ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview.

ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.

Use of a neutralizing antibody helps identify structural features critical for binding of Clostridium difficile toxin TcdA to the host cell surface.

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis.

Clostridium difficile TcdB harbors a glucosyltransferase that targets host Rho GTPases. However, the role of the enzyme activity in the induction of host intestinal disease has not been demonstrated. In this study, we established a mouse acute intestinal disease model by cecum injection of wild type and glucosyltransferase-deficient TcdB and a chronic model by delivering toxin intraluminally via engineered surrogate host Bacillus megaterium. We demonstrated, for the first time, that the glucosyltransferase activity of TcdB is essential for inducing disease symptoms and intestinal pathological responses that resemble human disease, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

Pyknotic cell death induced by Clostridium difficile TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity.

TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.

Repetitive domain of Clostridium difficile toxin B exhibits cytotoxic effects on human intestinal epithelial cells and decreases epithelial barrier function.

We have used recombinant repetitive domain of Clostridium difficile toxin B obtained from two different strains, rec-TcdB3(10463) and rec-TcdB3(8864) and a model intestinal epithelial cell line(s) to characterize their cytotoxic and cytopathic effect and influence on tight-junction organization. Both recombinant receptor binding domains caused intestinal epithelial cell damage, decreased transepithelial electrical resistance and induced translocation of ZO-1 from tight-junction proteins although less efficiently as holotoxins. Recombinant repetitive TcdB domains also caused stimulation of interleukin IL-8 synthesis in HT-29 cells. This is the first description of glucosyltransferase independent toxicity of TcdB and these C-terminal mediated effects may contribute to the pathophysiology of C. difficile infection.

The type III toxins of Pseudomonas aeruginosa disrupt epithelial barrier function.

The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to DeltaSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosa's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.

Comparison of wild type with recombinant Clostridium difficile toxin A.

Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest. We, therefore, expressed recombinant His-tagged toxin A using the Bacillus megaterium expression system. Specific antisera raised against the native toxin A from C. difficile and the recombinant toxin, respectively, showed identical sensitivity and specificity in Western blot and ELISA analyses towards both toxins. By comparison of both toxins in functional studies we showed that the recombinant toxin was about two times more cytotoxic than the native toxin, and the glucosyltransferase-activity of the recombinant toxin was even 10-fold increased. However, recombinant toxin A showed one essential difference to the classically purified one. The reported transferase-independent effect of toxin A to release cytochrome c from isolated mitochondria was not exhibited by the recombinant toxin A. This putative mitochondrial effect decreased with increased purity of toxin A, and was absent with recombinant toxin, strongly suggesting an clostridial contamination responsible. In summary, we tested the recombinant toxin A to be at least an adequate substitute for the native toxin, bearing the advantage of a rapid single-step purification and the absence of biological active contaminations.

Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection.

Pseudomonas aeruginosa utilizes a number of distinct pathways to secrete proteins that play various roles during infection. These include the type II secretion system, which is responsible for the secretion of the majority of exoproducts into the surrounding environment, including toxins and degradative enzymes. In contrast, the type III secretion system mediates the delivery of protein effectors directly into the cytoplasm of the host cell. Using tissue culture assays and a mouse acute-pneumonia model, we have determined the contribution of each of the type III effectors during infection. In strain PAK, ExoS is the major cytotoxin required for colonization and dissemination during infection. ExoT confers protection of tissue culture cells from type III-dependent lysis, while ExoY seemed to have little effect on cytotoxicity. ExoU is over 100-fold more cytotoxic than ExoS. The cytotoxicity of type II secretion was determined following deletion of the genes for the more toxic type III secretion system. The participation of these secretion systems during lifelong colonization of cystic fibrosis (CF) patients is unclear. By comparing clonal strains from the same patient isolated at the initial onset of P. aeruginosa infection and more than a decade later, after chronic colonization has been established, we show that initial strains are more cytotoxic than chronic strains that have evolved to reduce type III secretion. Constitutive expression of genes for the type III secretion system restored ExoS secretion but did not always reestablish cytotoxicity, suggesting that CF strains accumulate a number of mutations to reduce bacterial toxicity to the host.

Safety evaluation of an alpha-cyclodextrin glycosyltranferase preparation.

Alpha-cyclodextrin glucosyltransferase (alpha-CGTase, EC 2.4.1.19) is an amylolytic enzyme used for the production of alpha-cyclodextrin (alpha-CD), a novel, soluble dietary fiber, from food-grade starch. The safety of an alpha-CGTase preparation obtained by batch fermentation from a recombinant strain of Escherichia coli K12 harboring the alpha-CGTase gene from Klebsiella oxytoca strain M5a1 was examined. In a 13-week subchronic toxicity study in rats, the administration by gavage of the alpha-CGTase preparation at levels of up to 20 ml/kg bw/day, corresponding to a total organic solids dosage of 260 mg/kg bw/day, did not cause any systemic toxic effect. Some signs of irritation were observed in the respiratory tract which occurred, however, in one sex only and/or were not dose-related. Accordingly, these changes were considered to be an unspecific consequence of the reflux and aspiration of the dosing solution. There was no evidence of a genotoxic activity in Ames tests and a chromosome aberration test in cultured human lymphocytes. It is concluded that the examined alpha-CGTase preparation is safe when used for the production of alpha-CD.

Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia.

Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality. Although the intrinsic virulence of P. aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease. In this study, the virulence of 35 P. aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia. The virulence of individual isolates differed significantly from one to another in each of these assays. Increased virulence was associated with the secretion of ExoU, a toxin transported by the P. aeruginosa type III secretion system. Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence. Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P. aeruginosa.

UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii.

We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile. We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/10(6) cells) was lower than in the wild type (3.7 nmol/10(6) cells); and (iii) sensitivity to toxin B was restored upon microinjection of UDP-glucose. Using the mutant as indicator cell we also found that the related Clostridium sordellii lethal toxin is a glucosyltransferase which requires UDP-glucose as a cofactor. Like toxin B it glucosylated 21-23-kDa proteins in cell lysates, but Rho was not a substrate for lethal toxin.

Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase)

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.