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  • br an anaerobic gram positive spore forming bacterium that

    2020-02-14


    , an MK-2048 gram-positive, spore-forming bacterium that can induce fatal intestinal inflammatory disease, is the most prevalent cause of antibiotic-associated diarrhea and pseudomembranous colitis in nosocomial settings. Two exotoxins, toxin A (TcdA) and toxin B (TcdB), secreted by the bacterium, are primarily responsible for the induction of disease because strains lacking both toxins are avirulent. TcdA and TcdB are large homologous single-chain proteins that contain at least 4 distinct domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a putative translocation domain, and a C-terminal receptor binding domain (RBD). Although the exact method of toxin entry into target cells remains elusive, a model of toxin action is emerging: after receptor-mediated endocytosis, which may involve regions in both the translocation domain and RBD,, the CPD and GTD eventually are translocated into the cytosol where cysteine protease self-cleaves and releases GTD from the rest of the toxin. GTD inactivates Rho guanosine triphosphatases, leading to the intoxication of host cells. Thus, CPD plays a key role in the cytosolic delivery of GTD effectors. Autoprocessing-deficient TcdA or TcdB mutants are still cytotoxic to host cells inducing cell rounding, Rho guanosine triphosphatase glucosylation, and apoptosis, but such activity is reduced compared with their wild-type toxins., infection (CDI) is typified by diarrhea, intestinal inflammation, and tissue damage, and in severe cases pseudomembranous colitis. Toxin-induced intestinal inflammation, a hallmark of CDI, is characterized by the production of proinflammatory cytokines/chemokines, neutrophil infiltration, and intestinal epithelial damage. Both TcdA and TcdB are proinflammatory; however, TcdB induces markedly weaker inflammatory responses than TcdA. TcdA elicited neutrophil influx, extensive tissue damage, and fluid accumulation within a short incubation period in various animal gut loop models., , , These acute enterotoxic responses, key characteristics of pseudomembranous colitis, thus defined TcdA as a major virulence factor in these earlier studies, although recent studies found that isogenic strains that express only TcdB induced more severe diseases in animals than those that express only TcdA., , In contrast, TcdB did not show such acute enterotoxicity in animal gut loop models, , , , , even though TcdB was a more potent cytotoxin than TcdA., In a more recent study using a cecum injection model, TcdA but not TcdB could induce rapid cecal inflammation. The precise mechanism controlling such differential inflammatory activities of the 2 toxins is unknown. In this study, we identified a previously unknown function of CPD as an internal regulator of the proinflammatory activity of toxins. By using a series of chimeric and enzyme-deficient holotoxins in a well-established mouse ileal loop model that clearly differentiates between responses to TcdA and to TcdB, we determined that CPD-mediated autoprocessing regulates the acute inflammatory responses to the 2 toxins. Moreover, we validated this result in human intestinal tissues and immune cells. Our finding thus provides new understanding of the molecular mechanism regulating toxin biological functions and insight into the pathogenesis of CDI. Materials and Methods
    Results
    Discussion The functions of the domains in regulating cytotoxicity of the 2 toxins have been studied extensively,6, 7, 22, 30, 31 however, the underlying mechanism that regulates the proinflammatory activity of TcdA and TcdB remains elusive. Earlier studies using ligated ileal loop models and more recent studies using cecum or intrarectal toxin instillation have consistently shown that TcdA, but not TcdB, induced rapid enterotoxic responses resembling some characteristics of human CDI in multiple animal models.10, 11, 12, 14, 19, 32, 33, 34, 35, 36 The molecular mechanism that governs such a differential response is unknown, but several reports have shown that the expression and distribution of toxin receptors on target cells37, 38 and host intestinal epithelia may account for the different responses of the host to the 2 toxins. However, in this study, we found that swapping TcdA’s RBD for that of TcdB did not quell inflammation, but, more importantly, a single point mutation in the CPD or in the cysteine autocleavage site of TcdB (TcdB-C698S and TcdB-L543A, respectively) significantly enhanced the toxin’s proinflammatory activity. Moreover, chemical inhibition of TcdB autoprocessing converted wild-type TcdB into a proinflammatory enterotoxin in a mouse ileal loop model. These results indicated that the receptor binding of the toxins to host intestinal epithelia did not differentiate the inflammatory activities of the toxins because these point mutations within CPD and the chemicals are unlikely to affect TcdB-receptor binding activity. In addition to receptor binding, we examined glucosyltransferase (GT) activity. TcdB possesses a more potent GT than that of TcdA, which contributes to its stronger cytotoxicity. The acute intestinal inflammatory responses induced by TcdA were dependent on the toxin’s GT activity because GT-deficient aTcdA failed to induce any measurable responses. However, the disparate potency in GT activity between the 2 toxins cannot explain their difference in proinflammatory activity because the TxA-Bgt and TxB-ACPD chimeras that harbor GTD from TcdB were enterotoxigenic. These data indicated that neither the receptor interaction of the toxins with host intestinal epithelia nor the GT activity is likely the major factor that differentiates toxin-induced inflammatory responses.