Control of Clostridium difficile Physiopathology in Response to Cysteine Availability.
The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.
Thomas
T
Dubois
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.
Marie
M
Dancer-Thibonnier
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.
Marc
M
Monot
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.
Audrey
A
Hamiot
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
Laurent
L
Bouillaut
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France Université Paris 7-Denis Diderot, Paris, France.
Olga
O
Soutourina
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France Université Paris 7-Denis Diderot, Paris, France.
Isabelle
I
Martin-Verstraete
Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France bdupuy@pasteur.fr.
Bruno
B
Dupuy
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000502:physiology*; D003545:Cysteine / Q000378:metabolism*; D004734:Energy Metabolism / Q000235:genetics; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial; D006710:Homocysteine / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D019289:Pyruvic Acid / Q000378:metabolism; D014709:Vero Cells
D000596:Amino Acids; D001427:Bacterial Toxins; D006710:Homocysteine; D019289:Pyruvic Acid; D003545:Cysteine; D006862:Hydrogen Sulfide