Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined.

Botulinum neurotoxins (BoNTs) are the most potent toxins ever known. They are mostly produced by Clostridium botulinum but also by other clostridia. BoNTs associate with non-toxic proteins (ANTPs) to form complexes of various sizes. Toxin production is highly regulated through complex networks of regulatory systems involving an alternative sigma factor, BotR, and at least 6 recently described two-component systems (TCSs). TCSs allow bacteria to sense environmental changes and to respond to various stimuli by regulating the expression of specific genes at a transcriptional level. Several environmental stimuli have been identified to positively or negatively regulate toxin synthesis; however, the link between environmental stimuli and TCSs is still elusive. This review aims to highlight the role of TCSs as a central point in the regulation of toxin production in C. botulinum.

Clostridial neurotoxins, botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT), are potent toxins, which are responsible for severe neurological diseases in man and animals. BoNTs induce a flaccid paralysis (botulism) by inhibiting acetylcholine release at the neuromuscular junctions, whereas TeNT causes a spastic paralysis (tetanus) by blocking the neurotransmitter release (glycine, GABA) in inhibitory interneurons within the central nervous system. Clostridial neurotoxins recognize specific receptor(s) on the target neuronal cells and enter via a receptor-mediated endocytosis. They transit through an acidic compartment which allows the translocation of the catalytic chain into the cytosol, a prerequisite step for the intracellular activity of the neurotoxins. TeNT migrates to the central nervous system by using a motor neuron as transport cell. TeNT enters a neutral pH compartment and undergoes a retrograde axonal transport to the spinal cord or brain, where the whole undissociated toxin is delivered and interacts with target neurons. Botulism most often results from ingestion of food contaminated with BoNT. Thus, BoNT passes through the intestinal epithelial barrier mainly via a transcytotic mechanism and then diffuses or is transported to the neuromuscular junctions by the lymph or blood circulation. Indeed, clostridial neurotoxins are specific neurotoxins which transit through a transport cell to gain access to the target neuron, and use distinct trafficking pathways in both cell types.

Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation.