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Low pH-induced formation of ion channels by clostridium difficile toxin B in target cells
H Barth1, G Pfeifer, F Hofmann
1Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
Abstract:
Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH
Insights
Clostridium difficile toxin B enters cells via endosomal acidification, forming channels at low pH. This mechanism, involving structural changes and membrane insertion, is key for large clostridial cytotoxin activity.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Clostridium difficile toxin B (TcdB) causes antibiotic-associated diarrhea and pseudomembranous colitis.
- TcdB inactivates Rho GTPases through glucosylation, disrupting cellular functions.
- Understanding TcdB's cellular uptake and membrane interaction is crucial for developing interventions.
Purpose of the Study:
- To investigate the mechanism of TcdB uptake into eukaryotic cells.
- To elucidate the role of endosomal pH in TcdB-mediated cell damage.
- To determine the structural basis for TcdB's membrane interaction and channel formation.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells to study TcdB uptake.
- Employed bafilomycin A1 to inhibit endosomal acidification and assess its effect on TcdB uptake.
- Measured (86)Rb(+) release from preloaded cells to quantify membrane permeability changes.
- Investigated TcdB's effect on artificial lipid bilayer membranes.
- Used antibodies against TcdB holotoxin and its enzyme domain.
Main Results:
- Bafilomycin A1 blocked TcdB uptake, indicating dependence on endosomal acidification.
- Extracellular acidification (pH ≤ 5.2) induced TcdB uptake into the cytosol.
- Low pH (≤ 5.6) triggered TcdB-induced (86)Rb(+) release, dependent on toxin concentration and pH.
- Antibodies against holotoxin prevented channel formation, while those against the enzyme domain had no effect.
- A truncated TcdB fragment (aa 547-2366) also induced pH-dependent (86)Rb(+) efflux.
- TcdB and Clostridium sordellii lethal toxin formed pH-dependent channels in artificial membranes.
Conclusions:
- TcdB uptake is dependent on endosomal acidification.
- Low pH induces structural changes in TcdB, leading to membrane insertion and channel formation.
- This pH-dependent membrane interaction and channel formation is a conserved mechanism among large clostridial cytotoxins.