title: Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene.
abstract: The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed.
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors: Alexandra
authors: A
authors: Olling
authors: Sophie
authors: S
authors: Seehase
authors: Nigel P
authors: NP
authors: Minton
authors: Helma
authors: H
authors: Tatge
authors: Saskia
authors: S
authors: Schröter
authors: Saskia
authors: S
authors: Kohlscheen
authors: Andreas
authors: A
authors: Pich
authors: Ingo
authors: I
authors: Just
authors: Ralf
authors: R
authors: Gerhard
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D006801:Humans; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C431549:TcdE protein, Clostridium difficile; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile