Fig. 1

Tc toxin translocation mechanism and exchange of TccC HVRs. a Schematic of the Tc toxin translocation mechanism. The cocoon-like TcB-TcC component (blue-purple) encapsulates the autoproteolytically cleaved cytotoxic C-terminus of TcC known as the HVR (black). Upon binding of the TcB-TcC component to the pentameric TcA component (visible monomers in red, beige, orange, and green) via the TcA-binding domain of TcB (darker blue), the HVR is released into the central channel of the toxin (gray). Upon pH-induced prepore-to-pore transition at the cell membrane, the channel opens and the HVR is translocated into the cytoplasm. b Schematic of the experimental concept. The HVR in the cocoon is recombinantly replaced by an alternative protein to be translocated (green). c Effect of TccC3HVR to TccC5HVR replacement in the TcdB2-TccC3 cocoon on cytotoxicity. The ability of the TccC5HVR construct to kill HEK293T cells demonstrates that it is able to effectively translocate through the pore formed by the TcdA1 pentamer. A fourfold higher concentration of the TccC5HVR construct is needed to obtain a cytotoxic effect comparable to that of the original TccC3HVR. While this may indicate less-efficient translocation, the effect is more likely owing to TccC5HVR being a less-potent toxin than TccC3HVR, a finding confirmed by previous studies¹⁸. Experiments were performed in triplicates with qualitatively identical results. Scale bars: 100 μm