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Previous Bacterial Toxin Studies

The identification and characterisation of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholera.

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 have characterised a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells and crustaceans. 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 describe the high-resolution crystal structures of the multidomain toxin and its catalytic domain. 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.

TABLE 1: Comparison of the ADPRT kinetic parameters for ExoAc and cholixc toxins

The kinetic parameters were determined as described under “Experimental Procedures.” The values represent the mean_S.D. from three independent experiments.

cholix tabel

Figure 1

FIGURE 1. Cholix toxin is cytotoxic toward mouse fibroblasts. Cytotoxicity after _48 h toxin exposure. a, cells display several morphotypes with no toxin. b, with 50 ng/ml E581A toxin cells still reach normal density. c, with 1 ng/ml wild-type toxin, cells are normal. d, 10 ng/ml; e, 25 ng/ml; or f, 50 ng/ml of wild-type toxin clearly results in cytotoxic effects.

figure 2

FIGURE 2. Biological activity of cholix toxin. a, protein synthesis inhibition in mouse fibroblasts. Cholix toxin (closed circles) inhibits protein synthesis similarly to ExoA (open circles) but E581A mutant (closed squares) does not. b, protein synthesis inhibition by ExoA and cholix toxin inmouseembryonicfibroblast1 (LRP1_/_)andPEA13 (LRP1_/_) cells at 100 ng/ml. c, survival (%) of A. nauplii exposed to cholix toxin. Cholix toxin (closed circles) displays a dose-dependent effect on A. nauplii after 42–45 h. Cholix toxin E581A (open circle) displays no toxic effect. d, Western blot showing biotin-ADP-ribose labeling of eEF2 in CHO lysate. Lane 1, 0.25_g of eEF2 incubated with cholix toxin; lanes 2–4, various controls; lanes 5–7, labeling of CHO lysate by cholixc toxin, ExoAc, and diphtheria toxin catalytic fragment (DTA), respectively.

figure 3

FIGURE 3. The structure of cholix toxin. a, ribbon drawing of the cholix toxin structure (PDB entry 2Q5T): domain Ia, blue (1–264); domain II (265–386), red; domain Ib (387– 423), orange; and domain III (424–634), green. Disulfides and furin cut sites are shown as light green and blue spheres, respectively. b, superposition of ExoA onto the cholix toxin structure. Disulfides are indicated by light green and yellow spheres for cholix toxin and ExoA, respectively. c, ribbon drawing of the catalytic domain structure (PDB entry 2Q6M). The two PJ34 inhibitors are shown in black and grey ball-and-stick representation, and the catalytic loops (L1–L4) are shown in yellow, cyan, orange, and red, respectively. d, binding of PJ34 to the NAD binding pocket. The phenyl moiety of Tyr-504 forms stacking interactions with PJ34 (4 Å), and Tyr-493 is also adjacent to the hetero-ring system.

Last revised 15 November 2016