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 þ , 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 þ substrate adopts a conformation remarkably different from that of the NAD þ analogue, bTAD, 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.
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.Bacteria use virulence factors as tools to facilitate disease in plants, animals, and humans (14,26,30,34); one strategy to combat infection is to inhibit these factors by small-molecule therapy, thereby helping to neutralize the offending microbe (5,6,12,19,22). It is now generally appreciated that an antivirulence approach is a powerful alternative strategy for antibacterial treatment and vaccine development (27) and that it may require multiple tactics to resolve the current drug resistance dilemma (6,8). Antivirulence compounds offer significant advantages over conventional antibiotics since these inhibitors are directed toward specific mechanisms (targets) in the offending pathogen that promote infection rather than against an essential metabolic factor (12). Neutralizing the cytotoxic properties of virulence factors from microorganisms without threatening their survival offers reduced selection pressure, making the induction of drug resistance mutations less likely (6). Additionally, virulence-specific therapeutics avoid the undesirable effects on the host microbiota that are associated with current antibiotics.The mono-ADP-ribosyltransferase (mART) family is a group of toxic bacterial enzymes, some of which possess a long history against human civilization. The best-characterized and wellknown members of this lethal family are cholera toxin (CT) from Vibrio cholerae, diphtheria toxin (DT) produced by Corynebacterium diphtheriae, pertussis toxin (PT) from Bordella pertussis, heat-labile enterotoxin from Escherichia coli, C3-like exoenzyme produced by Clostridium botulinum and Clostridium limosum, and exotoxin A (ExoA) from Pseudomonas aeruginosa. These enzymes act on NAD ϩ and facilitate the scission of the glycosidic bond (C-N) between nicotinamide and its conjugated ribose followed by the transfer of the ADPribose group to a nucleophilic residue on a target macromolecule (35). This family can be divided into the CT and DT groups. The CT group consists of an ExoS-like subgroup (enzymatic A domain alone or paired with ano...
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.
Background: Cethrax toxin from B. cereus inactivates mammalian cells through cytoplasmic ADP-ribosyltransferase activity. Results: The crystal structure of Certhrax reveals that it has two domains, one that binds protective antigen and another that has ADP-ribosyltransferase activity. Conclusion: Good inhibitors against the ADP-ribosyltransferase activity have been developed. Significance: Certhrax may be an important virulence factor in B. cereus pathogenesis.
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 ␣-skeletal actin and nonmuscle -and ␥-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.
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