Characterization of Vis Toxin, a Novel ADP-Ribosyltransferase from Vibrio splendidus.

Vis toxin was identified by a bioinformatics strategy as a putative virulence factor produced by Vibrio splendidus with mono-ADP-ribosyltransferase activity. Vis was purified to homogeneity as a 28 kDa single-domain enzyme and was shown to possess NAD(+)-glycohydrolase [KM(NAD(+)) = 276 ± 12 µM] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [KM(agmatine) = 272 ± 18 mM]. Mass spectrometry analysis revealed that Vis labels l-arginine with ADP-ribose from the NAD(+) substrate at the amino nitrogen of the guanidinium side chain. Vis is toxic to yeast when expressed in the cytoplasm under control of the CUP1 promotor, and catalytic variants lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. Several small molecule inhibitors were identified from a virtual screen, and the most potent compounds were found to inhibit the transferase activity of the enzyme with Ki values ranging from 25 to 134 µM. Inhibitor compound M6 bears the necessary attributes of a solid candidate as a lead compound for therapeutic development. Vis toxin was crystallized, and the structures of the apoenzyme (1.4 Å) and the enzyme bound with NAD(+) (1.8 Å) and with the M6 inhibitor (1.5 Å) were determined. The structures revealed that Vis represents a new subgroup within the mono-ADP-ribosyltransferase toxin family.

Certhrax toxin, an anthrax-related ADP-ribosyltransferase from Bacillus cereus.

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD(+) with high affinity (K(D) = 52.3 ± 12.2 µM). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K(D) = 1.7 ± 0.2 µM, K(i) = 1.8 ± 0.4 µM) and PJ34 (K(D) = 5.8 ± 2.6 µM, K(i) = 9.6 ± 0.3 µM)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD(+) binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.

Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 µM, K(m)(actin) = 24 µM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 µM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.

Newly discovered and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase toxins.

The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.

ADP-ribosylation of cross-linked actin generates barbed-end polymerization-deficient F-actin oligomers.

Actin filament subunit interfaces are required for the proper interaction between filamentous actin (F-actin) and actin binding proteins (ABPs). The production of small F-actin complexes mimicking such interfaces would be a significant advance toward understanding the atomic interactions between F-actin and its many binding partners. We produced actin lateral dimers and trimers derived from F-actin and rendered polymerization-deficient by ADP-ribosylation of Arg-177. The degree of modification resulted in a moderate reduction in thermal stability. Calculated hydrodynamic radii were comparable to theoretical values derived from recent models of F-actin. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar those of wild-type actin, suggesting native or near-native interfaces on the oligomers. Changes in DNase I binding affinity under low and high ionic strength suggested a high degree of conformational flexibility in the dimer and trimer. Polymer nucleation activity was lost upon ADP-ribosylation and rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin, suggesting that interactions with actin binding proteins can overcome the inhibiting activities of ADP-ribosylation. The combined strategy of chemical cross-linking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds.

Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins.

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.

The nature and character of the transition state for the ADP-ribosyltransferase reaction.

Exotoxin A (ExoA) from Pseudomonas aeruginosa is an important virulence factor that belongs to a class of exotoxins that are secreted by pathogenic bacteria which cause human diseases such as cholera, diphtheria, pneumonia and whooping cough. We present the first crystal structures, to our knowledge, of ExoA in complex with elongation factor 2 (eEF2) and intact NAD(+), which indicate a direct role of two active-site loops in ExoA during the catalytic cycle. One loop moves to form a solvent cover for the active site of the enzyme and reaches towards the target residue (diphthamide) in eEF2 forming an important hydrogen bond. The NAD(+) substrate adopts a conformation remarkably different from that of the NAD(+) analogue, betaTAD, observed in previous structures, and fails to trigger any loop movements. Mutational studies of the two loops in the toxin identify several residues important for catalytic activity, in particular Glu 546 and Arg 551, clearly supporting the new complex structures. On the basis of these data, we propose a transition-state model for the toxin-catalysed reaction.