Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

Clostridium difficile is a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenic C. difficile strains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and in-flammation. C. difficile toxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdEmutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoformmay play a specific role in TcdE-controlled toxin release. We created plasmid constructs that ex-press only one of the three TcdE isoforms and complemented the TcdEmutant with these isoforms.Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166 was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms on C. difficile cell via-bility and toxin release were determined. Among the three isoforms, overexpression of TcdE166 and TcdE142 had a profound ef-fect on cell viability compared to the TcdE140 isoform. Similarly, TcdE166 and TcdE142 facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed. IMPORTANC