Molecular characterization of the Clostridium difficile toxin A gene

Abstract

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs has a mol% G+C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Galoa1-3Gal01-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Transcription Analysis of the Genes tcdA‐E of the Pathogenicity Locus of Clostridium Difficile

“…Characteristic features of the Tcd proteins. TcdA and TcdB, the enterotoxin A and the cytotoxin B, are well known and have been described and discussed in detail before (Dove et al, 1990;Barroso et a]., 1990;von Eichel-Streiber et al, 1992). The function of the other three proteins TcdC-E is yet to be defined.…”

Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typingClostridium difficilestrains

“…The presence of the toxin A gene was assayed by PCR amplification of the 484 bp fragment located between bases 302 and 785 [12] of the toxin A gene, using primers AM2: 5' GGATAG-GTGGAGAAGTCAGTG 3' and AMB: 5' CC-ACGATTTAACAACTCCTGAC 3' (Organic Chemistry Laboratory, Institut Pasteur, Paris, France).…”

Physical Characterization of Clostridium Difficile Toxins and Toxoids: Effect of the Formaldehyde Crosslinking on Thermal Stability

“…These toxins cause acute inflammation of the colonic mucosa in humans which results in a wide spectrum of symptoms including mild diarrhea to life-threatening pseudomembranous colitis. [2][3][4][5][6] C. difficile-associated disease (CDAD) is increasingly prevalent in hospitalized patients and no effective prevention method is available. Treatment with the antimicrobial agents metronidazole or vancomycin leads to recurrence of the disease.…”