Systemic Dissemination of Clostridium difficile Toxins A and B Is Associated With Severe, Fatal Disease in Animal Models

Steele, Jennifer; Chen, Kevin; Sun, Xingmin; Zhang, Yongrong; Wang, Haiying; Tzipori, Saul; Feng, Hanping

doi:10.1093/infdis/jir748

Cited by 79 publications

(95 citation statements)

References 34 publications

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“…Systemic toxicity by TcdB requires functional glucosyltransferase activity. To investigate the pathophysiological validity of our in vitro toxin findings, we used a well-characterized systemic TcdB toxemia model (55,58) to test enzyme-deficient toxin mutants. Mice challenged intraperitoneally with mutant TcdB demonstrated significantly attenuated systemic virulence responses.…”

Section: Resultsmentioning

confidence: 99%

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Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

Shi

Yang

et al. 2015

Infect Immun

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TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V H H) library raised against TcdB, we identified two V H H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD 50 ) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy. C lostridium difficile is an anaerobic Gram-positive bacterial species that can induce serious and potentially fatal inflammatory disease of the colon and is the most prevalent cause of antibioticassociated diarrhea and pseudomembranous colitis in nosocomial settings (1, 2). Disease in patients with C. difficile infection is strongly associated with the two exotoxins, TcdA and TcdB (3). Both toxins are large, homologous single-chain proteins that contain at least four distinct domains (4-6): the N terminus glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a C terminus receptor binding domain (RBD; also known as combined repetitive oligopeptides, or CROPs). A recent study suggests that there might also be an additional receptor binding region besides the N-terminal CROP region (7) although the specific region has yet to be identified. Both toxins exert cytopathic effects that include cell rounding after disruption of the actin cytoskeleton and tight junctions in human colonocytes (8, 9). Toxin exposure may also trigger potent cytotoxic and inflammatory effects leading to mucosal cell death, diarrhea, and colitis associated with C. difficile infections (10, 11). TcdB appears to be more clinically relevant for C. difficile virulence as it is invariably associated with clinically isol...

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Section: Resultsmentioning

confidence: 99%

“…Because TcdB disseminates systemically during C. difficile infection and causes toxemia (55,61,62), we investigated systemic toxicity of wild-type and mutant toxins by challenging mice intraperitoneally with defined toxin concentrations. The TcdB LD 50 in mice is 20 ng (1 g kg Ϫ1 ), with 100 ng causing fulminant disease in all animals.…”

Section: Discussionmentioning

confidence: 99%

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

Shi

Yang

et al. 2015

Infect Immun

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“…Unlike the hamster model of CDI where animals consistently develop a fulminant and lethal disease course (40), the recently established mouse CDI model develops a wider range of disease symptoms that more accurately resemble major aspects of the human disease (7). In this study, we chose to challenge mice with doses of C. difficile that recapitulate the full clinical spectrum of disease symptoms ranging from a self-limiting diarrhea to ful-minant systemic disease (53). We also conducted confirmatory experiments in hamsters in accordance with previous studies that mainly use mortality as a single disease parameter for evaluation (28,43).…”

Section: Discussionmentioning

confidence: 99%

A Chimeric Toxin Vaccine Protects against Primary and Recurrent Clostridium difficile Infection

et al. 2012

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fThe global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

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“…In some cases, a fatal outcome occurs despite total colectomy and eradication of the organism (35,36), further suggesting systemic effects. TcdA and TcdB have been detected in the blood of animals experimentally infected with C. difficile (37) and in human cases of C. difficile infection (38). TcdB has been shown to be a cardiotoxin (39).…”

Section: Clinical Significancementioning

confidence: 99%

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

Hunt

Ballard

2013

Microbiol Mol Biol Rev

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SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile , now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade.

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Systemic Dissemination of Clostridium difficile Toxins A and B Is Associated With Severe, Fatal Disease in Animal Models

Abstract: Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.

Cited by 79 publications

References 34 publications

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

A Chimeric Toxin Vaccine Protects against Primary and Recurrent Clostridium difficile Infection

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

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