Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.     

Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae

Many bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin cross-linking. However, identification and subsequent deletion of the actin cross-linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548-amino-acid region of RTX was identified to be responsible for the toxin-induced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho-modifying bacterial toxins.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.     

A Rho-related GTPase is involved in Ca(2+)-dependent neurotransmitter exocytosis

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca(2+)-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca(2+)-dependent neuroexocytosis.     

The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay

The cell cytoskeleton is widely acknowledged as a master for NK cell function. Specifically, actin filaments guide the NK cell binding to target cells, engendering the formation of the so-called immunological synapse, while microtubules direct the killer behavior. All these cytoskeleton-dependent activities are competently governed by the Rho GTPases, a family of regulatory molecules encompassing the three different subfamilies, Rho, Rac, and Cdc42. By using a Rac GTPase-activating bacterial protein toxin from Escherichia coli named cytotoxic necrotizing factor 1 (CNF1), we obtained results supporting the activation of Rac GTPase as a booster for effector cell-binding efficiency, recruitment ability, and, consequently, cytotoxicity. In particular, the augmented killer capacity of CNF1-treated NK cells was associated with the increased expression of certain cell adhesion or activation-associated molecules and the reshaping of the actin and microtubule networks. Importantly, CNF1 counteracted the activity exerted by toxins disrupting the cytoskeletal architecture. Hence, the activation of Rho GTPases, particularly Rac, induced by CNF1, appears to orchestrate a dynamic cross talk between microtubules and actin filaments, leading to a fruitful NK cell activity and polarization state. Our findings suggest that protein toxins might be viewed as modulators of NK cell cytotoxic activity and could possibly be regarded as useful pharmacological tools for certain Rho-linked immune diseases in the near future.     

Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1).

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.     

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.     

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.     

Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells

Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.     

An Escherichia coli cytotoxin increases superoxide anion generation via rac in epithelial cells

Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin from Escherichia coli that induces the activation of Rho, Rac, and Cdc42 GTPases, all involved in actin reorganization. Rac plays a further role in oxidase function. In epithelial cells, CNF1 has been reported to induce a phagocytic-like behavior in terms of a ruffle-driven ingestion of large material. We herein show that CNF1-activated epithelial cells may exert additional cell responses typical of professional phagocytes following stimulation, i.e., an increase in oxygen consumption and the generation of superoxide anions. Such effects were triggered by the contact of latex beads with epithelial cells and were significantly augmented by CNF1-induced Rac activation. Altogether our data indicate that Rac, one of the targets of CNF1, plays a pivotal role in these phenomena, suggesting the involvement in epithelial cells of a Rac-dependent NADPH-oxidase complex similar to that employed by professional phagocytes.     

Activation and proteasomal degradation of rho GTPases by cytotoxic necrotizing factor-1 elicit a controlled inflammatory response

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3alpha (MIP-3alpha) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3alpha, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.     

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.     

Small rho GTPases regulate antigen presentation in dendritic cells

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.     

JNK phosphorylation of paxillin, acting through the Rac1 and Cdc42 signaling cascade, mediates neurite extension in N1E-115 cells

Neurons extend neurites from the cell body before formation of the polarized processes of an axon and dendrites. Neurite outgrowth involves remodeling of the cytoskeletal components, which are initially regulated by small GTPases of the Rho family. Here we show that c-Jun N-terminal kinase (JNK), which is controlled by Rho GTPases Rac1 and Cdc42, is activated following neurite extension in mouse N1E-115 neuroblastoma cells as a model. The extension is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) and Clostridium difficile Toxin B, the inhibitor for Rho GTPases. Additionally, paxillin, the multifunctional focal adhesion protein, is phosphorylated at Ser 178 by upregulation of the Rac1/Cdc42/JNK cascade. Conversely, transfection of the paxillin construct harboring the Ser 178-to-Ala mutation into cells inhibits neurite extension. Taken together, these results suggest the novel role of the Rac1/Cdc42/JNK signaling cascade in neurite extension and indicate that the downstream target paxillin may be one of the convergent points of various signaling pathways underlying neurite extension.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42

To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.     

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. These authors contributed equally