Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864 – implications for transcription, expression and enzymatic activity of toxins A and B

Abstract

The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3' ends of tcdA and tcdC. Transcription of the tcdA-E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA-E genes that confirms our previous findings in strain 10463. Recombinant TcdA-8864 had UDP-glucose-glucosyltransferase activity, proving that the N-terminal 698 amino acids of the polypeptide represent the catalytic domain. However, this truncated TcdA molecule lacks a ligand and translocation domain. To assess the catalytic domain of TcdB-8864, the sequence of the 5' end of its gene was determined. TcdB-8864 shows high homology to TcdB-1470 but lower homology to TcdB-10463 within this domain. This fits well with the altered glucosylation specificity of TcdB-8864 (Rac1, Rap2 and Ra1). Having defined the variations of transcription, expression and enzymatic activity of toxins A and B, implications for the pathogenic potential of strain 8864 are discussed.

Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes

“…Subsequent analysis of toxin genes has revealed that two groups of these TcdA − B + strains exist ; the first group is represented by an atypical strain (8864 or CCUG 20309) and the second group includes strains from serogroups F and X (Borriello et al, 1992 ;Lyerly et al, 1992 ;Depitre et al, 1993 ;Rupnik et al, 1997 ;Soehn et al, 1998 ;von Eichel-Streiber et al, 1999). Recently, a PCR-RFLP method (known as toxinotyping) has been developed to analyse changes in the PaLoc and to type strains of C. difficile (Rupnik et al, 1998).…”

Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants

“…Many of the variant toxinotypes have polymorphisms within one or both of these genes (Rupnik et al, 1998Spigaglia & Mastrantonio, 2002) which may result in non-functional products that alter the rate of synthesis of toxins A and B. The toxin A-negative (AÀB+) strain 8864 which has an unusually high cytotoxicity has been found to possess mutations within gene tcdC that give rise to a grossly truncated TcdC polypeptide (Lyerly et al, 1992;Soehn et al, 1998).…”

“…The cytopathic effects caused by these toxin A-positive (A+B+) strains appear identical to those observed for toxin A-negative (AÀB+) strains from toxinotypes VIII and X. Toxin A-negative (AÀB+) strains are known to produce altered forms of toxin B with extensive sequence variations in the N-terminal enzymic domain (Sambol et al, 2000;von Eichel-Streiber et al, 1995). Toxin B of strain VPI 10463 glucosylates GTPases of the Rho subfamily only (Just et al, 1995), whereas toxin B of strains 8864 and 1470, in common with LT, additionally glucosylate Ras subfamily GTPases (ChavesOlarte et al, 1999;Popoff et al, 1996;Soehn et al, 1998). One of these Ras subfamily GTPases, R-Ras, is known to control integrin-extracellular matrix interactions (Chaves-Olarte et al, 1999) and its glucosylation may be responsible for the clumping of Vero cells.…”

“…Routine laboratories that carry out direct toxin testing of stools may identify infection by toxin A-negative (AÀB+) strains due to a negative toxin A immunoassay result, combined with a positive toxin A and B immunoassay result or a positive cytotoxicity test. Also, toxin A-negative (AÀB+) strains produce variant forms of toxin B with altered glucosyltransferase activity for small GTPases (Chaves-Olarte et al, 1999;Soehn et al, 1998) that cause atypical cytotoxicity in mammalian cell lines (Chaves-Olarte et al, 1999;Kuijper et al, 2001;von Eichel-Streiber et al, 1995). Due to their ease of detection, toxin A-negative (AÀB+) strains have been extensively characterized.…”

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

“…Thus, the two general mutations are one in which a premature stop codon leads to production of a severely truncated TcdC protein and one in which internal deletions reduce the size of the protein by 6 residues. Other strains of C. difficile known to produce variant forms of TcdB (e.g., strain 8864) have also been found to produce a truncated form of TcdC of only 22 amino acids (180).…”