Activation of the phospholipase D (PLD) pathway is a widespread response when cells are activated by agonists that bind receptors on the cell surface. A 16-kD cytosolic component can reconstitute guanosine triphosphate (GTP)-mediated activation of phospholipase D in HL60 cells depleted of their cytosol by permeabilization. This factor was purified and identified as two small GTP-binding proteins, ARF1 and ARF3. Recombinant ARF1 substituted for purified ARF proteins in the reconstitution assay. These results indicate that phospholipase D is a downstream effector of ARF1 and ARF3. The well-established role of ARF in vesicular traffic would suggest that alterations in lipid content by PLD are an important determinant in vesicular dynamics.

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Targeting to mitochondria is emerging as a common strategy that bacteria utilize to interact with these central executioners of apoptosis. Several lines of evidence have in fact indicated mitochondria as specific targets for bacterial protein toxins, regarded as the principal virulence factors of pathogenic bacteria. This work shows, for the first time, the ability of the Clostridium difficile toxin B (TcdB), a glucosyltransferase that inhibits the Rho GTPases, to impact mitochondria. In living cells, TcdB provokes an early hyperpolarization of mitochondria that follows a calcium-associated signaling pathway and precedes the final execution step of apoptosis (i.e. mitochondria depolarization). Importantly, in isolated mitochondria, the toxin can induce a calcium-dependent mitochondrial swelling, accompanied by the release of the proapoptogenic factor cytochrome c. This is consistent with a mitochondrial targeting that does not require the Rho-inhibiting activity of the toxin. Of interest, the mitochondrial ATP-sensitive potassium channels are also involved in the apoptotic response to TcdB and appear to be crucial for the cell death execution phase, as demonstrated by using specific modulators of these channels. To our knowledge, the involvement of these mitochondrial channels in the ability of a bacterial toxin to control cell fate is a hitherto unreported finding.

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.

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.

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT cyt ; amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT cyt to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT cyt . Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT cyt on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT cyt microinjection into various cells, but it does not affect the intrinsic activity of LT cyt on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT cyt for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

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.

Stimulation of phospholipase D (PLD) by cell surface receptors has been observed in many cell types. We have investigated the mechanism of activation of this enzyme in undifferentiated HL60 cells. GTP analogues and Ca2+ (buffered in the nanomolar to micromolar range) were introduced into HL60 cells in the presence of the permeabilizing agent, streptolysin O. We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is a potent activator of phospholipase D when Ca2+ is available at micromolar levels. Phorbol 12-myristate 13-acetate or Ca2+ alone can also stimulate PLD, but to a limited extent. The activation of PLD by GTP[S] can be partially dissociated from GTP[S]-stimulated phosphoinositide-specific phospholipase C, suggesting that a G-protein may be directly involved in regulating PLD. However, maximal activation of PLD only occurs under conditions that are permissive to phospholipase C stimulation. We conclude that PLD activation is under dual control, i.e. protein kinase C- as well as G-protein-mediated regulation. Synergistic activation occurs when both pathways are simultaneously stimulated. We conclude that full activation of PLD requires protein kinase C, increased Ca2+ and a GTP-binding protein. Evidence for cytosolic components that may also be involved in obtaining full activation of PLD is also presented.