Project description:Clostridium tetani produces the tetanus-causing tetanus toxin (TeNT), one of the most powerful bacterial toxins known to humankind. The regulation of toxin expression is complex and involves the alternative sigma factor TetR as well as other regulators. Here, we identified a novel regulatory molecule, a non-coding small regulatory RNA (sRNA), located in the 3’ untranslated region of the tent gene. We show with an antisense RNA approach and recombinant expression of the tent locus with and without the sRNA in C. tetani strains that the sRNA acts as a negative regulator of TeNT expression and modulates the growth pattern of C. tetani. Its possible role is to limit tetanus toxin levels in the exponential growth phase; thus, it might interlock bacterial growth and toxin production.
Project description:We used RNA-seq to determine Clostridium tetani gene expression changes in response to culture conditions and time. Changes in response to time were more pronounced than those in response to culture conditions. The tetanus toxin gene is always highly expressed but does show expression changes between culture conditions. These results may become part of an approach to reduce animal testing during vaccine manufacturing.
Project description:To develop a method for improved assessment of the genetic stability of bacterial vaccine strains, we applied next-generation sequencing to a Clostridium tetani model strain (including inter- and intra-lab replicates) and other strains. Data were processed to determine (gain or loss of) gene copy numbers. Strains could easily be distiguished based on gain/loss of prophage-like and CRISPR/Cas genes. We found that the model strain has multiple copies of the plasmid carrying the gene coding for tetanus toxin as well as several other genes. Data were reproducible within and between laboratories. The limit of detection of our method is an order of magnitude better than that of the pulsed-field gel electrophoresis (PFGE) currently used during manufacturing. This approach may part of an approach to reduce animal testing during vaccine manufacturing.
