Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDIReport as inadecuate




Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI - Download this document for free, or read online. Document in PDF available to download.

Proteome Science

, 9:48

First Online: 17 August 2011Received: 31 May 2011Accepted: 17 August 2011

Abstract

BackgroundThe 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.

ResultsAltogether 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.

ConclusionOur 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.

KeywordsC. difficile-associated diarrhea colonic cells ICPL™ relative quantification Toxin A AbbreviationsCDAD C. difficile-associated diarrhea

rTcdA recombinant toxin A

ICPL™ isotope-coded protein labeling.

Electronic supplementary materialThe online version of this article doi:10.1186-1477-5956-9-48 contains supplementary material, which is available to authorized users.

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Author: Nelli Jochim - Ralf Gerhard - Ingo Just - Andreas Pich

Source: https://link.springer.com/







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