Expression of Recombinant Clostridial Neurotoxin by C. tetani

Abstract

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 Clostridia. 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 tent evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by C. tetani, whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of C. botulinum. To assess whether the stability of TeNT is due to an innate property of the toxin or is a result of C. tetani's intra- and extra-cellular environment, both TeNT and complex-free BoNT/A1^ERY were expressed recombinantly in atoxic C. tetani and analyzed for expression and stability. The strong clostridial ferredoxin (fdx) promotor resulted in the expression of recombinant TeNT at greater levels and earlier time points than endogenously produced TeNT. Recombinant BoNT/A1^ERY was similarly expressed by atoxic C. tetani, although partial degradation was observed. The rBoNT/A1^ERY produced in C. tetani 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^ERY in spent media of C. tetani or C. botulinum was affected by growth media but not by pH. These data indicate that the distinct metabolism of C. tetani or C. botulinum under various growth conditions is a primary factor in creating a more or less favorable environment for complex-free CNT stability.

Keywords: Clostridium tetani; botulinum neurotoxin; expression system; protein stability; recombinant; tetanus toxin.

Figure 1

C. tetani 454 and 19406 express stable, full-length rTeNT at levels exceeding that of a wild-type strain under the ferredoxin promoter.

Figure 2

Full-length rBoNT/A1^ERY is expressed by C. tetani 454 but is only partially proteolytically processed to its dichain form. (A) Western blots depict the stability of rBoNT/A^ERY over time when the expression strains are cultured in toxin production media (TPM). C. botulinum Hall A Hyper/tox-(82152-A1^ERY) is known to strongly express stable BoNT/A and was therefore used as an expression control to compare against C. tetani 454 (82153-A1^ERY). (B) Whole culture samples were collected at the noted times and either reduced with 100 mM DTT or left unreduced. The 50 ng purified BoNT/A1 was used as a loading control. The locations of the ~150 kDa holotoxin (HC + LC/A), the ~100 kDa heavy chain (HC/A), and the ~50 kDa light chain (LC/A) are indicated.

Figure 3

Full-length rBoNT/A1^ERY is expressed by C. tetani 454 in modified Mueller Miller media but degrades over time. (A) Western blots of C. botulinum Hall A hyper/tox- (83152-A1^ERY) and C. tetani 454 (82153-A1^ERY) cultured in modified Mueller Miller media (+0.5 g/L reduced iron powder). Whole culture samples were taken at 24 and 96 h and either reduced with 100 mM DTT or left unreduced. (B) Western blot of C. tetani 454 (82153-A1^ERY) expression on modified Mueller Miller agar plates without the addition of reduced iron powder or any alternative iron source. Whole cell growth (WC) on the plate was resuspended at the indicated time points and centrifuged into cell pellet (pellet) and supernatant (SN). Then, 50 ng purified BoNT/A1 was loaded as a control. The locations of the ~150 kDa holotoxin (HC + LC/A), the ~100 kDa heavy chain (HC/A), and the ~50 kDa light chain (LC/A) are indicated.

Figure 4

Comparative stability of clostridial neurotoxins in spent media under varying pH conditions. Static cultures of the atoxic strains C. tetani 454 (A,C) and C. botulinum LNT01 (B,D) were incubated at 37 °C for 96 h in either modified Mueller Miller media (supplemented with 0.5 g/L of reduced iron powder) or toxin production media (TPM). Culture supernatant was collected and pH adjusted to pH 5, 6, 7, and 8 via 1 M NaOH or 1 M HCl. The pH adjusted spent media, spent media with no pH adjustment, and media which had not supported bacterial growth (fresh media) were sterile filtered through a 0.22 uM PVDF membrane. Purified 150 kDa BoNT/A1 (A,B) or TeNT (C,D) were added to each sample, incubated for 24 h at 37 °C, and then analyzed via SDS-PAGE and Western blot. The ~150 kDa protein bands, indicating the amount of full-length toxin remaining, were analyzed via densitometry. The mean concentration (n = 3) of remaining holotoxin is represented as a percentage relative to the amount of toxin initially added to the sample.