Clostridioides difficile is an anaerobic gram-positive bacterium that can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study we aimed to obtain topological information on the C-terminus of TcdC and demonstrate that the C-terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and mutating this residue results in release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors. Importance Transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. Activity of TcdC has been mapped to its C-terminus and the N-terminus serves as membrane anchor. Acting as anti-sigma factor requires a cytoplasmic localization of the C-terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C-terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 results in release of TcdC from the bacteria. The use of the HiBiTopt system for topology determination of membrane proteins is a valuable tool, increasing the range of available systems to tackle important aspects of the C. difficile development.

21Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large 22 clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also 23 encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative 24 negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several 25 studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC 26 function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated 27 protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is 28 mediated through the OB-fold domain present at the C-terminus of the protein. This domain was 29 previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating 30 a cytoplasmic localization of the C-terminal OB-fold. 31In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning 32 Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of 33 TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of 34 the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action 35 that is different from characterized anti-sigma factors. 363 Importance 37Transcription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma 38 factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.