Toxin production in clostridium difficile

Abstract: Clostridium difficile is a is a Gram potential human pathogen. It causing symptoms ranging from mild C. difficile associated diarrhea (CDAD) to severe inflammation of the colon including pseudomembranous colitis and fulminant colitis. Over 300 000 and about 10 000 CDAD episodes are diagnosed annually in the USA and Sweden, respectively. Onset of CDAD is typically associated with antibiotic therapy. These drugs may disrupt the colonization resistance of the colon microflora and allow for overgrowth of C. difficile. Pathogenic strains of C. difficile usually produce two difficile toxins, A and B. These main virulence factors cause severe tissue damage due to their ability to both disrupt the assembly of the cytoskeleton in enterocytes and to induce inflammation in the colon. In this study several novel factors influencing toxin production in C. difficile were identified and further investigated. A specific subset of amino acids and particularly cysteine was found to suppress the induction of the toxin yields in C. difficile by 100 to 1000-fold. Glucose also lowered toxin synthesis and furthermore, reduced the uptake and metabolism of certain amino acids. When toxin synthesis was already initiated amino acids stopped toxin synthesis much more efficiently than glucose, indicating that amino acids are more directly sensed and linked to C. difficile toxin expression, whereas glucose has a more indirect effect. The induction of toxin synthesis in media lacking a carbohydrate source and with limiting levels of key amino acids was preceded by the expression of several alternative metabolic pathways involved in energy production as shown by proteomic analysis. When these pathways apparently were no longer able to supply energy sufficient for growth, toxin synthesis was induced. Examples of candidate regulatory proteins sensing metabolic stress (ATP/GTP and amino acid levels) are CodY, LepA, Obg and Tex. The C difficile toxins were also found to be temperature regulated with maximum expression at 37 degrees C. This effect was dependent on the auto-regulated alternative sigma factor TcdD. Toxins were released by an export mechanism and not, as previously postulated, through lysis of the bacteria. During high toxin production and export a ToIC-like protein accumulated in the growth medium. This putative pore-forming protein could represent a novel protein secretion machinery in Gram-positive bacteria and be involved in toxin release by C difficile.