Intoxication strategy of Helicobacter pylori VacA toxin.

VacA toxin from the cancer-inducing bacterium Helicobacter pylori is currently classified as a pore-forming toxin but is also considered a multifunctional toxin, apparently causing many pleiotropic cell effects. However, an increasing body of evidence suggests that VacA could be the prototype of a new class of monofunctional A-B toxins in which the A subunit exhibits pore-forming instead of enzymatic activity. Thus, VacA may use a peculiar mechanism of action, allowing it to intoxicate the human stomach.

Molecular cross-talk between Helicobacter pylori and human gastric mucosa.

Helicobacter pylori (H. pylori) has co-evolved with humans to be transmitted from person to person and to colonize the stomach persistently. A well-choreographed equilibrium between the bacterial effectors and host responses permits microbial persistence and health of the host, but confers a risk for serious diseases including gastric cancer.

Helicobacter pylori counteracts the apoptotic action of its VacA toxin by injecting the CagA protein into gastric epithelial cells.

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.

Structural basis for the NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes.

C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps.

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C.

Specificity of immunomodulator secretion in urinary samples in response to infection by alpha-hemolysin and CNF1 bearing uropathogenic Escherichia coli.

Escherichia coli are the most common etiological agents of urinary tract infections (UTIs). Uropathogenic E. coli (UPECs) produce specific toxins including the cytotoxic necrotizing factor-1 (CNF1) and the alpha-hemolysin (alpha-Hly).

Differential requirement for the translocation of clostridial binary toxins: iota toxin requires a membrane potential gradient.

Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2-II, respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I, respectively) with ADP-ribosyltransferase activity that depolymerizes the actin cytoskeleton. After internalization, it was found that C2 and Iota toxins were not routed to the Golgi apparatus and exhibited differential sensitivity to inhibitors of endosome acidification. While the C2-I component of C2 toxin was translocated into the cytosol from early endosomes, translocation of the Ia component of Iota toxin occurred between early and late endosomes, was dependent on more acidic conditions, and uniquely required a membrane potential gradient.

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells.

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA.

Urinary interleukin-8 is elevated in urinary tract infections independently of the causative germs.

Interleukin-8 elevation in urine during urinary tract infections (UTIs) has been documented for different uropathogenic germs in 85 patients. We showed that for 17 different isolates, IL-8 was increased in 92% of UTIs with an average value of 627 pg/ml for infected, as compared to 45 pg/ml for uninfected patients. We suggest that the high negative predictive value of the IL-8 urine assay could be useful to eliminate UTIs in routine screenings.

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes.

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells.

E. coli CNF1 toxin: a two-in-one system for host-cell invasion.

The cytotoxic necrotizing factor-1 (CNF1), a bacterial toxin of uropathogenic bacteria (UPEC), hijacks cellular Rho proteins of the Ras GTPase super-family. Recently, we have made three important findings. First, we have established that, following Rho protein activation by deamidation, these cellular proteins are ubiquitylated and degraded by the proteasome.

Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells.

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level.

Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis?

In the past few years, an important question in microbiology has arisen from reports indicating that several pathogenic bacteria have evolved virulence factors directed towards a Ras subfamily of GTPases, namely the Rho GTPases. Progress made in studying both the virulence factors and the signaling pathways involving Rho GTPases has shed light on this crosstalk. One central question is raised by the findings that both activating and inactivating virulence factors that target Rho GTPases coexist in some pathogenic bacteria.

To be helped or not helped, that is the question.

Diphtheria toxin (DT)* is the paradigm of the powerful A-B toxins. These bacterial poisons bind to cells, are endocytosed, and inject their catalytic domain into the cytosol causing the irreversible modification of a key component of the the host cellular machinery. The mechanism by which the hydrophilic enzymatic fragment of DT crosses the endosomal membrane and is released into the cytosol remains controversial.

Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein.

Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD-cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co-regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine-Dalgarno sequence present in the cnf1 coding sequence [fold-back inhibition (cnf1 fbi)].

Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function.

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function.

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion.

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac.

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells.

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells.

NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme.

We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively.

Strategies for the structural determination of the catalytic domain of Escherichia coli cytotoxic necrotizing factor 1.

Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa toxin produced by certain pathogenic strains of Escherichia coli, constitutively activates members of the Rho GTPase family, leading to cytopathic effects. The toxin inhibits GTP turnover by Rho proteins through site-specific deamidation of a Rho glutamine required for GTP hydrolysis. To understand the basis for catalytic activity and target specificity of CNF1, the structure of a catalytically active fragment of CNF1 was sought.