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... Show moreClostridioides 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, which positively regulates toxin gene expression, and TcdC, which is 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 the tcdC genotype and toxin production. Consequently, the 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 oligonucleotide/oligosaccharide binding fold (0B-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 that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors.IMPORTANCE The 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. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an 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 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiT(opt)) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development. Show less
Novakova, E.; Kotlebova, N.; Gryndlerova, A.; Novak, M.; Vladarova, M.; Wilcox, M.; ... ; Krutova, M. 2020
We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert(R)... Show moreWe aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert(R) C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes' detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Delta 117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Delta 117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping. Show less
