Clostridiumnovyi's Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells.

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/ß-catenin pathway. However, the mechanism by which SMG alters the Wnt/ß-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/ß-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/ß-catenin and Wnt/ß-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/ß-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.

The Essential Role of Rac1 Glucosylation in Clostridioides difficile Toxin B-Induced Arrest of G1-S Transition.

Clostridioides difficile infection (CDI) in humans causes pseudomembranous colitis (PMC), which is a severe pathology characterized by a loss of epithelial barrier function and massive colonic inflammation. PMC has been attributed to the action of two large protein toxins, Toxin A (TcdA) and Toxin B (TcdB). TcdA and TcdB mono-O-glucosylate and thereby inactivate a broad spectrum of Rho GTPases and (in the case of TcdA) also some Ras GTPases. Rho/Ras GTPases promote G1-S transition through the activation of components of the ERK, AKT, and WNT signaling pathways. With regard to CDI pathology, TcdB is regarded of being capable of inhibiting colonic stem cell proliferation and colonic regeneration, which is likely causative for PMC. In particular, it is still unclear, the glucosylation of which substrate Rho-GTPase is critical for TcdB-induced arrest of G1-S transition. Exploiting SV40-immortalized mouse embryonic fibroblasts (MEFs) with deleted Rho subtype GTPases, evidence is provided that Rac1 (not Cdc42) positively regulates Cyclin D1, an essential factor of G1-S transition. TcdB-catalyzed Rac1 glucosylation results in Cyclin D1 suppression and arrested G1-S transition in MEFs and in human colonic epithelial cells (HCEC), Remarkably, Rac1-/- MEFs are insensitive to TcdB-induced arrest of G1-S transition, suggesting that TcdB arrests G1-S transition in a Rac1 glucosylation-dependent manner. Human intestinal organoids (HIOs) specifically expressed Cyclin D1 (neither Cyclin D2 nor Cyclin D3), which expression was suppressed upon TcdB treatment. In sum, Cyclin D1 expression in colonic cells seems to be regulated by Rho GTPases (most likely Rac1) and in turn seems to be susceptible to TcdB-induced suppression. With regard to PMC, toxin-catalyzed Rac1 glucosylation and subsequent G1-S arrest of colonic stem cells seems to be causative for decreased repair capacity of the colonic epithelium and delayed epithelial renewal.

Evaluation of Immunomodulatory Responses and Changed Wound Healing in Type 2 Diabetes-A Study Exploiting Dermal Fibroblasts from Diabetic and Non-Diabetic Human Donors.

The dermis is the connective layer between the epidermis and subcutis and harbours nerve endings, glands, blood vessels, and hair follicles. The most abundant cell type is the fibroblast. Dermal fibroblasts have a versatile portfolio of functions within the dermis that correspond with different types of cells by either direct contact or by autocrine and paracrine signalling. Diabetic skin is characterized by itching, numbness, ulcers, eczema, and other pathophysiological changes. These pathogenic phenotypes have been associated with the effects of the reactive glucose metabolite methylglyoxal (MGO) on dermal cells. In this study, dermal fibroblasts were isolated from diabetic and non-diabetic human donors. Cultured dermal fibroblasts from diabetic donors exhibited reduced insulin-induced glucose uptake and reduced expression of the insulin receptor. This diabetic phenotype persists under cell culture conditions. Secretion of IL-6 was increased in fibroblasts from diabetic donors. Increased secretion of IL-6 and MIF was also observed upon the treatment of dermal fibroblasts with MGO, suggesting that MGO is sufficient for triggering these immunomodulatory responses. Remarkably, MIF treatment resulted in decreased activity of MGO-detoxifying glyoxalase-1. Given that reduced glyoxalase activity results in increased MGO levels, these findings suggested a positive-feedback loop for MGO generation, in which MIF, evoked by MGO, in turn blocks MGO-degrading glyoxalase activity. Finally, secretion of procollagen Type I C-Peptide (PICP), a marker of collagen production, was reduced in fibroblast from diabetic donors. Remarkably, treatment of fibroblasts with either MGO or MIF was sufficient for inducing reduced PICP levels. The observations of this study unravel a signalling network in human dermal fibroblasts with the metabolite MGO being sufficient for inflammation and delayed wound healing, hallmarks of T2D.

Low Density Lipoprotein Receptor-Related Protein-1 (LRP1) Is Involved in the Uptake of Clostridioides difficile Toxin A and Serves as an Internalizing Receptor.

Toxin producing Clostridioides difficile strains cause gastrointestinal infections with the large glucosylating protein toxins A (TcdA) and B (TcdB) being major virulence factors responsible for the onset of symptoms. TcdA and TcdB enter their target cells via receptor-mediated endocytosis. Inside the cell, the toxins glucosylate and thereby inactivate small GTPases of the Rho-/Ras subfamilies resulting in actin reorganization and cell death. The receptors of TcdA are still elusive, glycoprotein 96 (gp96), the low density lipoprotein receptor family (LDLR) and sulfated glycosaminoglycans (sGAGs) have most recently been suggested as receptors for TcdA. In this study, we provide evidence on rapid endocytosis of Low density lipoprotein Receptor-related Protein-1 (LRP1) into fibroblasts and Caco-2 cells by exploiting biotinylation of cell surface proteins. In contrast, gp96 was not endocytosed either in the presence or absence of TcdA. The kinetics of internalization of TfR and LRP1 were comparable in the presence and the absence of TcdA, excluding that TcdA facilitates its internalization by triggering internalization of its receptors. Exploiting fibroblasts with a genetic deletion of LRP1, TcdA was about one order of magnitude less potent in LRP1-deficient cells as compared to the corresponding control cells. In contrast, TcdB exhibited a comparable potency in LRP1-proficient and -deficient fibroblasts. These findings suggested a role of LRP1 in the cellular uptake of TcdA but not of TcdB. Correspondingly, binding of TcdA to the cell surface of LRP1-deficient fibroblasts was reduced as compared with LRP1-proficient fibroblasts. Finally, TcdA bound to LRP1 ligand binding type repeat cluster II (amino acid 786-1,165) and cluster IV (amino acid 3332-3779). In conclusion, LRP1 appears to serve as an endocytic receptor and gp96 as a non-endocytic receptor for TcdA.

Expression and (Lacking) Internalization of the Cell Surface Receptors of Clostridioides difficile Toxin B.

Toxin-producing strains of Clostridioides difficile and Clostridium perfringens cause infections of the gastrointestinal tract in humans and ruminants, with the toxins being major virulence factors, essential for the infection, and responsible for the onset of severe symptoms. C. difficile toxin A (TcdA) and toxin B (TcdB), and the large cytotoxin (TpeL) from C. perfringens are single chain bacterial protein toxins with an AB-like toxin structure. The C-terminal delivery domain mediates cell entry of the N-terminal glycosyltransferase domain by receptor-mediated endocytosis. Several cell surface proteins have been proposed to serve as toxin receptors, including chondroitin-sulfate proteoglycan 4 (CSPG4), poliovirus receptor-like 3 (PVRL3), and frizzled-1/2/7 (FZD1/2/7) for TcdB and LDL-receptor-related protein-1 (LRP1) for TpeL. The expression of the TcdB receptors was investigated in human intestinal organoids (HIOs) and in cultured cell lines. HIOs from four human donors exhibited a comparable profile of receptor expression, with PVRL3, LRP1, and FZD7 being expressed and CSPG4 and FZD2 not being expressed. In human epithelial Caco-2 cells and HT29 cells as well as in immortalized murine fibroblasts, either receptor FZD2/7, CSPG4, PVRL3, and LRP1 was expressed. The question whether the toxins take advantage of the normal turnover of their receptors (i.e., constitutive endocytosis and recycling) from the cell surface or whether the toxins activity induce the internalization of their receptors has not yet been addressed. For the analysis of receptor internalization, temperature-induced uptake of biotinylated toxin receptors into immortalized mouse embryonic fibroblasts (MEFs) and Caco-2 cells was exploited. Solely LRP1 exhibited constitutive endocytosis from the plasma membrane to the endosome, which might be abused by TpeL (and possibly TcdB as well) for cell entry. Furthermore, internalization of CSPG4, PVRL3, FZD2, and FZD7 was observed neither in MEFs nor in Caco-2 cells. FZD2/7, CSPG4, and PVRL3 did thus exhibit no constitutive recycling. The presence of TcdB and the p38 activation induced by anisomycin were not able to induce or enhance CSPG4 or PVRL3 uptake in MEFs. In conclusion, FZD2/7, CSPG4, and PVRL3 seem to serve as cell surface binding receptors rather than internalizing receptors of TcdB.

