24).…”