TY - JOUR

T1 - Effects of Clostridium difficile Toxin A on the proteome of colonocytes studied by differential 2D electrophoresis

AU - Zeiser, Johannes J.

AU - Klodmann, Jennifer

AU - Braun, Hans Peter

AU - Gerhard, Ralf

AU - Just, Ingo

AU - Pich, Andreas

N1 - Funding information: This work was supported by a grant to AP and IJ from the Deutsche Forschungsgemeinschaft ( Pi278/2-2 ) and to RG by the Deutsche Forschungsgemeinschaft ( SFB621 , project B5). The authors thank Helma Tatge, Angelika Neumann and Dagmar Lewejohann for excellent technical assistance and Christina Rode for help with Delta2D.

PY - 2011/8/26

Y1 - 2011/8/26

N2 - Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain, multi-domain toxin A (TcdA) and B (TcdB). Their glucosyltransferase domain selectively inactivates Rho proteins leading to a reorganization of the cytoskeleton. To study exclusively glucosyltransferase-dependent molecular effects of TcdA, human colonic cells (Caco-2) were treated with recombinant wild type TcdA and the glucosyltransferase deficient variant of the toxin, TcdA gd for 24h. Changes in the protein pattern of the colonic cells were investigated by 2-D DIGE and LCMS/MS methodology combined with detailed proteome mapping. gdTcdA did not induce any detectable significant changes in the protein pattern. Comparing TcdA-treated cells with a control group revealed seven spots of higher and two of lower intensity (p<0.05). Three proteins are involved in the assembly of the cytoskeleton (β-actin, ezrin, and DPYL2) and four are involved in metabolism and/or oxidative stress response (ubiquitin, DHE3, MCCB, FABPL) and two in regulatory processes (FUBP1, AL1A1). These findings correlate well to known effects of TcdA like the reorganization of the cytoskeleton and stress the importance of Rho protein glucosylation for the pathogenic effects of TcdA.

AB - Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain, multi-domain toxin A (TcdA) and B (TcdB). Their glucosyltransferase domain selectively inactivates Rho proteins leading to a reorganization of the cytoskeleton. To study exclusively glucosyltransferase-dependent molecular effects of TcdA, human colonic cells (Caco-2) were treated with recombinant wild type TcdA and the glucosyltransferase deficient variant of the toxin, TcdA gd for 24h. Changes in the protein pattern of the colonic cells were investigated by 2-D DIGE and LCMS/MS methodology combined with detailed proteome mapping. gdTcdA did not induce any detectable significant changes in the protein pattern. Comparing TcdA-treated cells with a control group revealed seven spots of higher and two of lower intensity (p<0.05). Three proteins are involved in the assembly of the cytoskeleton (β-actin, ezrin, and DPYL2) and four are involved in metabolism and/or oxidative stress response (ubiquitin, DHE3, MCCB, FABPL) and two in regulatory processes (FUBP1, AL1A1). These findings correlate well to known effects of TcdA like the reorganization of the cytoskeleton and stress the importance of Rho protein glucosylation for the pathogenic effects of TcdA.

KW - 2D-DIGE

KW - Caco-2

KW - Clostridium difficile

KW - Proteome map

KW - Toxin A

UR - http://www.scopus.com/inward/record.url?scp=82355173202&partnerID=8YFLogxK

U2 - 10.1016/j.jprot.2011.08.012

DO - 10.1016/j.jprot.2011.08.012

M3 - Article

C2 - 21890007

AN - SCOPUS:82355173202

VL - 75

SP - 469

EP - 479

JO - Journal of proteomics

JF - Journal of proteomics

SN - 1874-3919

IS - 2

ER -