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.

Exploring the inhibitory potential of the antiarrhythmic drug amiodarone against Clostridioides difficile toxins TcdA and TcdB

“…Castanospermine, a compound described as a potent inhibitor of the GTD of TcdB, was used as positive control in this assay. 14 , 24 Whereas 10 mM castanospermine showed, as expected, robust inhibition of the glucosyltransferase activity of TcdB (~50% reduction), the same approach with 30 and 100 µM amiodarone yielded no inhibition ( Figure 6b ).…”

“…The reaction was performed with UDP-glucose as co-substrate and with the GTPase Rac1 as substrate, which was recombinantly purified from Escherichia coli as described previously. 24 In brief, all reactions were performed for 1 h at 37°C in a total volume of 15 µl of glucosylation buffer (50 mM HEPES, 100 mM KCl, 2 mM MgCl 2 , 1 mM MnCl 2 , 100 mg/L BSA, pH 7.5), including 200 pM TcdB, 100 µM UDP-glucose, 5 µM Rac1, and, optionally, amiodarone (30 or 300 µM) or castanospermine (10 mM). Next, 10 µl of each reaction was transferred to a 96-well half-area microplate (Greiner, #675075, Austria).…”

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation

“…Native TcdB from C. difficile VPI 10,463 was expressed and purified as described earlier [ 48 ]. Rac1 was expressed and purified as a recombinant GST-tagged protein as described previously [ 49 ]. Native TcdA was obtained from List (Campbell, CA, USA).…”

Clostridioides difficile, a New “Superbug”

“…A newer option is blocking the toxins. Calcium aluminosilicate and human serum albumin have shown toxin-binding activity in vitro; some researchers report that aspirin has a good effect, and recent data has shown that human alpha-defensins and ambroxol have antitoxic activity [112,113]. The information for the important treatment options is shown in Table 1.…”