Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.     

Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.     

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.     

Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalysed by Clostridium sordellii lethal toxin

Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptotic cell death. One yet unsolved problem in studies on TcsL is the lack of a method allowing the specific detection of (H/K/N)Ras glucosylation. In this study, we identify the Ras(Mab 27H5) antibody as a glucosylation-sensitive antibody capable for the immunoblot detection of (H/K/N)Ras glucosylation in TcsL-treated cells. Alternative Ras antibodies including the K-Ras(Mab F234) antibody or the v-H-Ras(Mab Y13-159) antibody recognize Ras proteins regardless of glucosylation. (H/K)Ras are further shown to be more efficaciously glucosylated by TcsL than Rac1 in rat basophilic leukemia cells as well as in a cell-free system.

Structured summary

MINT-7261742: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) H-RAS (uniprotkb:P01112) by enzymatic studies (MI:0415)MINT-7261729: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) Rac1 (uniprotkb:P63000) by enzymatic studies (MI:0415)MINT-7261772: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) K-RAS (uniprotkb:P01116) by enzymatic studies (MI:0415)MINT-7261784: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) N-RAS (uniprotkb:P01111) by enzymatic studies (MI:0415)     

High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types

Background

Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.

Results

Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages.

Conclusions

Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0361-4) contains supplementary material, which is available to authorized users.     

Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556). Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.     

Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry

During mitotic entry, the centrosomes provide a scaffold for initial activation of the CyclinB/Cdk1 complex, the mitotic kinase Aurora A, and the Aurora A-activating kinase p21-activated kinase (PAK). The activation of PAK at the centrosomes is yet regarded to happen independently of the Rho-GTPases Rac/Cdc42. In this study, Rac1 (but not RhoA or Cdc42) is presented to associate with the centrosomes from early G₂ phase until prometaphase in a cell cycle-dependent fashion, as evidenced by western blot analysis of prepared centrosomes and by immunolabeling. PAK associates with the G₂/M-phase centrosomes in a Rac1-dependent fashion. Furthermore, specific inhibition of Rac1 by C. difficile toxinB-catalyzed glucosylation or by knockout results in inhibited activation of PAK1/2, Aurora A, and the CyclinB/Cdk1 complex in late G₂ phase/prophase and delayed mitotic entry. Inhibition of PAK activation at late G₂-phase centrosomes caused by Rac1 inactivation coincides with impeded activation of Aurora A and the CyclinB/Cdk1 complex and delayed mitotic entry.     

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling     

CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.     

Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864.

Here we report data describing some principles of the interaction between small GTP-binding proteins and large Clostridial cytotoxins (LCTs). Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL-1522, TcdB-1470 and TcdB-8864. Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates. Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N-terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated. Within this stretch, three regions differ significantly in Rac1 and RhoA. Regions containing amino acids 41-45 and 50-54 had no effect on toxin induced glucosylation, whereas amino acids 22-27 had a drastic impact on the potential of all three toxins to covalently modify the GTPases. Point mutations K25T of RhoA (numbering according to Rac1) and K27A of Cdc42 significantly increased glucosylation by the cytotoxins; introduction of lysines at the equivalent positions of Rac1 hindered modification. Our experiments demonstrate the influence of this charged residue on GTPase-LCT interactions. Amino acids 22-27 are part of the transition between the alpha1-helix to the switch I region of small GTP-binding proteins; both are known structures for specificity determination of the interactions with physiologic partners. Comparing these structures with data from our investigation we suggest that TcsL-1522, TcdB-1470 and TcdB-8864 mimic aspects of the physiologic interactions of small GTP-binding proteins.     

The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1

The rapid migration of intestinal epithelial cells is important to the healing of mucosal ulcers and wounds. This cell migration requires the presence of polyamines and the activation of RhoA. RhoA activity, however, is not sufficient for migration because polyamine depletion inhibited the migration of IEC-6 cells expressing constitutively active RhoA. The current study examines the role of Rac1 and Cdc42 in cell migration and whether their activities are polyamine-dependent. Polyamine depletion with alpha-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of alpha-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vector-transfected cells, whereas cells expressing dominant negative Rac1 and Cdc42 migrated more slowly. Polyamine depletion had no effect on the migration of cells expressing Rac1 and only partially inhibited the migration of those expressing Cdc42. Although polyamine depletion caused the disappearance of actin stress fibers in cells transfected with empty vector, it had no effect on cells expressing Rac1. Constitutively active Rac1 increased RhoA and Cdc42 activity in both normal and polyamine-depleted cells. These results demonstrate that Rac1, RhoA, and Cdc42 are required for optimal epithelial cell migration and that Rac1 activity is sufficient for cell migration in the absence of polyamines due to its ability to activate RhoA and Cdc42 as well as its own effects on the process of cell migration. These data imply that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream from Rac1.     

Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells

Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. Here we report that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. We detected that stimulation of DCC receptors with netrin-1 resulted in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development.     

SPECs, small binding proteins for Cdc42

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.     

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.