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

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.

Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

“…Thus, the degree of glucosylation was determined by the change in abundance of the glucosylation specific peptide normalized to the general GTPase regulation. 27 It is also in line with previous reports applying gdTcdA to investigate possible glucosyltransferase independent effects of TcdA. 44,45 After treatment with recombinant TcdA, 872 proteins exhibited a significant change in abundance (p < 0.05, foldchange >40%) compared to control and gdTcdA treatment, respectively.…”

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

“…Sample preparation and processing (liquid chromatography, LC) for mass spectrometry (MS) analysis was performed as described previously [52] with the exception that proteins were alkylated by addition of 2% acrylamide. Peptide samples were separated with a nano-flow ultra-high pressure liquid chromatography system (RSLC, Thermo Scientific).…”

Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients

“…The ICPL TM technique allows high-throughput quantitative proteome profiling on a global scale. It is used for protein profile comparison [21,22]. …”