Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in Clostridium difficile

Abstract

It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Åkerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n ‫؍‬ 6) and clinical isolates (n ‫؍‬ 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.

Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism

“…For quantitative real-time PCR (qRT-PCR) analysis, 0.5 ml culture aliquots collected at ODs between 0.2 and 1.3 were mixed with 1 ml RNAprotect Bacteria Reagent (Qiagen) and centrifuged for 10 min; the supernatant was removed and pellets were stored at -70 uC until RNA extraction (below). For details regarding the preparation of growth media, dilutions, culture conditions and toxin determination see Karlsson et al (1999Karlsson et al ( , 2000.…”

“…A detailed 2D PAGE procedure is described in Karlsson et al (2000). In brief, frozen cells were thawed, suspended in 1 ml ice-cold MilliQ water and disrupted by sonication (26 30 s, 100 W, Labsonic 1510, B. Braun), whereafter cellular debris was removed by centrifugation (16 000 g, 10 min, 4 uC).…”

“…Toxin synthesis is normally upregulated during entry into the stationary growth phase in tryptone/peptone-yeast media (Dupuy & Sonenshein, 1998;Karlsson et al, 1999), and is associated with the induction of many other C. difficile proteins that are repressed together with the toxins by the presence of glucose, cysteine and proline (Karlsson et al, 2000). In addition, arginine, glutamine, biotin and carbonate influence toxin production, further supporting a link between carbon/amino acid metabolism and toxin production (Ikeda et al, 1998;Karasawa et al, 1997;Karlsson et al, 1999;Maegawa et al, 2002;Yamakawa et al, 1996Yamakawa et al, , 1998.…”

“…Here, we sought to identify additional genes and intracellular processes associated with toxin production in C. difficile. The results expand the knowledge of C. difficile metabolism and toxin production (Karlsson et al, 2000(Karlsson et al, , 2003, and are discussed in relation to global regulators in other Gram-positive bacteria.…”

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city, Brazil

“…is part of the intestinal indigenous microbiota of young children and they can produce several endogenous infections (Van der Vorm 1999). C. difficile is considered the etiologic agent of PMC and the major cause of antibiotic-associated diarrhea in adults, but the attribution of this organism to enteric diseases in children is less clear (Karlsson et al 2000).…”