Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epitheli... more Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epithelia. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. Two members of the Ras superfamily of small GTPases, K-Ras and RhoA, activate ENaC. K-Ras activates ENaC via a signaling pathway involving phosphatidylinositol 3-kinase and production of phosphatidylinositol 3,4,5-trisphosphate with the phospholipid directly interacting with the channel to increase open probability. How RhoA increases ENaC activity is less clear. Here we report that RhoA and K-Ras activate ENaC through independent signaling pathways and final mechanisms of action. Activation of RhoA signaling rapidly increases the membrane levels of ENaC likely by promoting channel insertion. This process dramatically increases functional ENaC current, resulting in tight spatial-temporal control of these channels. RhoA signals to ENaC via a transduction pathway, including the downstream effectors Rho kinase and phosphatidylinositol-4-phosphate 5-kinase. Phosphatidylinositol 4,5-biphosphate produced by activated phosphatidylinositol 4-phosphate 5-kinase may play a role in targeting vesicles containing ENaC to the plasma membrane.
The International Journal of Biochemistry Cell Biology, May 31, 2013
E. coli cytotoxic necrotizing factor 1 induces expression of the cell death-regulating GTPase Rho... more E. coli cytotoxic necrotizing factor 1 induces expression of the cell death-regulating GTPase RhoB.RhoB expression is based on transcriptional activation and involves Rac1-c-Myc signaling.RhoB suppresses cell cycle progression and cell death in CNF1-treated cells.RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB−/− mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB+/− MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.
TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Bo... more TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin co...
Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but... more Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.
The International Journal of Biochemistry & Cell Biology, 2013
RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediat... more RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB(-/-) mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB(+/-) MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.
Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucos... more Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h, a steady state was reached, with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.
Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical sur... more Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptosis. In this study, TcsL and K-Ras knock-down by siRNA are presented to result in expression of the cell death-regulating small GTPase RhoB. TcsL-induced RhoB expression is based on transcriptional activation involving p38(alpha) MAP kinase. Newly synthesized RhoB protein is rapidly degraded in a proteasome- and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a Rho family protein. Although often characterised as a pro-apoptotic protein, RhoB suppresses caspase-3 activation in TcsL-treated fibroblasts. The finding on the cytoprotective activity of RhoB in TcsL-treated cells re-enforces the concept that RhoB exhibits cytoprotective rather than pro-apoptotic activity in a cellular background of inactive Ras.
Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associ... more Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associated diarrhoea (CDAD). TcdA and TcdB mono-glucosylate small GTPases of the Rho family, thereby causing actin re-organisation in colonocytes, resulting in the loss of colonic barrier function. The hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) is an approved drug for the treatment of cholestasis and biliary cirrhosis. In this study, TUDCA-induced activation of Akt1 is presented to increase cellular levels of pS71-Rac1/Cdc42 in human hepatocarcinoma (HepG2) cells, showing for the first time that bile acid signalling affects the activity of Rho proteins. Rac1/Cdc42 phosphorylation, in turn, protects Rac1/Cdc42 from TcdB-catalysed glucosylation and reduces the TcdB-induced cytopathic effects in HepG2 cells. The results of this study indicate that TUDCA may prove useful as a therapeutic agent for the treatment of CDAD.
Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, inclu... more Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P(1-5)) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S1P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S1P, a phenomenon that correlated to the up-regulation of S1P1 and S1P3 in maturing DC. The same pattern of S1P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S1P could be severely hampered by application of the S1P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S1P-mediated signaling plays a pivotal role in the life cycle of DC.
The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the out... more The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy.
Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium d... more Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.
Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epitheli... more Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epithelia. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. Two members of the Ras superfamily of small GTPases, K-Ras and RhoA, activate ENaC. K-Ras activates ENaC via a signaling pathway involving phosphatidylinositol 3-kinase and production of phosphatidylinositol 3,4,5-trisphosphate with the phospholipid directly interacting with the channel to increase open probability. How RhoA increases ENaC activity is less clear. Here we report that RhoA and K-Ras activate ENaC through independent signaling pathways and final mechanisms of action. Activation of RhoA signaling rapidly increases the membrane levels of ENaC likely by promoting channel insertion. This process dramatically increases functional ENaC current, resulting in tight spatial-temporal control of these channels. RhoA signals to ENaC via a transduction pathway, including the downstream effectors Rho kinase and phosphatidylinositol-4-phosphate 5-kinase. Phosphatidylinositol 4,5-biphosphate produced by activated phosphatidylinositol 4-phosphate 5-kinase may play a role in targeting vesicles containing ENaC to the plasma membrane.
The International Journal of Biochemistry Cell Biology, May 31, 2013
E. coli cytotoxic necrotizing factor 1 induces expression of the cell death-regulating GTPase Rho... more E. coli cytotoxic necrotizing factor 1 induces expression of the cell death-regulating GTPase RhoB.RhoB expression is based on transcriptional activation and involves Rac1-c-Myc signaling.RhoB suppresses cell cycle progression and cell death in CNF1-treated cells.RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB−/− mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB+/− MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.
TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Bo... more TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin co...
Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but... more Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.
The International Journal of Biochemistry & Cell Biology, 2013
RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediat... more RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB(-/-) mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB(+/-) MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.
Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucos... more Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h, a steady state was reached, with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.
Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical sur... more Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptosis. In this study, TcsL and K-Ras knock-down by siRNA are presented to result in expression of the cell death-regulating small GTPase RhoB. TcsL-induced RhoB expression is based on transcriptional activation involving p38(alpha) MAP kinase. Newly synthesized RhoB protein is rapidly degraded in a proteasome- and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a Rho family protein. Although often characterised as a pro-apoptotic protein, RhoB suppresses caspase-3 activation in TcsL-treated fibroblasts. The finding on the cytoprotective activity of RhoB in TcsL-treated cells re-enforces the concept that RhoB exhibits cytoprotective rather than pro-apoptotic activity in a cellular background of inactive Ras.
Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associ... more Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associated diarrhoea (CDAD). TcdA and TcdB mono-glucosylate small GTPases of the Rho family, thereby causing actin re-organisation in colonocytes, resulting in the loss of colonic barrier function. The hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) is an approved drug for the treatment of cholestasis and biliary cirrhosis. In this study, TUDCA-induced activation of Akt1 is presented to increase cellular levels of pS71-Rac1/Cdc42 in human hepatocarcinoma (HepG2) cells, showing for the first time that bile acid signalling affects the activity of Rho proteins. Rac1/Cdc42 phosphorylation, in turn, protects Rac1/Cdc42 from TcdB-catalysed glucosylation and reduces the TcdB-induced cytopathic effects in HepG2 cells. The results of this study indicate that TUDCA may prove useful as a therapeutic agent for the treatment of CDAD.
Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, inclu... more Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P(1-5)) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S1P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S1P, a phenomenon that correlated to the up-regulation of S1P1 and S1P3 in maturing DC. The same pattern of S1P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S1P could be severely hampered by application of the S1P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S1P-mediated signaling plays a pivotal role in the life cycle of DC.
The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the out... more The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy.
Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium d... more Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.
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Papers by Harald Genth