Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

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.

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies

“…The side-chain oxygen of Asn 110 is situated 2.9 Å away from the tertiary amine of the R-group of PJ34, which forms another important H-bond. As reported earlier in other mART toxin structures with PJ34 (25,35,36), the electron density of the hetero-ring system of the inhibitor is clearly defined; however, the electron density is weaker for the R-group (tertiary amine) (supplemental Fig. S1), suggesting some flexibility in the "tail" of the inhibitor.…”

“…Several potent inhibitors were identified, including a previously characterized inhibitor of mART toxins known as PJ34 (25,35,36). The inhibitor structures are stabilized within the active site of Scabin, largely through hydrophobic interactions combined with a few critical H-bonds.…”

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

“…Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig.…”

The 1.8 Å Cholix Toxin Crystal Structure in Complex with NAD+ and Evidence for a New Kinetic Model

“…Structurally, it is a three-domain A/B toxin. It consists of a receptor binding domain (R) for receptor-mediated endocytosis, a membrane translocation domain (T) for translocation to the host cytoplasm, and the catalytic domain (C) (11).…”

“…Cholix is the third member of the DT group. It is most similar to ExoA with respect to structure, enzyme activity, and inhibitor specificity (2,11). Structurally, it is a three-domain A/B toxin.…”