scholarly journals Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells

2008 ◽  
Vol 57 (6) ◽  
pp. 765-770 ◽  
Author(s):  
Ralf Gerhard ◽  
Stefanie Nottrott ◽  
Janett Schoentaube ◽  
Helma Tatge ◽  
Alexandra Olling ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Small Gtpases ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Single Chain ◽  
Toxin A ◽  
Induced Apoptosis ◽  
The Impact

The intestinal epithelial cell line HT-29 was used to study the apoptotic effect of Clostridium difficile toxin A (TcdA). TcdA is a 300 kDa single-chain protein, which glucosylates and thereby inactivates small GTPases of the Rho family (Rho, Rac and Cdc42). The effect of TcdA-catalysed glucosylation of the Rho GTPases is well known: reorganization of the actin cytoskeleton with accompanying morphological changes in cells, leading to complete rounding of cells and destruction of the intestinal barrier function. Less is known about the mechanism by which apoptosis is induced in TcdA-treated cells. In this study, TcdA induced the activation of caspase-3, -8 and -9. Apoptosis, as estimated by the DNA content of cells, started as early as 24 h after the addition of TcdA. The impact of Rho glucosylation was obvious when mutant TcdA with reduced or deficient glucosyltransferase activity was applied. TcdA mutant W101A, with 50-fold reduced glucosyltransferase activity, induced apoptosis only at an equipotent concentration compared with wild-type TcdA at a 50 % effective concentration of 0.2 nM. The enzyme-deficient mutant TcdA D285/287N was not able to induce apoptosis. Apoptosis induced by TcdA strictly depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Destruction of the actin cytoskeleton by latrunculin B was not sufficient to induce apoptosis, indicating that apoptosis induced by TcdA must be due to another mechanism. In summary, TcdA-induced apoptosis (cytotoxic effect) depends on the glucosylation of Rho GTPases, but is not triggered by destruction of the actin cytoskeleton (cytopathic effect).

Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases

APOPTOSIS ◽  
2007 ◽  
Vol 12 (8) ◽  
pp. 1443-1453 ◽  
Author(s):  
Stefanie Nottrott ◽  
Janett Schoentaube ◽  
Harald Genth ◽  
Ingo Just ◽  
Ralf Gerhard
Keyword(s):  
Clostridium Difficile ◽  
Rho Gtpases ◽  
Toxin A ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin A ◽  
Induced Apoptosis

scholarly journals Burkholderia pseudomallei as an Enteric Pathogen: Identification of Virulence Factors Mediating Gastrointestinal Infection

2020 ◽  
Vol 89 (1) ◽  
pp. e00654-20
Author(s):  
Javier I. Sanchez-Villamil ◽  
Daniel Tapia ◽  
Grace I. Borlee ◽  
Bradley R. Borlee ◽  
David H. Walker ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Burkholderia Pseudomallei ◽  
Enteric Pathogen ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Gastrointestinal Infection ◽  
Content Type ◽  
Route Of Infection ◽  
The Impact

ABSTRACTBurkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei.

scholarly journals Intrarectal Instillation of Clostridium difficile Toxin A Triggers Colonic Inflammation and Tissue Damage: Development of a Novel and Efficient Mouse Model of Clostridium difficile Toxin Exposure

2012 ◽  
Vol 80 (12) ◽  
pp. 4474-4484 ◽  
Author(s):  
Simon A. Hirota ◽  
Vadim Iablokov ◽  
Sarah E. Tulk ◽  
L. Patrick Schenck ◽  
Helen Becker ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Clostridium Difficile ◽  
Inflammatory Mediators ◽  
Tissue Damage ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial ◽  
Toxin A ◽  
Colonic Inflammation ◽  
Content Type ◽  
Clostridium Difficile Toxin

ABSTRACTClostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ ofC. difficileinfection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

scholarly journals ARHGAP25, a novel Rac GTPase-activating protein, regulates phagocytosis in human neutrophilic granulocytes

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Roland Csépányi-Kömi ◽  
Gábor Sirokmány ◽  
Miklós Geiszt ◽  
Erzsébet Ligeti
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Active Form ◽  
Cell Types ◽  
Small Gtpases ◽  
Negative Regulator ◽  
Phagocytic Cells ◽  
Rac Gtpase

Members of the Rac/Rho family of small GTPases play an essential role in phagocytic cells in organization of the actin cytoskeleton and production of toxic oxygen compounds. GTPase-activating proteins (GAPs) decrease the amount of the GTP-bound active form of small GTPases, and contribute to the control of biologic signals. The number of potential Rac/RhoGAPs largely exceeds the number of Rac/Rho GTPases and the expression profile, and their specific role in different cell types is largely unknown. In this study, we report for the first time the properties of full-length ARHGAP25 protein, and show that it is specifically expressed in hematopoietic cells, and acts as a RacGAP both in vitro and in vivo. By silencing and overexpressing the protein in neutrophil model cell lines (PLB-985 and CosPhoxFcγR, respectively) and in primary macrophages, we demonstrate that ARHGAP25 is a negative regulator of phagocytosis acting probably via modulation of the actin cytoskeleton.

