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

Clostridium difficile is
the major cause of intestinal infections in hospitals. The major
virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong
to the group of clostridial glucosylating toxins (CGT) that inactivate
small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient
mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic
cells (Caco-2) were analyzed using high-resolution LC–MS/MS
and the SILAC technique. The changes in abundance of more than 5100
proteins were quantified. Nearly 800 toxin-responsive proteins were
identified that were involved in cell cycle, cell structure, and adhesion
as well as metabolic processes. Several proteins localized to mitochondria
or involved in lipid metabolism were consistently of higher abundance
after TcdA treatment. All changes of protein abundance depended on
the glucosyltransferase activity of TcdA. Glucosylation of the known
targets of TcdA such as RhoA, RhoC, RhoG was detected by LC–MS/MS.
In addition, an almost complete glucosylation of Rap1­(A/B), Rap2­(A/B/C)
and a partial glucosylation of Ral­(A/B) and (H/K/N)­Ras were detected.
The glucosylation pattern of TcdA was compared to that of other CGT
like TcdB, the variant TcdB from C. difficile strain
VPI 1470 (TcdBF), and lethal toxin from C. sordellii (TcsL).