Simulated Microgravity Reduces Focal Adhesions and Alters Cytoskeleton and Nuclear Positioning Leading to Enhanced Apoptosis via Suppressing FAK/RhoA-Mediated mTORC1/NF-κB and ERK1/2 Pathways.

Simulated-microgravity (SMG) promotes cell-apoptosis. We demonstrated that SMG inhibited cell proliferation/metastasis via FAK/RhoA-regulated mTORC1 pathway. Since mTORC1, NF-κB, and ERK1/2 signaling are important in cell apoptosis, we examined whether SMG-enhanced apoptosis is regulated via these signals controlled by FAK/RhoA in BL6-10 melanoma cells under clinostat-modelled SMG-condition. We show that SMG promotes cell-apoptosis, alters cytoskeleton, reduces focal adhesions (FAs), and suppresses FAK/RhoA signaling. SMG down-regulates expression of mTORC1-related Raptor, pS6K, pEIF4E, pNF-κB, and pNF-κB-regulated Bcl2, and induces relocalization of pNF-κB from the nucleus to the cytoplasm. In addition, SMG also inhibits expression of nuclear envelope proteins (NEPs) lamin-A, emerin, sun1, and nesprin-3, which control nuclear positioning, and suppresses nuclear positioning-regulated pERK1/2 signaling. Moreover, rapamycin, the mTORC1 inhibitor, also enhances apoptosis in cells under 1 g condition via suppressing the mTORC1/NF-κB pathway. Furthermore, the FAK/RhoA activator, toxin cytotoxic necrotizing factor-1 (CNF1), reduces cell apoptosis, restores the cytoskeleton, FAs, NEPs, and nuclear positioning, and converts all of the above SMG-induced changes in molecular signaling in cells under SMG. Therefore, our data demonstrate that SMG reduces FAs and alters the cytoskeleton and nuclear positioning, leading to enhanced cell apoptosis via suppressing the FAK/RhoA-regulated mTORC1/NF-κB and ERK1/2 pathways. The FAK/RhoA regulatory network may, thus, become a new target for the development of novel therapeutics for humans under spaceflight conditions with stressed physiological challenges, and for other human diseases.

Simulated microgravity inhibits cell focal adhesions leading to reduced melanoma cell proliferation and metastasis via FAK/RhoA-regulated mTORC1 and AMPK pathways.

Simulated microgravity (SMG) was reported to affect tumor cell proliferation and metastasis. However, the underlying mechanism is elusive. In this study, we demonstrate that clinostat-modelled SMG reduces BL6-10 melanoma cell proliferation, adhesion and invasiveness in vitro and decreases tumor lung metastasis in vivo. It down-regulates metastasis-related integrin α6ß4, MMP9 and Met72 molecules. SMG significantly reduces formation of focal adhesions and activation of focal adhesion kinase (FAK) and Rho family proteins (RhoA, Rac1 and Cdc42) and of mTORC1 kinase, but activates AMPK and ULK1 kinases. We demonstrate that SMG inhibits NADH induction and glycolysis, but induces mitochondrial biogenesis. Interestingly, administration of a RhoA activator, the cytotoxic necrotizing factor-1 (CNF1) effectively converts SMG-triggered alterations and effects on mitochondria biogenesis or glycolysis. CNF1 also converts the SMG-altered cell proliferation and tumor metastasis. In contrast, mTORC inhibitor, rapamycin, produces opposite responses and mimics SMG-induced effects in cells at normal gravity. Taken together, our observations indicate that SMG inhibits focal adhesions, leading to inhibition of signaling FAK and RhoA, and the mTORC1 pathway, which results in activation of the AMPK pathway and reduced melanoma cell proliferation and metastasis. Overall, our findings shed a new light on effects of microgravity on cell biology and human health.

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018.

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis.

Large clostridial toxins mono-O-glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [14 C]glucosylation, sequential [32 P]ADP-ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS-based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo-2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R-Ras2.

Impaired glyoxalase activity is associated with reduced expression of neurotrophic factors and pro-inflammatory processes in diabetic skin cells.

