Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile

Abstract

Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.

Genomic reconstruction of σ54 regulons in Clostridiales

“…6a) strongly affects production of toxins in these species [38,39]. Recent studies have shown that σ 54 and CdsR mediate the cysteine-dependent repression of toxin production in C. difficile [40,41]. We identified a putative σ 54 promoter and UAS site of CsdR upstream of cdsB gene involved in cysteine catabolism in C. difficile, C. botulinum, C. sporogenes, and C. scatologenes ( Fig.…”

Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile

“…These results are not surprising since most of the genes transcribed by RNAP associated to SigL respond to specific inducers through their associated EBP activators sensing these signals (Francke et al, 2011;Nie et al, 2019). For example, cysteine or proline strongly induces the expression of cdsB and prdC genes and the prdA operon, respectively, in a CdsR-or a PrdR-dependent manner (Bouillaut et al, 2013;Gu et al, 2017). We did not detect a SigL-dependent control of cdsB expression as previously observed (Gu et al, 2017) and only a two-fold decrease of expression was detected for three genes of the prdA operon in the sigL::erm mutant ( Supplementary Table S7).…”

“…Interestingly, the genes for 24 transcriptional activators (named EBP for enhancer binding proteins activating SigLdependent promoters) containing a AAA + domain, which is responsible for ATP hydrolysis and their interaction with SigL (Francke et al, 2011), are present in the genome of C. difficile. Only two EBPs, CsdR and PrdR, controlling cysteine and proline catabolism, respectively, have been experimentally characterized in C. difficile (Bouillaut et al, 2013;Gu et al, 2017;Gu et al, 2018). By combining in silico analysis, RNA-seq and genomewide promoter mapping, we have recently identified more than 200 non-coding RNAs (ncRNAs) in C. difficile from different functional classes including riboswitches, transand cis-acting antisense RNAs (Soutourina et al, 2013).…”

“…The regulons of several RNAP sigma factors have been recently defined in C. difficile including sporulation specific sigma factors, the general stress-response sigma factor, SigB and the motility-associated sigma factor, SigD ( Saujet et al, 2011 ; El Meouche et al, 2013 ; Pereira et al, 2013 ; Saujet et al, 2013 ; Kint et al, 2017 ). Only two studies have previously shown a role of SigL, belonging to the Sigma 54 family, in the degradation of cysteine associated with a control of toxin production ( Dubois et al, 2016 ; Gu et al, 2017 ; Nie et al, 2019 ). Interestingly, the genes for 24 transcriptional activators (named EBP for enhancer binding proteins activating SigL-dependent promoters) containing a AAA + domain, which is responsible for ATP hydrolysis and their interaction with SigL ( Francke et al, 2011 ), are present in the genome of C. difficile .…”

“…Interestingly, the genes for 24 transcriptional activators (named EBP for enhancer binding proteins activating SigL-dependent promoters) containing a AAA + domain, which is responsible for ATP hydrolysis and their interaction with SigL ( Francke et al, 2011 ), are present in the genome of C. difficile . Only two EBPs, CsdR and PrdR, controlling cysteine and proline catabolism, respectively, have been experimentally characterized in C. difficile ( Bouillaut et al, 2013 ; Gu et al, 2017 ; Gu et al, 2018 ).…”