Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502

Abstract

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD 600 ]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum. We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature.KEYWORDS Clostridium botulinum ATCC 3502, botulinum neurotoxin A1 (BoNT/A1), botulinum toxin complex (TC), arginine, glucose, spore formation, nutritional regulation

Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E

“…The only known negative regulator of BoNT synthesis, CBO0787/CBO0786, is a TCS whose regulator component CBO0786 binds specifically to the promoter of ntnh-botA and inhibits transcription of this operon by blocking the binding of the RNA polymerase-BotR complex to its target promoter DNA (Zhang et al, 2013b). The nature of the environmental signals activating the neurotoxinregulating TCSs is scarcely known, but availability of certain carbohydrates (Bonventre and Kempe, 1959) and amino acids (Patterson-Curtis and Johnson, 1989;Leyer and Johnson, 1990;Fredrick et al, 2017) and temperature (Selby et al, 2017), influence neurotoxin production and provide interesting hypotheses for further TCS studies. Not only the extracellular environment but also the intracellular state, such as the metabolic status of C. botulinum cells, affects BoNT production via the transition-state central regulator CodY .…”

Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A- hyper

“…Despite arginine being an essential amino acid, previous studies showed that an excess of arginine in the medium dramatically decreased BoNT formation up to 1,000-fold ( 35 , 39 ), which is much higher than many other transcriptional control mechanisms that have been demonstrated in toxin-forming pathogens. Similarly, some Clostridioides difficile strains have poor growth but enhanced toxin production in medium with low arginine levels ( 40 ).…”

Vaccine Production to Protect Animals Against Pathogenic Clostridia

“…Nutritional studies in C. botulinum showed that maltose, fructose, mannose, and sucrose are fermented by Group II, while only some strains in Group I ferment maltose and fructose [26]. Glucose is metabolized by both groups and is needed to promote toxin production in Group I [60]. Amino acids, such as arginine and tryptophan, are required by Group I and II, respectively, for growth and toxin synthesis.…”

“…Amino acids, such as arginine and tryptophan, are required by Group I and II, respectively, for growth and toxin synthesis. However, the oversupply of these amino acids has a negative impact on BoNT production [60]. Chemically defined media have been investigated with low success.…”