Truncation in the tcdC region of the Clostridium difficile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A.
BACKGROUND
The increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. To assess the role of hyper-production of Toxins A and B in clinical isolates of Clostridium difficile, two NAP1-related and five NAP1 non-related strains were compared.


METHODS
Sequencing was performed on tcdC, tcdR, and tcdE to determine if there were differences that might account for hyper-production of Toxin A and Toxin B in NAP1-related strains. Biological activity of Toxin B was evaluated using the HFF cell CPE assay and Toxin A biological activity was assessed using the Caco-2 Trans-membrane resistance assay.


RESULTS
Our results confirm that Toxin A and Toxin B production in NAP1-related strains and ATCC 43255 occurs earlier in the exponential growth phase compared to most NAP1-nonrelated clinical isolates. Despite the hyper-production observed in ATCC 43255 it had no mutations in tcdC, tcdR or tcdE. Analysis of the other clinical isolates indicated that the kinetics and ultimate final concentration of Toxin A and B did not correlate with the presence or lack of alterations in tcdC, tcdR or tcdE.


CONCLUSIONS
Our data do not support a direct role for alterations in the tcdC gene as a predictor of hyperproduction of Toxin A and B in NAP1-related strains.
Murray
Ruth
R
St. Boniface Research Centre, Winnipeg, MB, Canada. margaretmurray207@hotmail.com
Boyd
Dave
D
Levett
Paul N
PN
Mulvey
Michael R
MR
Alfa
Michelle J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D015373:Bacterial Typing Techniques; D018938:Caco-2 Cells; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D012097:Repressor Proteins; C105615:TcdC protein, Clostridium difficile; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile