The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin. The ADP-ribosyltransferase domain is located within the C terminus part of ExoS. Recent studies showed that the N terminus part of ExoS (amino acid residues 1-234, ExoS(1-234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells. Here we studied the effects of ExoS(1-234) on nucleotide binding and hydrolysis by Rho GTPases. ExoS(1-234) (100 -500 nM) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis. A similar increase in GTPase activity was stimulated by full-length ExoS. Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10 -11 nM ExoS(1-234), respectively. We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins. These data identify ExoS as a GTPase-activating protein for Rho GTPases.Rho GTPases, Rho, Rac, and Cdc42 are involved in the regulation of the actin cytoskeleton by cell membrane-bound receptors and act as molecular switches in a large array of signaling processes (1, 2). Recent studies indicate that the GTPases are the preferred eukaryotic substrates of various bacterial protein toxins and exoenzymes (3,4). C3-like exoenzymes (e.g. Clostridium botulinum exoenzyme C3) ADP-ribosylate RhoA, B, and C at asparagine 41, inhibiting the biological functions of the GTPases (5-7). Large clostridial cytotoxins (e.g. Clostridium difficile toxins A and B) monoglycosylate Rho threonine 37 and Rac and Cdc42 at threonine 35 (8, 9) and induce rounding up of cells and redistribution of the actin cytoskeleton. Escherichia coli cytotoxic necrotizing factors (CNF 1 and 2) 1 and dermonecrotic toxin DNT of Bordetella species (10, 11, 18) activate Rho family GTPases by increasing the lifetime of the active GTP-bound form of the Rho protein. CNF and DNT deamidate and/or transglutaminate glutamine 63 of Rho (glutamine 61 of Rac and Cdc42), 2 which inhibits the intrinsic and GTPase-activating protein (GAP)-stimulated GTP hydrolysis, resulting in a constitutively activated form of the GTPases.Pseudomonas aeruginosa produces two ADP-ribosyltransferases: exotoxin A, which ADP-ribosylates elongation factor 2, or exoenzyme S (ExoS), which ADP-ribosylates Ras (13). ExoS (453 amino acids) is secreted and translocated into eukaryotic target cells by a type III secretion mechanism, which requires bacterial to eukaryotic cell contact. The ADP-ribosyltransferase activity by ExoS is dependent upon a eukaryotic cofactor (FAS, factor activating exoenzyme S), which is a member of the 14-3-3 protein family (14). Ras and several other proteins are preferred eukaryotic substrates of ExoS (15). ExoS ADP-ribosylates Ras at arginine 41 and arginine 128. ADP-ribosylation at arginine 41 blocks the activation of Ras by its guanine nucleotide exchange factor, thereby interfering with Ras-mediated signal transduction (16). The ADP-ribosyltransferase domain is located within the C-...

Preface Bacterial ADP-ribosyltransferase toxins (bARTTs) transfer ADP-ribose to eukaryotic proteins to promote bacterial pathogenesis. In this review we use prototype bARTTs, such as diphtheria and pertussis toxins, as references for the characterization of several new bARTTs from human, insect, and plant pathogens, which were identified recently through bioinformatic analyses. Several of these toxins, including Cholix toxin from Vibrio cholerae, SpyA from Streptococcus pyogenes, HopU1 from Pseudomonas syringae, and the Tcc toxins from Photorhabdus luminescens, ADP-ribosylate novel substrates and possess unique organizations, which distinguish them from the reference toxins. The characterization of these toxins extends our appreciation for the variety of structure-function properties possessed by bARTTs and their roles in bacterial pathogenesis.

ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains. The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42. Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis. Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity. Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates. While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins. Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.

Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.