Blandine Geny

Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins.

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly-disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.

Bacterial protein toxins and lipids : pore formation or toxin entry into cells

Lipids are hydrophobic molecules which play critical functions in cells, in particular, they are essential constituents of membranes, whereas bacterial toxins are mainly hydrophilic proteins. All bacterial toxins interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Most bacterial toxins are PFTs (pore‐forming toxins) which oligomerize and insert into the lipid bilayer. A common mechanism of action involves the formation of a β‐barrel structure, resulting from the assembly of individual β‐hairpin(s) from individual monomers. An essential step for intracellular active toxins is to translocate their enzymatic part into the cytosol. Some toxins use a translocation mechanism based on pore formation similar to that of PFTs, others undergo a yet unclear ‘chaperone’ process.

Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes GM2 as receptor and forms anion-selective channels.

Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis

Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.1M KCl in lipid bilayer membranes, which is typical for a large diffusion pore. Septicum alpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion selectivity. Septicum alpha-toxin caused a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induced a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induced a rapid K(+) efflux and ATP depletion. Incubation of the cells in K(+)-enriched medium delayed cell death caused by septicum alpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K(+) represents an early signal of pore-forming toxins-mediated cell necrosis.

Identification of the channel forming domain of Clostridium perfringens epsilon-toxin (ETX)

Epsilon-toxin (ETX) is a potent toxin produced by Clostridium perfringens strains B and D. The bacteria are important pathogens in domestic animals and cause edema mediated by ETX. This toxin acts most likely by heptamer formation and rapid permeabilization of target cell membranes for monovalent anions and cations followed by a later entry of calcium. In this study, we compared the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A-B-types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and found a remarkable homology to amino acids 151-180 of ETX. Site-directed mutagenesis of amino acids within the putative channel-forming domain resulted in changes of cytotoxicity and effects on channel characteristics in lipid bilayer experiments including changes of selectivity and partial channel block by methanethiosulfonate (MTS) reagents and antibodies against His(6)-tags from the trans-side of the lipid bilayer membranes.

Lethal toxin from Clostridium sordellii induces apoptotic cell death by disruption of mitochondrial homeostasis in HL-60 cells

Lethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac. In the present study we show that LT‐IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell‐line HL‐60. LT‐IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol. Time‐course studies of caspase activation revealed that caspase‐9 and caspase‐3 were activated before caspase‐8. Moreover, although LT‐IP82‐induced cell death was abrogated by caspase‐inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria. Protection of mitochondria by Bcl‐2 overexpression prevented mitochondrial changes as well as apoptosis induction. Furthermore, evidence is provided that LT‐IP82‐induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process. Cell exposure to LT‐IP82 leads to a co‐localization of the toxin with a mitochondrial marker within 2 h. Therefore, we suggest that LT‐IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.

Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins

Clostridium sordellii lethal toxin (LT) is a glucosyltransferase which inactivates small GTPases from the Rho and Ras families. In the present work, we studied the effects of two variants, LT82 and LT9048, on the integrity of epithelial cell barrier using polarized MCCD (Mouse Cortical Collecting Duct) and MDCK (Madin‐Darby Canine Kidney) cells. Our results demonstrate for the first time that LTs have very limited effects on tight junctions. In contrast, we show that both toxins modified the paracellular permeability within 2–4 h. Concomitantly LT82 and LT9048 induced a disorganization of basolateral actin filaments, without modifying apical actin. Both toxins mainly altered adherens junctions by removing E‐cadherin‐catenin complexes from the membrane to the cytosol. Similar effects on adherens junctions have been observed with other toxins, which directly or indirectly depolymerize actin. Thereby, Rac, a common substrate of both LTs, might play a central role in LT‐dependent adherens junction alteration. Here, we show that adherens junction perturbation induced by LTs results neither from a direct effect of toxins on adherens junction proteins nor from an actin‐independent Rac pathway, but rather from a Rac‐dependent disorganization of basolateral actin cytoskeleton. This further supports that a dynamic equilibrium of cortical actin filaments is essential for functional E‐cadherin organization in epithelia.

G-protein-stimulated Phospholipase D Activity Is Inhibited by Lethal Toxin from Clostridium sordellii in HL-60 Cells

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5′-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Cα was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.

Bacterial protein toxins and lipids: role in toxin targeting and activity

All bacterial toxins, which globally are hydrophilic proteins, interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Intracellular active toxins follow various trafficking pathways, the sorting of which is greatly dependent on the nature of the receptor, notably lipidic receptor or receptor embedded into a distinct environment such as lipid microdomains. Numerous other toxins act locally on cell membrane. Indeed, phospholipase activity is a common mechanism shared by several membrane‐damaging toxins. In addition, many toxins active intracellularly or on cell membrane modulate host cell phospholipid pathways. Unusually, a few bacterial toxins require a lipid post‐translational modification to be active. Thereby, lipids are obligate partners of bacterial toxins.

Activation of a c-Jun-NH2-terminal kinase pathway by the lethal toxin from Clostridium sordellii, TcsL-82, occurs independently of the toxin intrinsic enzymatic activity and facilitates small GTPase glucosylation.

In the present study, we show that lethal toxin from Clostridium sordellii (TcsL‐82) activates the three MAP kinase pathways, but that only a permeable and specific c‐Jun‐NH2‐terminal kinase (JNK) inhibitor, JNK inhibitor II, prevents toxin‐dependent actin depolymerization and cell rounding. We show that JNK activation is dependent on entry of the toxin N‐terminal domain into the cytosol as bafilomycin A1, which prevents acidification of endocytic vesicle and subsequent cytosolic translocation of the toxin N‐terminal domain, prevents JNK activation. Inhibition of JNK activity delays small GTPase glucosylation generated by N‐terminal domain catalytic activity. Using a cell line mutant deficient in UDP‐glucose, we observed that activation of JNK occurs even in the absence of small GTPase glucosylation and, thus, is independent of the toxin intrinsic catalytic activity. Facilitation of target glucosylation by JNK activation appeared to be restricted to TcsL‐82 and was not a general feature of large clostridial toxins. Indeed, it was not observed with Toxin B from Clostridium difficile although this toxin also activates JNK.