The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface

Abstract

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.

Proteolytic activity of surface exposed HtrA determines its expression level and is needed to survive acidic conditions inClostridioides difficile

“…The bars represent an average of four independent biological replicates with standard deviations. Data for SrtB and HupA were previously published [24] but stem from the same experiments as the HtrA experiments . * Significant difference T-test, P<0.001.…”

“…As controls, we included HiBiT-tagged HupA protein (CD3496), a cytoplasmic, nucleoid-associated protein [21] and HiBiT-tagged sortase (CD2718), a cell wall located protein [22,23]. Data for SrtB and HupA were previously published [24] but stem from the same experiments as the HtrA experiments . * Significant difference T-test, P<0.001.…”

“…Primers CD3284-S217AF and CD3284-S217AR were used to create pJC115 through QuikChange mutagenesis To create pJC113 (encoding HtrA-HiBiT), we used oDB0067 and CD3284R6 to amplify the htrA coding sequence. The amplicon was cut with SacI and XhoI and ligated into pAF302 [24] that was cut with the same enzymes.…”

Clustering Disease of Clostridioides Difficile Infection: Implication for the Management in Internal Medicine

“…Closely related to the production of the two toxins, there is also the regulatory activity of TcdC which should repress the production of TcdA and TcdB, but in conjunction with exposure to antibiotics or changes in the intestinal microflora, loses its function on the gene expression of the toxins. As a result, the expression of TcdA and TcdB is upregulated, leading to increased toxin production, which can cause more severe disease symptoms [172,173].…”

The TcdE holin drives toxin secretion and virulence inClostridioides difficile

“…1a) 10 . TcdR is an alternative sigma factor required for PaLoc gene expression, while TcdC has been proposed to negatively regulate transcription as an antisigma factor [11][12][13][14][15] . TcdE shares homology with bacteriophage holins and has been shown to promote toxin release 8,16 .…”