Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector.
Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.
Institute for Medical Microbiology and Epidemiology of Infectious Diseases, University of Leipzig, Liebigstrasse 24, 04103 Leipzig, Germany. ackermg@medizin.uni-leipzig.de
G
G
Ackermann
B
B
Löffler
Y J
YJ
Tang-Feldman
S H
SH
Cohen
J
J
Silva
A C
AC
Rodloff
D001427:Bacterial Toxins / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D016133:Polymerase Chain Reaction
D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile