Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani

Abstract

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a “fermentation map”, which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions

“…Despite its differential expression by culture conditions and time, expression of tetX was always high and relatively stable during and between cultures (Figure 2), which shows that growth phase and conditions only affected tetX expression to a limited extent. This contrasts with the findings described by Licona-Cassani et al [6], which were later confirmed by Orellana et al [20]. Licona-Cassani et al found that tetX expression is triggered by the transition from amino acid consumption to peptide consumption, corresponding to a switch to a slower growth rate at around 10 h of culture.…”

“…In other Clostridium species, as well as other bacteria, prophage genes can influence cellular lysis (which leads to the release of TeNT into the medium) as well as regulate toxin gene transcription [23,24]. In fact, Orellana et al found that two genes (CTC_RS05470 and CTC_RS07675) located in different prophage insertion regions in the C. tetani genome clustered with the transcription of the tetX transcriptional regulator BotR [20]. It is therefore conceivable that further research on this prophage region may lead to new insights into TeNT regulation.…”

Regulatory Networks Controlling Neurotoxin Synthesis in Clostridium botulinum and Clostridium tetani

“…In addition to variations in toxin production according to different media, variations in BoNT or TeNT yields are often observed from batch to batch of the same culture medium, even using the same bacterial strain. The transcription of neurotoxin genes and toxin synthesis occur mainly within a short time interval between the late exponential growth and early stationary growth phase [33,85,86]. Thus, nutritional and environmental factors influence the regulation of toxin synthesis in C. botulinum and C. tetani, which takes place in a restricted phase of bacterial growth.…”

“…Peptides and amino acids appear to be important regulatory factors at the transcriptional and posttranscriptional levels. Indeed, large amounts (0.8-0.9 g/L) of amino acids (aspartate, glutamate, serine, histidine, threonine) downregulate tetR and tent by a yet non-identified regulatory pathway [86]. Arginine is an essential amino acid for C. botulinum growth, but an excess of arginine represses BoNT production in proteolytic group I C. botulinum [87].…”

“…The transition from free amino acid to peptide consumption is associated with increased pH due to the reduction of organic acids to alcohols and solvents, and the production of ammonia from peptide metabolism. During phase II, tetR and tent are highly expressed, as well as codY, the TCS that positively regulates tent, and two additional sigma factors located on the large plasmid containing tent, resulting in TeNT synthesis [54,82,86,93]. In complex media, glucose is consumed during the first phase of growth, leading to rapid bacterial multiplication and pH decrease.…”