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Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Published on: December 30, 2016
gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A
1Division of Gastroenterology, Beth Israel Deaconess Medical Center, Dana 501, 330 Brookline Ave., Boston, MA 02215, USA.
Infection and Immunity
|April 16, 2008
Summary
Glycoprotein 96 (gp96) on human colonocytes binds Clostridium difficile toxin A (TxA). This interaction enhances toxin entry, promotes inflammatory signaling, and increases cytotoxicity, identifying gp96 as a key mediator in colitis.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Clostridium difficile toxin A (TxA) is a primary cause of antibiotic-associated colitis.
- TxA internalization by colonocytes necessitates binding to a cell surface receptor.
Purpose of the Study:
- To identify novel plasma membrane proteins on human colonocytes that bind TxA.
- To elucidate the role of these binding proteins in TxA-mediated cellular effects.
Main Methods:
- Biotinylation and cross-linking of TxA to HT29 human colonic epithelial cells.
- Coimmunoprecipitation and mass spectrometry to identify TxA binding proteins.
- Fluorescence immunostaining and in vitro assays to confirm binding and translocation.
- Small interfering RNA (siRNA) to reduce gp96 expression and assess cytotoxicity.
Main Results:
- Glycoprotein 96 (gp96) was identified as the primary TxA binding protein on colonocytes.
- TxA binding to gp96 facilitates translocation from the cell membrane to the cytoplasm.
- Antibody or siRNA targeting gp96 reduced TxA binding, cell rounding, and cytotoxicity.
- gp96 deficiency decreased TxA-induced inflammatory signaling (p38) and apoptosis (BAK activation, DNA fragmentation).
Conclusions:
- Human colonocyte gp96 functions as a plasma membrane receptor for Clostridium difficile toxin A.
- gp96 mediates TxA cellular entry, contributes to inflammatory signaling, and promotes cytotoxicity.
- Targeting gp96 may offer a therapeutic strategy against C. difficile-associated colitis.
