Die Rolle der Hc-Fragmente der Botulinum Neurotoxine A, B, C und D : Charakterisierung der Bindung an die nicht-toxische nicht-hämagglutinierende Komponente und neuronale Rezeptoren

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Isolation and Functional Characterization of the Novel Clostridium botulinum Neurotoxin A8 Subtype

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins

The transcriptional regulatory network of Corynebacterium jeikeium K411 and its interaction with metabolic routes contributing to human body odor formation

Barzantny H, Schröder J, Strotmeier J, Fredrich E, Brune I, Tauch A. The transcriptional regulatory network of Corynebacterium jeikeium K411 and its interaction with metabolic routes contributing to human body odor formation. Journal of biotechnology. 2012;159(3):235-248.Lipophilic corynebacteria are involved in the generation of volatile odorous products in the process of human body odor formation by degrading skin lipids and specific odor precursors. Therefore, these bacteria represent appropriate model systems for the cosmetic industry to examine axillary malodor formation on the molecular level. To understand the transcriptional control of metabolic pathways involved in this process, the transcriptional regulatory network of the lipophilic axilla isolate Corynebacterium jeikeium K411 was reconstructed from the complete genome sequence. This bioinformatic approach detected a gene-regulatory repertoire of 83 candidate proteins, including 56 DNA-binding transcriptional regulators, nine two-component systems, nine sigma factors, and nine regulators with diverse physiological functions. Furthermore, a cross-genome comparison among selected corynebacterial species of the taxonomic cluster 3 revealed a common gene-regulatory repertoire of 44 transcriptional regulators, including the MarR-like regulator Jk0257, which is exclusively encoded in the genomes of this taxonomical subline. The current network reconstruction comprises 48 transcriptional regulators and 674 gene-regulatory interactions that were assigned to five interconnected functional modules. Most genes involved in lipid degradation are under the combined control of the global cAMP-sensing transcriptional regulator GlxR and the LuxR-family regulator RamA, probably reflecting the essential role of lipid degradation in C. jeikeium. This study provides the first genome-scale in silico analysis of the transcriptional regulation of metabolism in a lipophilic bacterium involved in the formation of human body odor. Copyright 2012 Elsevier B.V. All rights reserved

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

AbstractBotulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties

Substrates and Controls for the Quantitative Detection of Active Botulinum Neurotoxin in Protease-Containing Samples

Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT “Assay with a Large Immunosorbent Surface Area” that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis–Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ∼3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 μg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin

Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

<div><p>Botulinum neurotoxins (BoNTs) are produced by <i>Clostridium botulinum</i> and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.</p></div