Targeted delivery of an ADP-ribosylating bacterial toxin into cancer cells.

The actin cytoskeleton is an attractive target for bacterial toxins. The ADP-ribosyltransferase TccC3 from the insect bacterial pathogen Photorhabdus luminescence modifies actin to force its aggregation. We intended to transport the catalytic part of this toxin preferentially into cancer cells using a toxin transporter (Protective antigen, PA) which was redirected to Epidermal Growth Factor Receptors (EGFR) or to human EGF receptors 2 (HER2), which are overexpressed in several cancer cells. Protective antigen of anthrax toxin forms a pore through which the two catalytic parts (lethal factor and edema factor) or other proteins can be transported into mammalian cells. Here, we used PA as a double mutant (N682A, D683A; mPA) which cannot bind to the two natural anthrax receptors. Each mutated monomer is fused either to EGF or to an affibody directed against the human EGF receptor 2 (HER2). We established a cellular model system composed of two cell lines representing HER2 overexpressing esophageal adenocarcinomas (EACs) and EGFR overexpressing esophageal squamous cell carcinomas (ESCCs). We studied the specificity and efficiency of the re-directed anthrax pore for transport of TccC3 toxin and established Photorhabdus luminescence TccC3 as a toxin suitable for the development of a targeted toxin selectively killing cancer cells.

Clostridial Rho-inhibiting protein toxins.

Rho proteins are master regulators of a large array of cellular functions, including control of cell morphology, cell migration and polarity, transcriptional activation, and cell cycle progression. They are the eukaryotic targets of various bacterial protein toxins and effectors, which activate or inactivate the GTPases. Here Rho-inactivating toxins and effectors are reviewed, including the families of large clostridial cytotoxins and C3-like transferases, which inactivate Rho GTPases by glucosylation and ADP-ribosylation, respectively.

Rho-modifying C3-like ADP-ribosyltransferases.

C3-like exoenzymes comprise a family of seven bacterial ADP-ribosyltransferases, which selectively modify RhoA, B, and C at asparagine-41. Crystal structures of C3 exoenzymes are available, allowing novel insights into the structure-function relationships of these exoenzymes. Because ADP-ribosylation specifically inhibits the biological functions of the low-molecular mass GTPases, C3 exoenzymes are established pharmacological tools to study the cellular functions of Rho GTPases. Recent studies, however, indicate that the functional consequences of C3-induced ADP-ribosylation are more complex than previously suggested. In the present review the basic properties of C3 exoenzymes are briefly summarized and new findings are reviewed.

Requirement of Rac activity for maintenance of capillary endothelial barrier properties.

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice.

Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca2+-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng x ml(-1)) inactivated 50-90% of all endothelial Rho proteins within 60-90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 microM) for 90-120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (L(p)) but did not attenuate the transient increase in L(p) of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 microM) for up to 100 min reduced basal L(p) but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells.

First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Recombinant Yersinia YopT leads to uncoupling of RhoA-effector interaction.

Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis deliver different Yop (Yersinia outer proteins) effector proteins into mammalian cells by a type III secretion mechanism. Recently, it was shown that Yersinia producing YopT leads to disruption of the actin cytoskeleton of HeLa cells (M. Iriarte and G. R. Cornelis, Mol. Microbiol. 29:915-929, 1998). To analyze the molecular mechanism of YopT, we cloned and expressed YopT as a glutathione S-transferase fusion protein. Recombinant YopT caused rounding up of embryonic bovine lung cells and redistribution of the actin cytoskeleton rapidly after microinjection. The Escherichia coli cytotoxic necrotizing factor (CNF1), which constitutively activates Rho proteins, was not able to inhibit or revert YopT-induced cell rounding. YopT caused release of RhoA from embryonic bovine lung membranes and released recombinant isoprenylated RhoA from artificial PE or PE/PIP2 vesicles. Incubation of lysate or cytosol with YopT caused inhibition of the RhoA-rhotekin interaction but led to increased RhoA-RhoGDI interaction. It is suggested that inhibition of the interaction between RhoA and effectors is the underlying mechanism of the YopT action on the cytoskeleton.

Lysine and polyamines are substrates for transglutamination of Rho by the Bordetella dermonecrotic toxin.

Bordetella dermonecrotic toxin (DNT) catalyzes the transglutamination of glutamine-63/61 of Rho GTPases, thereby constitutively activating Rho proteins. Here we identified second substrates for transglutamination of RhoA by DNT. The enzymatically active fragment of DNT (residues 1136 to 1451, DeltaDNT) induced the incorporation of L-[(14)C]lysine in RhoA in a concentration-dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by DeltaDNT. Transglutamination of the GTPase with L-lysine inhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contrast to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT increased the incorporation of L-[(14)C]lysine into embryonic bovine lung cells. Microinjection of GST-RhoA together with the enzymatically active DNT fragment into Xenopus oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or spermidine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed that lysine is a preferred second substrate at least in vitro.

Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile.

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1-240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244-263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208-413 but loss of activity of several N-terminally deleted C2I proteins including C2I103-431, C2I190-431, and C2I30-431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress.

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin. Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival.

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.

Regulation by rho family GTPases of IL-1 receptor induced signaling: C3-like chimeric toxin and Clostridium difficile toxin B inhibit signaling pathways involved in IL-2 gene expression.

In this study the participation of Rho family GTPases in the regulation of IL-1-activated protein kinase cascades controlling IL-2 synthesis was investigated in murine EL-4 thymoma cells. The recombinant C3-like chimeric toxin, which consists of the C3 toxin of Clostridium limosum and the N-terminal part of Clostridium botulinum C2 toxin (C2IN-C3) interacting with the C2II binding subunit to facilitate uptake into cells, and selectively inactivates Rho A by ADP-ribosylation, prevented IL-1-stimulated activation of Jun-NH2-terminal-kinases (JNK) and p38 mitogen-activated-protein kinases (MAPK). UDP-monoglucosylation and concomitant inactivation of Rho A and of Rac-2 by Clostridium difficile toxin B also inhibited IL-1-induced activation of JNK and p38 MAPK, but additionally inhibited activation of the extracellular-regulated-kinase pathway and DNA binding of the transcription factor NFkappaB. Accordingly, pre-treatment of cells with C21N-C3 fusion toxin only decreased IL-1-stimulated IL-2 synthesis by 50%, while in C. difficile toxin B-treated cells IL-1-induced IL-2 secretion was reduced by 90%. These results imply that together with Rho A an additional member of the Rho family G proteins, i.e. Rac-2, is critically involved as an upstream regulator in IL-1-induced activation of different MAPK, stress-activated protein kinases, and in NFkappaB activation controlling IL-2 gene expression in response to IL-1, acting in close proximity to the IL-1-receptor complex.

Biological activity of a C-terminal fragment of Pasteurella multocida toxin.

The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cbeta. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.

Cellular uptake of the Clostridium perfringens binary iota-toxin.

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH < or = 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 +/- 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor.

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10(5)-10(6) molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca(2+)-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1.

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.