scholarly journals Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton

2000 ◽  
Vol 355 (1399) ◽  
pp. 965-970 ◽  
Author(s):  
Alan Hall ◽  
Catherine D. Nobes
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Fundamental Property ◽  
Small Gtpases ◽  
Membrane Receptors ◽  
Embryonic Cells ◽  
Filamentous Actin ◽  
Rho Family ◽  
Plasma Membrane Receptors

The actin cytoskeleton plays a fundamental role in all eukaryotic cells—it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. W ork in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.

scholarly journals Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes

2016 ◽  
Vol 60 (6) ◽  
pp. 3519-3523 ◽  
Author(s):  
Bradley T. Endres ◽  
Eugénie Bassères ◽  
Mohammed Khaleduzzaman ◽  
M. Jahangir Alam ◽  
Laurent Chesnel ◽  
...  
Keyword(s):  
Immune Response ◽  
Clostridium Difficile ◽  
Morphological Changes ◽  
Toxin Production ◽  
Morphological Data ◽  
Growth Media ◽  
Toxin A ◽  
Content Type ◽  
Cyclic Lipopeptide ◽  
The Impact

Surotomycin is a cyclic lipopeptide in development forClostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure onC. difficiletoxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treatedC. difficilegrowth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference,P< 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P< 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a “deflated” phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects onC. difficilethat results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

scholarly journals The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1815-1823 ◽  
Author(s):  
Keith S. Robinson ◽  
Aurelie Mousnier ◽  
Cordula Hemrajani ◽  
Neil Fairweather ◽  
Cedric N. Berger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clostridium Difficile ◽  
Rho Gtpases ◽  
Nucleotide Exchange ◽  
Nuclear Condensation ◽  
Apoptotic Pathway ◽  
Small Rho Gtpases ◽  
Clostridium Difficile Toxin B ◽  
Apoptotic Activity ◽  
Induced Apoptosis

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction

2003 ◽  
Vol 284 (4) ◽  
pp. C1073-C1082 ◽  
Author(s):  
Tom S. Liu ◽  
Mark W. Musch ◽  
Kazunori Sugi ◽  
Margaret M. Walsh-Reitz ◽  
Mark J. Ropeleski ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Atp Release ◽  
Protective Role ◽  
Atp Depletion ◽  
Intestinal Epithelial ◽  
Toxin A ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin A

We determined whether the cytoprotective heat shock protein HSP72 protects against the injurious effects of Clostridium difficile toxin A (TxA) on intestinal epithelial cells. Colonic epithelial Caco-2/bbe (C2) cells were stably transfected with HSP72 antisense (C2AS) or vector only (C2VC), resulting in low and high HSP72 expression, respectively. Measurements of epithelial barrier integrity, mitochondrial function, and apoptosis activation were assessed after TxA exposure. HSP72 and RhoA interactions were evaluated with immunoprecipitations. In C2AS cells, TxA was associated with a greater decrease in transepithelial resistance (TER), an increase in [3H]mannitol flux, and increased dissociation of perijunctional actin. Although HSP72 binds RhoA, it failed to prevent RhoA glucosylation. TxA caused a more rapid decrease in ATP, release of cytochrome c, and activation of caspase-9 in C2AS cells. To determine whether ATP depletion decreases TER, we treated cells with antimycin A, which caused a decline in TER. We conclude that HSP72 may protect intestinal epithelial cells from TxA-mediated damage through several mechanisms, including actin stabilization, mitochondrial protection, and inhibition of apoptosis activation, but not by prevention of RhoA glucosylation.

Cell motility: can Rho GTPases and microtubules point the way?

2001 ◽  
Vol 114 (21) ◽  
pp. 3795-3803 ◽  
Author(s):  
Torsten Wittmann ◽  
Clare M. Waterman-Storer
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Cell Types ◽  
Small Gtpases ◽  
Cell Polarization ◽  
Microtubule Cytoskeleton ◽  
Filament Bundles

Migrating cells display a characteristic polarization of the actin cytoskeleton. Actin filaments polymerise in the protruding front of the cell whereas actin filament bundles contract in the cell body, which results in retraction of the cell’s rear. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42. Although the microtubule cytoskeleton is also polarized in a migrating cell, and microtubules are essential for the directed migration of many cell types, their role in cell motility is not well understood at a molecular level. Here, we discuss the potential molecular mechanisms for interplay of microtubules, actin and Rho GTPase signalling in cell polarization and motility. Recent evidence suggests that microtubules locally modulate the activity of Rho GTPases and, conversely, Rho GTPases might be responsible for the initial polarization of the microtubule cytoskeleton. Thus, microtubules might be part of a positive feedback mechanism that maintains the stable polarization of a directionally migrating cell.

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