title: Toxin A of the nosocomial pathogen Clostridium difficile induces primary effects in the proteome of HEp-2 cells.
abstract: This study was carried out to investigate the impact of high concentrations of Clostridium difficile toxin A (TcdA) on the proteome of human cells. It should also be examined whether a catalytically deficient mutant (TcdANXN) has an effect on target cells.
  
  
  
  Proteome changes were investigated after treatment of HEp-2 cells with 20 nM TcdA for 8 h using a triplex SILAC labeling method and shotgun proteomics. Proteins from differently labeled and treated cells were combined for analysis using an HPLC coupled to an Orbitrap mass spectrometer.
  
  
  
  Nearly 4000 proteins were identified in each replicate and 3500 could be quantified by SILAC triplicate analysis. 51 proteins exhibited an altered abundance with 29 up-regulated and 22 down-regulated proteins. In contrast, TcdANXNhad no provable impact on the protein profile of HEp-2 cells. Data analysis of regulated proteins revealed that mainly plasma membrane, cell death, cell proliferation and actin cytoskeleton proteins were affected by TcdA treatment.
  
  
  
  This proteome analysis showed novel insights of TcdA impact onepithelial cells. Comparison with long-term treatment studies reveals distinctions in affected cellular processes that will improve the understanding of TcdA functions and might help to find new tools for diagnosis and treatment of CDI.
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Junemann
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Gurbet
authors.initials: G
authors.lastname: Birgin
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Jelena
authors.initials: J
authors.lastname: Erdmann
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Schröder
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.affiliation: Hannover Medical School, Institute of Toxicology, Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001427:Bacterial Toxins / Q000633:toxicity*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004768:Enterotoxins / Q000633:toxicity*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D020543:Proteome / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001427:Bacterial Toxins; D004768:Enterotoxins; D020543:Proteome; C041348:tcdA protein, Clostridium difficile
keywords: Clostridium difficile; SILAC; Shotgun proteomics; TcdA; TcdANXN