Patrice Boquet

Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine

Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells,. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho, and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.

The N‐terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release

The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp‐2 cells with DNAs encoding either the N‐terminal (p34) or the C‐terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA–GFP and p34–GFP, but not GFP–VacA or GFP–p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34–GFP or VacA–GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP–ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co‐transfection of DNA encoding Bcl‐2, known to block mitochondria‐dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.

Large clostridial cytotoxins — a family of glycosyltransferases modifying small GTP-binding proteins

Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

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. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.

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. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure, but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.

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. Further studies on this peculiar aspect of the bacteria-host cell interactions, which leads to the outbreak of infectious diseases, might clarify whether this aspect of Rho GTPase activation or inactivation represents a finely adapted response of the pathogen for its own benefit or might lead to a reaction of the host against the bacteria.

Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell‐binding and catalytic domains

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cells, the development of stress fibres via the GTPase Rho pathway. We showed that CNF1 is able to modify Rho both in vitro and in vivo. Recombinant N‐terminal 33 kDa (CNF1Nter) and C‐terminal 14.8–31.5 kDa (CNF1Cter) regions of the CNF1 protein allowed us to demonstrate that the N‐terminal region contains the cell‐binding domain of the toxin and that the C‐terminal region is responsible for its catalytic activity. CNF1Nter lowered the activity of CNF1 when provided to cells before the toxin whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C‐terminal domain lost its catalytic activity when deleted of various subdomains, suggesting a scattered distribution of catalytic‐site amino acids. Elucidation of the CNF1 functional organization and analysis of amino acid homologies between CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonecrotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in their C‐terminus, and that CNFs and PMT probably bind to analogous cell receptors.

Dissection of Pathways Implicated in Integrin-mediated Actin Cytoskeleton Assembly INVOLVEMENT OF PROTEIN KINASE C, RHO GTPase, AND TYROSINE PHOSPHORYLATION

A panel of antibodies to the αIIbβ3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the αIIbβ3 fibrinogen receptor. While some αIIbβ3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster β1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxicnecrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1), a Toxin That Activates the Rho GTPase

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.

Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH (‘activation’) results in structural changes in the protein and a marked enhancement of its cell‐vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]‐VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]‐VacA to cells. Binding of acid‐activated [125I]‐VacA to cells at 4°C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non‐specifically or to an abundant, low‐affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS–PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time‐dependent process at 37°C but not at 4°C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]‐VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2‐deoxy‐d‐glucose. These results indicate that acid or alkaline pH‐induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.