Overexpression of the Endosomal Anion/Proton Exchanger ClC-5 Increases Cell Susceptibility toward Clostridium difficile Toxins TcdA and TcdB

Abstract

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.

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate

“…EIPA-treated cells completely rounded up, which indicated only reduced uptake instead of complete abolishment, as it can be achieved by bafilomycin A. The role in toxin uptake of the chloride/ proton antiporter ClC-5, which directly acidifies endosomes, was reported before (Ruhe et al, 2017;Smith & Lippiat, 2010). All strategies gave results that were in line with our hypothesis of a Rac1dependent effect mediated by intracellular flush of toxin.…”

“…This was also proven for TcdB from strain R20291, where even the complete CROP domain except four amino acids was removed (TcdB R20291 1–1836 Figure S4). We recently observed that the ClC‐5 anion/proton exchanger makes cells more susceptible to TcdA and TcdB (Ruhe et al, ). ClC‐5 supports the v‐ATPase in acidification of endosomes and by generating a beneficial electrochemical gradient, thereby accelerating uptake and translocation of toxins (Hara‐Chikuma, Wang, Guggino, Guggino, & Verkman, ; Novarino, Weinert, Rickheit, & Jentsch, ; Piwon, Gunther, Schwake, Bosl, & Jentsch, ; Wang et al, ).…”

“…This was also proven for TcdB from strain R20291, where even the complete CROP domain except four amino acids was removed (TcdB R20291 1-1836 Figure S4). We recently observed that the ClC-5 anion/proton exchanger makes cells more susceptible to TcdA and TcdB (Ruhe et al, 2017 were more cytotoxic than their glucosyltransferase-proficient counterparts. The chimeras with trunk of toxin from hypervirulent strains were more cytotoxic than their counterparts from reference strain (mean ± SD, n = 8; p < 0.01).…”

Impairment of lysosomal function by Clostridioides difficile TcdB

“…Uptake into cells solely by frizzled 1,2,7 does not provoke this effect in HEp‐2 cells as shown in Figure by using TcdB1‐1832, which does not interact with CSPG4 (Henkel et al., 2020; Tao et al., 2016). Beside receptors, facilitation of acidification of the endosomal lumen by sodium and chloride flux also supports toxin translocation, thereby enhancing cytopathic and cytotoxic effects especially of TcdB (Beer et al., 2018a, 2018b; Ruhe et al., 2017). The early cell death can only be executed in the presence of signaling competent Rac1 and NADPH oxidase and is triggered by necrotic calcium signaling (Beer et al., 2018a, 2018b; Farrow et al., 2013, 2020).…”