Expression of Recombinant Clostridial Neurotoxin by <i>C. tetani</i>
Tetanus neurotoxins (TeNT) and botulinum neurotoxins (BoNTs) are closely related ~150 kDa protein toxins that together comprise the group of clostridial neurotoxins (CNTs) expressed by various species of <i>Clostridia</i>. While TeNT is expressed as a single polypeptide, BoNTs are always...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-12-01
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| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/12/12/2611 |
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| Summary: | Tetanus neurotoxins (TeNT) and botulinum neurotoxins (BoNTs) are closely related ~150 kDa protein toxins that together comprise the group of clostridial neurotoxins (CNTs) expressed by various species of <i>Clostridia</i>. While TeNT is expressed as a single polypeptide, BoNTs are always produced alongside multiple non-toxic proteins that form a stabilizing complex with BoNT and are encoded in a conserved toxin gene cluster. It is unknown how <i>tent</i> evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by <i>C. tetani</i>, whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of <i>C. botulinum</i>. To assess whether the stability of TeNT is due to an innate property of the toxin or is a result of <i>C. tetani</i>’s intra- and extra-cellular environment, both TeNT and complex-free BoNT/A1<sup>ERY</sup> were expressed recombinantly in atoxic <i>C. tetani</i> and analyzed for expression and stability. The strong clostridial ferredoxin (<i>fdx</i>) promotor resulted in the expression of recombinant TeNT at greater levels and earlier time points than endogenously produced TeNT. Recombinant BoNT/A1<sup>ERY</sup> was similarly expressed by atoxic <i>C. tetani</i>, although partial degradation was observed. The rBoNT/A1<sup>ERY</sup> produced in <i>C. tetani</i> was also partially proteolytically processed to the dichain form. Investigations of bacterial growth media and pH conditions found that the stability of rTeNT and rBoNT/A1<sup>ERY</sup> in spent media of <i>C. tetani</i> or <i>C. botulinum</i> was affected by growth media but not by pH. These data indicate that the distinct metabolism of <i>C. tetani</i> or <i>C. botulinum</i> under various growth conditions is a primary factor in creating a more or less favorable environment for complex-free CNT stability. |
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| ISSN: | 2076-2607 |