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.

scholarly journals Upregulation of the Host SLC11A1 Gene by Clostridium difficile Toxin B Facilitates Glucosylation of Rho GTPases and Enhances Toxin Lethality

2013 ◽  
Vol 81 (8) ◽  
pp. 2724-2732 ◽  
Author(s):  
Yanan Feng ◽  
Stanley N. Cohen
Keyword(s):  
Clostridium Difficile ◽  
Rho Gtpases ◽  
Rna Binding ◽  
Divalent Cations ◽  
Bacterial Toxin ◽  
Content Type ◽  
Small Rho Gtpases ◽  
Protein Toxins ◽  
Mechanistic Basis ◽  
Clostridium Difficile Toxin B

ABSTRACTPseudomembranous enterocolitis associated withClostridium difficileinfection is an important cause of morbidity and mortality in patients being treated with antibiotics. Two closely related large protein toxins produced byC. difficile, TcdA and TcdB, which act identically but at different efficiencies to glucosylate low-molecular-weight Rho GTPases, underlie the microbe's pathogenicity. Using antisense RNA encoded by a library of human expressed sequence tags (ESTs), we randomly inactivated host chromosomal genes in HeLa cells and isolated clones that survived exposure to ordinarily lethal doses of TcdB. This phenotypic screening and subsequent analysis identified solute carrier family 11 member 1 (SLC11A1; formerly NRAMP1), a divalent cation transporter crucial to host defense against certain microbes, as an enhancer of TcdB lethality. Whereas SLC11A1 normally is poorly expressed in human cells of nonmyeloid lineage, TcdB increased SLC11A1 mRNA abundance in such cells through the actions of the RNA-binding protein HuR. We show that short hairpin RNA (shRNA) directed against SLC11A1 reduced TcdB glucosylation of small Rho GTPases and, consequently, toxin lethality. Consistent with the previously known role of SLC11A1 in cation transport, these effects were enhanced by elevation of Mn2+in media; conversely, they were decreased by treatment with a chelator of divalent cations. Our findings reveal an unsuspected role for SLC11A1 in determiningC. difficilepathogenicity, demonstrate the novel ability of a bacterial toxin to increase its cytotoxicity, establish a mechanistic basis for these effects, and suggest a therapeutic approach to mitigate cell killing byC. difficiletoxins A and B.

scholarly journals Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors

2010 ◽  
Vol 78 (4) ◽  
pp. 1417-1425 ◽  
Author(s):  
Richard Bulgin ◽  
Benoit Raymond ◽  
James A. Garnett ◽  
Gad Frankel ◽  
Valerie F. Crepin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rho Gtpases ◽  
Shigella Flexneri ◽  
Small Gtpases ◽  
Effector Proteins ◽  
Actin Dynamics ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
T3ss Effector

ABSTRACT Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

Pyrrolo-1,5-benzoxazepines: a new class of apoptotic agents

2001 ◽  
Vol 29 (6) ◽  
pp. 704-706 ◽  
Author(s):  
D. M. Zisterer ◽  
M. M. McGee ◽  
G. Campiani ◽  
A. Ramunno ◽  
C. Fattorusso ◽  
...  
Keyword(s):  
Cellular Response ◽  
Caspase 3 ◽  
Signal Transduction Pathway ◽  
Benzodiazepine Receptor ◽  
Cell Types ◽  
Dependent Manner ◽  
Apoptotic Pathway ◽  
Leukaemia Cell Line ◽  
Apoptotic Activity ◽  
Induced Apoptosis

Some members of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) potently induce apoptosis in a number of human cancerous cell lines including HL-60 cells and the drug-resistant chronic myelogenous leukaemia cell line, K562. The apoptotic induction seems to be independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR), which binds these PBOXs with high affinity, due to a lack of correlation between their affinities for the receptor and their apoptotic potencies and their high apoptotic activity in PBR-deficient cells. PBOX-6, a potent member of the series, induces a transient activation of c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which correlates with induction of apoptosis. Expression of a cytoplasmic inhibitor of the JNK signal transduction pathway, Jip-1, prevents JNK activity and significantly reduces the extent of apoptosis induced by PBOX-6. This demonstrates the requirement for JNK in the cellular response to this apoptotic agent. In addition, PBOX-6 activates caspase-3-like proteases in K562 and HL-60 cells. The caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), blocks caspase-3-like protease activity in both cell types but only prevents PBOX-6-induced apoptosis in HL-60 cells, suggesting that the requirement for caspase-3-like proteases in the apoptotic pathway is dependent on the cell type.

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).

Rho protein inactivation induced apoptosis of cultured human endothelial cells

2002 ◽  
Vol 283 (4) ◽  
pp. L830-L838 ◽  
Author(s):  
Stefan Hippenstiel ◽  
Bernd Schmeck ◽  
Phillipe Dje N'Guessan ◽  
Joachim Seybold ◽  
Matthias Krüll ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Human Umbilical Cord ◽  
Caspase 9 ◽  
Toxin B ◽  
Protein Levels ◽  
Endothelial Apoptosis ◽  
Clostridium Difficile Toxin B ◽  
Induced Apoptosis ◽  
Camp Elevation

Small GTP-binding Rho GTPases regulate important signaling pathways in endothelial cells, but little is known about their role in endothelial cell apoptosis. Clostridial cytotoxins specifically inactivate GTPases by glucosylation [ Clostridium difficile toxin B-10463 (TcdB-10463), C. difficile toxin B-1470 (TcdB-1470)] or ADP ribosylation ( C. botulinum C3 toxin). Exposure of human umbilical cord vein endothelial cells (HUVEC) to TcdB-10463, which inhibits RhoA/Rac1/Cdc42, or to C3 toxin, which inhibits RhoA, -B, -C, resulted in apoptosis, whereas inactivation of Rac1/Cdc42 with TcdB-1470 was without effect, suggesting that Rho inhibition was responsible for endothelial apoptosis. Disruption of endothelial microfilaments as well as inhibition of p160ROCK did not induce endothelial apoptosis. Exposure to TcdB-10463 resulted in activation of caspase-9 and -3 but not caspase-8 in HUVEC. Moreover, Rho inhibition reduced expression of antiapoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid but had no effect on Bax or FLIP protein levels. Caspase-3 activity and apoptosis induced by TcdB-10463 were abolished by cAMP elevation. In summary, inhibition of Rho in endothelial cells activates caspase-9- and -3-dependent apoptosis, which can be antagonized by cAMP elevation.

Clostridium difficile Toxin B as a Probe for Rho GTPases

2008 ◽  
pp. 159-168 ◽  
Author(s):  
I. Just ◽  
J. Selzer ◽  
F. Hofmann ◽  
K. Aktories
Keyword(s):  
Clostridium Difficile ◽  
Rho Gtpases ◽  
Toxin B ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin B

The association between RhoB and caspase-2: changes with lovastatin-induced apoptosis

2005 ◽  
Vol 83 (5) ◽  
pp. 608-619 ◽  
Author(s):  
Jennifer Y Kong ◽  
Simon W Rabkin
Keyword(s):  
Clostridium Difficile ◽  
Reductase Inhibitor ◽  
Nuclear Fraction ◽  
Toxin B ◽  
Clostridium Difficile Toxin ◽  
Hmg Coa Reductase Inhibitor ◽  
Clostridium Difficile Toxin B ◽  
Caspase 2 ◽  
Induced Apoptosis ◽  
Coa Reductase

Because cytoskeletal actin is regulated, in part, by Rho, and because Rho and caspases are involved in apoptosis, we sought to determine whether there was an association between RhoB and caspase-2. A RhoB–caspase-2 association was consistently demonstrated in neonatal mouse cardiomyocytes with Western Blotting, either after im mun o precipitation with RhoB followed by immunoblotting with caspase-2, or in reciprocal experiments after immuno precipitation with caspase-2 and immunoblotting with RhoB (n = 14). Although the RhoB–caspase-2 complex was constitutively present, the link between RhoB and caspase-2 may be operative in apoptosis because the HMG-CoA reductase inhibitor lovastatin increased the RhoB–caspase complex, especially in the nuclear fraction of the cell, with a peak occurrence 2 h after treatment. This association was unaffected by the caspase-2 inhibitor zVDVAD. Lovastatin produced apoptosis that was accompanied by an activation of caspase-2, as demonstrated by its immunohistochemistry and by the fact that the caspase-2 inhibitor zVDVAD reduced lovastatin-induced apoptosis. Lovastatin induced dramatic changes in cell morphology and a reduction in F-actin. Immunoblotting for actin suggests that lovastatin does not induce a degradation of the actin molecule, but rather affects filamentous F-actin. Caspase-2 inhibition with zVDVAD reduced lovastatin-induced alteration in cytoskeletal F-actin. The Rho inhibitor, Clostridium difficile toxin B, blunted the ability of lovastatin to induce apoptosis. In summary, these data show a previously unrecognized association between RhoB and caspase-2 in the cytosolic and nuclear fractions, which has ramifications for processes regulated by RhoB and caspase-2, including apoptosis.Key words: actin, apoptosis, caspase-2, cardiomyocyte, heart, lovastatin.

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
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