title: Overexpression of the Endosomal Anion/Proton Exchanger ClC-5 Increases Cell Susceptibility towardClostridium difficileToxins TcdA and TcdB.
abstract: VirulentC. difficiletoxins 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 ofC. difficiletoxins. 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 ofC. difficiletoxins 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 virulenceC. difficiletoxins. 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.
authors.affiliation: Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
authors.forename: Frederike
authors.initials: F
authors.lastname: Ruhe
authors.affiliation: Institute for Toxicology, Hannover Medical School Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Olling
authors.affiliation: Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Abromeit
authors.affiliation: Institute for Toxicology, Hannover Medical School Hannover, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Rataj
authors.affiliation: Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Grieschat
authors.affiliation: Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
authors.forename: Andre
authors.initials: A
authors.lastname: Zeug
authors.affiliation: Institute for Toxicology, Hannover Medical School Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.affiliation: Institute for Neurophysiology, Hannover Medical School Hannover, Germany.
authors.forename: Alexi
authors.initials: A
authors.lastname: Alekov
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001896:Boron Compounds; D002460:Cell Line; D002470:Cell Survival; D018118:Chloride Channels / Q000378:metabolism*; D016360:Clostridioides difficile / Q000472:pathogenicity*; D004768:Enterotoxins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001896:Boron Compounds; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; C057908:trimethylaminocarboxyldihydroboran
keywords: C. difficile; CLC proteins; CLC transport; ClC-5; TcdA; TcdB; endocytosis; endosomal acidification