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 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 Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab

2014 ◽  
Vol 83 (2) ◽  
pp. 822-831 ◽  
Author(s):  
Zhiyong Yang ◽  
Jeremy Ramsey ◽  
Therwa Hamza ◽  
Yongrong Zhang ◽  
Shan Li ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Standard Of Care ◽  
Disease Recurrence ◽  
Health Care Facilities ◽  
High Rate ◽  
Gastrointestinal Infections ◽  
Disease Symptoms ◽  
Content Type ◽  
Mechanistic Basis ◽  
Developed World

Clostridium difficileinfection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated withC. difficilein these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

scholarly journals Clostridium difficile Toxin A Attenuates Wnt/β-Catenin Signaling in Intestinal Epithelial Cells

2014 ◽  
Vol 82 (7) ◽  
pp. 2680-2687 ◽  
Author(s):  
Bruno Bezerra Lima ◽  
Bárbara Faria Fonseca ◽  
Nathália da Graça Amado ◽  
Débora Moreira Lima ◽  
Ronaldo Albuquerque Ribeiro ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Rho Gtpases ◽  
Small Gtpases ◽  
Host Cells ◽  
Reporter Assay ◽  
Content Type ◽  
Protein Levels ◽  
Dependent Inhibition

ABSTRACTClostridium difficiletoxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/β-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of β-catenin and Western blotting for β-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the β-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/β-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3β inhibitor), or constitutively active β-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.

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

scholarly journals Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

2015 ◽  
Vol 84 (2) ◽  
pp. 395-406 ◽  
Author(s):  
Kasper Krogh Andersen ◽  
Nika M. Strokappe ◽  
Anna Hultberg ◽  
Kai Truusalu ◽  
Imbi Smidt ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Neutralizing Antibody ◽  
Treated Group ◽  
Western World ◽  
Hamster Model ◽  
Toxin B ◽  
Content Type ◽  
Vhh Antibodies ◽  
Clostridium Difficile Toxin B

Clostridium difficileis the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors ofC. difficileare two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineeredLactobacillusstrains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdBin vitrowere isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form inLactobacillus paracaseiBL23, they were able to neutralize the cytotoxic effect of the toxin in anin vitrocell-based assay. Prophylactic treatment with a combination of two strains of engineeredL. paracaseiBL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA−TcdB+strain ofC. difficile(P< 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized withC. difficilefor up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

scholarly journals Effective Sequestration of Clostridium difficile Protein Toxins by Calcium Aluminosilicate

2015 ◽  
Vol 59 (12) ◽  
pp. 7178-7183 ◽  
Author(s):  
Joseph M. Sturino ◽  
Karina Pokusaeva ◽  
Robert Carpenter
Keyword(s):  
Clostridium Difficile ◽  
Preclinical Study ◽  
Etiologic Agent ◽  
Toxin B ◽  
Content Type ◽  
Antibiotic Associated Diarrhea ◽  
Calcium Aluminosilicate ◽  
Protein Toxins ◽  
Novel Drug

ABSTRACTClostridium difficileis a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible forC. difficileinfection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors forClostridium difficilepathogenesis. Given the toxin-centric mechanism by whichC. difficilepathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treatC. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdBin vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic- or chemotherapy-inducedC. difficileinfection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB.

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