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Inhibition of Clostridioides difficile Toxins TcdA and TcdB by Ambroxol.

ABSTRACT: Clostridioides (C.) difficile produces the exotoxins TcdA and TcdB, which are the predominant virulence factors causing C. difficile associated disease (CDAD). TcdA and TcdB bind to target cells and are internalized via receptor-mediated endocytosis. Translocation of the toxins' enzyme subunits from early endosomes into the cytosol depends on acidification of endosomal vesicles, which is a prerequisite for the formation of transmembrane channels. The enzyme subunits of the toxins translocate into the cytosol via these channels where they are released after auto-proteolytic cleavage. Once in the cytosol, both toxins target small GTPases of the Rho/Ras-family and inactivate them by mono-glucosylation. This in turn interferes with actin-dependent processes and ultimately leads to the breakdown of the intestinal epithelial barrier and inflammation. So far, therapeutic approaches to treat CDAD are insufficient, since conventional antibiotic therapy does not target the bacterial protein toxins, which are the causative agents for the clinical symptoms. Thus, directly targeting the exotoxins represents a promising approach for the treatment of CDAD. Lately, it was shown that ambroxol (Ax) prevents acidification of intracellular organelles. Therefore, we investigated the effect of Ax on the cytotoxic activities of TcdA and TcdB. Ax significantly reduced toxin-induced morphological changes as well as the glucosylation of Rac1 upon intoxication with TcdA and TcdB. Most surprisingly, Ax, independent of its effects on endosomal acidification, decreased the toxins' intracellular enzyme activity, which is mediated by a catalytic glucosyltransferase domain. Considering its undoubted safety profile, Ax might be taken into account as therapeutic option in the context of CDAD.

SUBMITTER: Heber S

PROVIDER: S-EPMC8764291 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Virulent C. difficile toxins TcdA and TcdB invade host intestinal epithelia by endocytosis and use the acidic environment of intracellular vesicles for further processing and activation. We investigated the role of ClC-5, a chloride/proton exchanger expressed in the endosomes of gastrointestinal epithelial cells, in the activation and processing of C. difficile toxins. Enhanced intoxication by TcdA and TcdB was observed in cells expressing ClC-5 but not ClC-4, another chloride/proton exchanger with similar function but different localization. In accordance with the established physiological function of ClC-5, its expression lowered the endosomal pH in HEK293T cells by approximately 0.6 units and enhanced approximately 5-fold the internalization of TcdA. In colon HT29 cells, 34% of internalized TcdA localized to ClC-5-containing vesicles defined by colocalization with Rab5, Rab4a, and Rab7 as early and early-to-late of endosomes but not as Rab11-containing recycling endosomes. Impairing the cellular uptake of TcdA by deleting the toxin CROPs domain did not abolish the effects of ClC-5. In addition, the transport-incompetent mutant ClC-5 E268Q similarly enhanced both endosomal acidification and intoxication by TcdA but facilitated the internalization of the toxin to a lower extent. These data suggest that ClC-5 enhances the cytotoxic action of C. difficile toxins by accelerating the acidification and maturation of vesicles of the early and early-to-late endosomal system. The dispensable role of electrogenic ion transport suggests that the voltage-dependent nonlinear capacitances of mammalian CLC transporters serve important physiological functions. Our data shed light on the intersection between the endocytotic cascade of host epithelial cells and the internalization pathway of the large virulence C. difficile toxins. Identifying ClC-5 as a potential specific host ion transporter hijacked by toxins produced by pathogenic bacteria widens the horizon of possibilities for novel therapies of life-threatening gastrointestinal infections.

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Inhibition of Clostridioides difficile Toxins TcdA and TcdB by Ambroxol.

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