Patients suffering from type II diabetes develop several skin manifestations including cutaneous infections, diabetic dermopathy, diabetic bullae and acanthosis nigricans. Diabetic micro- and macroangiopathy as well as diabetic neuropathy are believed to play a crucial role in the development of diabetic skin disorders. A reduced cutaneous nerve fibre density was reported in diabetic subjects, which subsequently leads to impaired sensory nerve functions. Using an innervated skin model, we investigated the impact of human diabetic dermal fibroblasts and keratinocytes on porcine sensory neurons. Diabetic skin cells showed a reduced capacity to induce neurite outgrowth due to a decreased support with neurotrophic factors, such as NGF. Furthermore, diabetic keratinocytes displayed insulin resistance and increased expression of pro-inflammatory cytokines demonstrating the persistent effect of diabetes mellitus on human skin cells. Dysregulations were related to a significantly reduced glyoxalase enzyme activity in diabetic keratinocytes as experimentally reduced glyoxalase activity mimicked the increase in pro-inflammatory cytokine expression and reduction in NGF. Our results demonstrate an impaired crosstalk of diabetic skin cells and sensory neurons favouring hypo-innervation. We suggest that reduced methylglyoxal detoxification contributes to an impaired neurocutaneous interaction in diabetic skin.

Role of p38alpha/beta MAP Kinase in Cell Susceptibility to Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B.

Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38alpha-/- MSCV empty vector MEFs and the corresponding cell line with reconstituted p38alpha expression (p38alpha-/- MSCV p38alpha MEFs). Genetic deletion of p38alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38-/- MSCV empty vector MEFs and in p38alpha-/- MSCV p38alpha MEFs, suggesting that inhibition of p38beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38alpha deletion. In conclusion, the protective effects of SB203580 and of p38alpha deletion are likely not based on inhibition of the toxins' glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells.

Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B.

Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the "Large clostridial glycosylating toxins." These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB-together with Toxin A (TcdA)-is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn(2+) > Co(2+) > Mg(2+) >> Ca(2+), Cu(2+), Zn(2+). TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn(2+) and 180 µM for Mg(2+). TcsL and TcdB further require co-stimulation by monovalent K⁺ (not by Na⁺). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K⁺ and Mg(2+) (rather than Mn(2+)) in mammalian target cells.

DXD motif-dependent and -independent effects of the chlamydia trachomatis cytotoxin CT166.

The Gram-negative, intracellular bacterium Chlamydia trachomatis causes acute and chronic urogenital tract infection, potentially leading to infertility and ectopic pregnancy. The only partially characterized cytotoxin CT166 of serovar D exhibits a DXD motif, which is important for the enzymatic activity of many bacterial and mammalian type A glycosyltransferases, leading to the hypothesis that CT166 possess glycosyltransferase activity. CT166-expressing HeLa cells exhibit actin reorganization, including cell rounding, which has been attributed to the inhibition of the Rho-GTPases Rac/Cdc42. Exploiting the glycosylation-sensitive Ras(27H5) antibody, we here show that CT166 induces an epitope change in Ras, resulting in inhibited ERK and PI3K signaling and delayed cell cycle progression. Consistent with the hypothesis that these effects strictly depend on the DXD motif, CT166 with the mutated DXD motif causes neither Ras-ERK inhibition nor delayed cell cycle progression. In contrast, CT166 with the mutated DXD motif is still capable of inhibiting cell migration, suggesting that CT166 with the mutated DXD motif cannot be regarded as inactive in any case. Taken together, CT166 affects various fundamental cellular processes, strongly suggesting its importance for the intracellular survival of chlamydia.

UV radiation induces CXCL5 expression in human skin.

CXCL5 has recently been identified as a mediator of UVB-induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose-dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non-immune skin cells also contribute to UV-induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte-keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV-radiated human skin and the essential role of fibroblast-keratinocyte interaction in the regulation of epidermal CXCL5.

Yersinia enterocolitica exploits different pathways to accomplish adhesion and toxin injection into host cells.

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 ß1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to ß1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on ß1 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 ß1 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.

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins.

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.

Pyknotic cell death induced by Clostridium difficile TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity.

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 condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.

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.

The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases.

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.