Time-resolved cellular effects induced by TcdA from Clostridium difficile.

RATIONALE: 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).

Secretome analysis of Clostridium difficile strains.

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.

Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI.

BACKGROUND: The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI). RESULTS: Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results. CONCLUSION: Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation.