Time-resolved cellular effects induced by TcdA from Clostridium difficile.
BACKGROUND
The anaerobe Clostridium difficile is a common pathogen that causes infection of the colon leading to diarrhea or pseudomembranous colitis. Its major virulence factors are toxin A (TcdA) and toxin B (TcdB), which specifically inactivate small GTPases by glucosylation leading to reorganization of the cytoskeleton and finally to cell death. In the present work a quantitative proteome analysis using the isotope-coded protein label (ICPL) approach was conducted to investigate proteome changes in the colon cell line Caco-2 after treatment with recombinant wild-type TcdA (rTcdA-wt) or a glucosyltransferase-deficient mutant TcdA (rTcdA-mut).


METHODS
Proteins from crude cell lysates or cellular subfractions were identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Two time points (5 h, 24 h) of toxin treatment were analyzed and about 4000 proteins were identified in each case.


RESULTS
After 5 h treatment with rTcdA-wt, 150 proteins had a significantly altered abundance; rTcdA-mut caused regulation of 50 proteins at this time point. After 24 h treatment with rTcdA-wt changes in abundance of 61 proteins were observed, but no changes in protein abundance were detected after 24 h if cells were treated with rTcdA-mut. TcdA affected several proteins involved in signaling events, cytoskeleton and cell-cell contact organization, translation, and metabolic processes. The ICPL-dependent quantification was verified by label-free targeted MS techniques based on multiple reaction monitoring (MRM) and triple quadrupole mass spectrometry.


CONCLUSIONS
LC/MS-based proteome analyses and the ICPL approach revealed comprehensive and reproducible proteome date and provided new insights into the cellular effects of clostridial glucosylating toxins (CGT).
Hannover Medical School, Institute of Toxicology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Nelli
N
Jochim
Ralf
R
Gerhard
Ingo
I
Just
Andreas
A
Pich
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D018938:Caco-2 Cells; D002467:Cell Nucleus / Q000737:chemistry; D002853:Chromatography, Liquid; D003600:Cytosol / Q000737:chemistry; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D006801:Humans; D009154:Mutation; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000187:drug effects*; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D020543:Proteome; C041348:tcdA protein, Clostridium difficile