Positive Regulation of Botulinum Neurotoxin Gene Expression by CodY in Clostridium botulinum ATCC 3502

Abstract

Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G؉C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status. Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin, which is the most poisonous biological substance known to mankind. Botulinum neurotoxin blocks neurotransmission in cholinergic nerves (1, 2) in humans and animals to cause botulism, a potentially lethal flaccid paralysis. Despite its extreme toxicity, botulinum neurotoxin is widely utilized as a powerful therapeutic agent to treat numerous neurological disorders (3, 4).Seven antigenically distinct botulinum neurotoxin types (A to G), and several subtypes therein, have been identified (5-9). Moreover, a novel toxin type H was recently proposed (10) and awaits further characterization (11). Type A1 neurotoxin is well characterized as a consequence both of its frequent involvement in human botulism worldwide and of its use as a therapeutic agent (12). Type A1 neurotoxin is produced as a complex containing the neurotoxin itself and associated nontoxic proteins (ANTPs) that comprise a nontoxic nonhemagglutinin protein (NTNH) and three hemagglutinin proteins (HAs; HA17, HA33, and HA70) (13-15). The NTNH protects the neurotoxin from low pH-and protease-induced inactivation in the gastrointestinal tract (16), while the HAs assist the neurotoxin absorption, probably by interacting with oligosaccharides and E-cadherin on intestinal epithelial cells (17).In C. botulinum type A1, the genes encoding the neurotoxin (botA) and ANTPs (ntnh, ha17, ha33, ha70) are located in a gene cluster and are organized in two operons, namely, the ntnh-botA and ha operons (18). Within the neurotoxin gene cluster, botR, located between the two operons, encodes an alternative sigma factor that is a member of group 5 of the sigma 70 family, including Clostridium difficile TcdR, Clostridium perfringens UviA, and Clostridium tetani TetR. BotR directly controls the transcription of both the ntnh-botA and ha operons (19,20). An Agr-like quorum sensing system was found to be involved in positive regulation of the neurotoxin production (21), suggesting that the cell density-dependent signals control neurotoxin production. Also, the CLC_10...

An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production

“…A recent report by Zhang and colleagues [29] demonstrated the role of the global regulator protein CodY in toxin synthesis and elements of this observation provide support for a plausible picture of the nutrition-related effects on toxigenesis in C . botulinum Group I type A1:…”

“…Positively, through the participation of BotR [17], Agr quorum sensing system [28], CodY [29] and CLC_1094/CLC_1093 (equivalent to CBO_1042/CBO_1041), CLC_1913/ CLC_1914 (equivalent to CBO_1967/ CBO_1968) and CLC_0663/CLC_0661 (equivalent to CBO_0608/CBO_0607) two component signal transduction systems [30]. Negatively, through the participation of CBO0787/CBO0786 (equivalent to CLC_0843/CLC_0842) [31] which is also a two component signal transduction system [32].…”

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

“…That previous observation (12) provided the first evidence for positive CodY regulation of toxin gene expression in a pathogenic Clostridium species and represented one of relatively few examples at that time of positive gene expression regulation by CodY. However, since our report, CodY was also shown to positively regulate botulinum neurotoxin gene expression by C. botulinum (18). The current study now implicates CodY in NanI-mediated positive control of ETX production, since a nanI null mutation decreases codY gene transcription levels, coincident with less ETX production.…”

“…For example, in log-phase cultures growing in rich media, CodY represses the tcdA and tcdB genes, which encode Clostridium difficile toxins A and B (15), as well as the Staphylococcus aureus hysA gene encoding the secreted enzyme hyaluronidase (16). Conversely, there is increasing evidence that CodY can positively regulate expression of some toxins made by low-GϩC-content Gram-positive bacteria, including production of anthrax toxin by Bacillus anthracis (17), botulinum neurotoxin by Clostridium botulinum (18), and ETX by C. perfringens (12).…”

CodY Regulates Expression of the Bacillus subtilis Extracellular Proteases Vpr and Mpr

“…In pathogenic species, key virulence genes are also under CodY control (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). CodY can control transcription by binding in the vicinity of the promoter region of the target genes, by competing with a positive regulator for binding, or by serving as a roadblock to RNA polymerase (24).…”