The composition of gut microbiota has been associated with host metabolic phenotypes, but it is not known if gut bacteria may influence host appetite. Here we show that regular nutrient provision stabilizes exponential growth of E. coli, with the stationary phase occurring 20 min after nutrient supply accompanied by bacterial proteome changes, suggesting involvement of bacterial proteins in host satiety. Indeed, intestinal infusions of E. coli stationary phase proteins increased plasma PYY and their intraperitoneal injections suppressed acutely food intake and activated c-Fos in hypothalamic POMC neurons, while their repeated administrations reduced meal size. ClpB, a bacterial protein mimetic of α-MSH, was upregulated in the E. coli stationary phase, was detected in plasma proportional to ClpB DNA in feces, and stimulated firing rate of hypothalamic POMC neurons. Thus, these data show that bacterial proteins produced after nutrient-induced E. coli growth may signal meal termination. Furthermore, continuous exposure to E. coli proteins may influence long-term meal pattern.

Clostridium difficile intestinal disease is mediated largely by the actions of toxins A (TcdA) and B (TcdB), whose production occurs after the initial steps of colonization involving different surface or flagellar proteins. In B. subtilis, the sigma factor SigD controls flagellar synthesis, motility, and vegetative autolysins. A homolog of SigD encoding gene is present in the C.difficile 630 genome. We constructed a sigD mutant in C. difficile 630 ∆erm to analyze the regulon of SigD using a global transcriptomic approach. A total of 103 genes were differentially expressed between the wild-type and the sigD mutant, including genes involved in motility, metabolism and regulation. In addition, the sigD mutant displayed decreased expression of genes involved in flagellar biosynthesis, and also of genes encoding TcdA and TcdB as well as TcdR, the positive regulator of the toxins. Genomic analysis and RACE-PCR experiments allowed us to characterize promoter sequences of direct target genes of SigD including tcdR and to identify the SigD consensus. We then established that SigD positively regulates toxin expression via direct control of tcdR transcription. Interestingly, the overexpression of FlgM, a putative anti-SigD factor, inhibited the positive regulation of motility and toxin synthesis by SigD. Thus, SigD appears to be the first positive regulator of the toxin synthesis in C. difficile.

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A 2 pm 3 3 A 2 pm 3 (A 2 pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala 4 3 A 2 pm 3 cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala 4 . Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt cd1 and ldt cd2 , were inactivated. The inactivation of either ldt cd1 or ldt cd2 significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt cd1 -and ldt cd2 -encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.Clostridium difficile, a Gram-positive spore-forming bacterium, is the major cause of intestinal diseases associated with antibiotic therapy such as ampicillin, clindamycin, and cephalosporins, which disrupt the barrier intestinal flora and allow C. difficile colonization (1, 2). Clinical manifestations range from asymptomatic colonization or mild diarrhea to pseudomembranous colitis (3). The main virulence factors have been identified as toxin A and B (4). Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new highly virulent strain (BI/NAP1/027) of C. difficile.Antibiotic treatment of C. difficile-associated disease requires metronidazole or vancomycin therapy.Peptidoglycan ( (9), which were originally detected in Enterococcus faecium (Ldt fm ) (10) and then in other Gram-positive bacteria (11,12), in mycobacteria (13-15), and in Escherichia coli (16,17). Ldts use acyl donors containing a tetrapeptide stem (9) and were consequently expected to confer resistance to ␤-lactams (10, 18).Another possible variation of the PG structure is the occurrence of N-deacetylation or O-acetylation of glycan strands, either on GlcNAc or on MurNAc residues (19,20). N-Deacetylation in Listeria monocytogenes (21) or Streptococcus pneumoniae (22) and O-acetylation in Staphylococcus aureus have been linked to lysozyme resistance (23).The PGs of C. difficile should have some specificities regarding the effect of antibiotics inhibiting PG biosynthesis; C. difficile, although susceptible to ␤-lactams, exhibits higher minimal inhibitory concentrations than in oth...

The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection.