No abstract

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC 50 ‫؍‬ 4.6 ؎ 0.4 ng/ml) and crustaceans (Artemia nauplii LD 50 ‫؍‬ 10 ؎ 2 g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1-and 1.25-Å resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through 54 and associated transcriptional regulators.Bacterial cells synthesize myriad small organic molecules to signal and adapt to environmental, physiological, and population structure changes. These molecules include extracellular signals such as acyl homoserine lactones, butyrolactones, quinolones, a furanosyl borate diester, oligopeptides, 3-hydroxypalmitic acid methyl ester, and a hydroxyketone and intracellular signals including cyclic nucleotides and ppGpp (25; reviewed in reference 11). The phenotypic response to signaling compounds often involves traits that are beneficial under adverse conditions, such as biofilm formation, virulence, motility, bioluminescence, sporulation, competence, modification of carbon and energy utilization, and macromolecule biosynthesis.Indole is a relatively recent addition to the list of signaling molecules used by bacteria and is produced as a by-product of the breakdown of tryptophan by the enzyme tryptophanase (TnaA) (18). Since the expression of the tnaA gene is controlled by catabolite repression, it is transcribed only during carbon limitation (57). As a result of this regulation, large quantities of indole are produced during the stationary phase of growth. Indole has long been known to act as a chemorepellent of Escherichia coli (52), but only more recently has indole also been shown to control the expression of a wide assortment of genes and phenotypes unrelated to chemotaxis in many different bacteria. For example, in E. coli indole controls the expression of genes involved in amino acid metabolism (54), plasmid maintenance (13), and quorum sensing (32), among other functions (14). Additionally, indole may function as an interspecies signal contributing to biofilm formation in an assortment of different bacteria known to carry a copy of the tryptophanase gene (36). In E. coli specifically, the indoledriven transcriptional response, which controls subsequent biofilm formation, is known to be controlled by the quorumsensing transcriptional regulator SdiA (32).It was previously shown that control of biofilm formation by indole also extends to the etiological agent of the ...