Characterization of the role of tcdC in the production of toxins A and B in vitro by Clostridium difficile.

摘要

Clostridium difficile is the most common infectious cause of nosocomial antibiotic associated diarrhea. The more severe manifestations of C. difficile associated disease include psendomembranous colitis, toxic megacolon and death. Two large clostridial toxins, toxin A (enterotoxin) and toxin B (cytotoxin), are responsible for the pathogenesis of C. difficile. A third toxin, that shares homology to the clostridial family of binary toxins, has been identified in some strains of C. difficile. While the role the C. difficile binary toxin in disease is not clear, evidence suggests that C. difficile strains with binary toxin cause more severe disease. While the binary toxin has been identified in all NAP1 strains of C. diffiicile the role of this toxin in the pathogenesis of CDAD has yet to be fully explained. We amplified the binary toxin cdtB locus from 12.5% of all C. difficile isolates obtained between 2000 and 2001. Binary toxin was not amplified from the clinical isolates that caused the two outbreaks that occurred during this period. Our data indicates that the presence of the binary toxin alone is not the sole virulence factor that determines whether a C. difficile strain will cause a nosocomial outbreak or not.; Recent outbreaks of C. difficile in Quebec have been linked to a hypervirulent strain of C. difficile. The ability of this strain to cause outbreaks has been linked to an 18 by and 1 bp mutation in the open reading frame of the putative negative regulator of toxin production and to the presence of the binary toxin locus. The putative negative regulator of toxin production, TcdC, is expressed during early logarithmic growth. In our study we found that mutations to the tcdC gene were not a predictor of toxin hyper production in C. difficile broth culture for NAP1-related and non NAP1-related strains evaluated. Our data demonstrated that the NAP1-related strain produced increased levels of biologically active toxin B (4log10 cpeU/mL) in broth culture as compared to non-NAP1 clinical isolates (2log10 cpeU/mL). However, the hyper toxin producing ATCC strain produced higher levels of biologically active toxin B (5log10 cpeU/mL) as compared to the NAP1-related strain while a non-NAP1 strain with a truncated talC gene did not show increased toxin production in broth culture.; By evaluating stool from CDAD patients within 4 hours of disease diagnosis, we demonstrated a correlation between toxin B in the stools of patients with CDAD and toxin produced by corresponding clinical isolates in broth cultures.; A novel finding from our research was that there was an increase in the efficiency of sporulation, as measured by initiation and rate of spore conversion in broth culture for the NAP1-related strain when compared to a non-NAP1 strain. The combination of increased sporulation efficiency and increased toxin production in the NAP1-related strains may result in an increase in spread and pathogenesis in this strain.; In summary our data provide invaluable insight into the role of the TcdC protein in hyperproduction of toxin by the NAP1 strain and a novel aspect we demonstrated was the efficient sporulation ability of this strain. The data from our study furthers our understanding of the virulence of this unique strain of C. difficile.