Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from <i>Clostridioides difficile</i>

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of &gt;99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile

High-resolution structure of native toxin A from Clostridioides difficile

Clostridioides difficile infections have emerged as the leading cause of healthcare‐associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 Å resolution cryo‐EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor‐binding domain, which likely serves to shield the C‐terminal part of the hydrophobic pore‐forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the N‐terminal part of the pore‐forming region suggest that the globular subdomain shields the upper part of the pore‐forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore‐forming region at neutral pH, as well as the pH‐induced inter‐domain dynamics

Vaccine safety of a new toxoid-based vaccine against Clostridioides difficile infection in rabbits

National audienceClostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea. The main virulence factors responsible for the disease’s symptoms are two secreted cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB). Treatment with antibiotics is difficult, as recurrent episodes of CDI usually occur in around 20-30% of patients after antibiotic therapy has ended. Several vaccines based on detoxified or recombinant forms of these toxins have been or are currently being tested in clinical trials. One of them is inactivated by formaldehyde, a method with obvious drawbacks. A new metal-catalyzed oxidation (MCO) method was used to detoxify the toxins while preserving the neutralizing epitopes. The aim of the project was therefore to combine these detoxified TxA and TxB toxins with a potent LiteVax adjuvant (LVA) to demonstrate vaccine safety and generate high levels of antibody responses in rabbits.For each study, 42 female rabbits, aged 8 weeks, were divided into 6 groups of 7 animals, each group vaccinated with different toxin concentrations (TxA between 5 and 20 µg and TxB between 20 and 80 µg) and different adjuvants (placebo, LVA adjuvant or alum). Each group was vaccinated three times at 14-day intervals by intramuscular route. Every day, the rabbits were observed for behavioral and feeding problems. Weekly kinetics were performed to determine serum and neutralizing antibody titers (against TcdA and TcdB), blood counts (cell populations) and inflammatory cytokines.White blood cell count and neutrophil percentage increased one day after immunization with LVA, but little or not with alum. Antibody titers were similar to those obtained with a conventional formaldehyde-inactivated CDI vaccine adjuvanted with alum. Alum enhanced antibody responses against TcdA but not against TcdB. LVA adjuvant generated significantly higher antibody responses against TcdB compared to alum. Histopathological analysis of the injection sites 42 days after the last injection showed no abnormal tissue reaction. In conclusion, MCO is an alternative method for the production of safe and immunogenic toxoid antigens, and TxA and TxB in combination with LVA adjuvant is a very interesting vaccine candidate against CDI