Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.

To analyse the transcription pattern of the five tcdA-E genes of the pathogenicity locus (PaLoc) of Clostridium difficile a protocol was established to purify RNA from strain VPI10463. Transcription analysis of the five tcdA-E genes showed that they were all transcribed. In the early exponential phase, a high level of tcdC and low levels of tcdA,B,D,E transcripts were detectable; this was inverted in the stationary phase, suggesting that TcdC might have a negative influence on transcription of the other genes. Three transcription initiation sites, one for tcdA and two for tcdB were determined by primer extension analysis. Readthrough transcripts from outside the locus were not obtainable, s…

Morphological changes in adherent cells induced by Clostridium difficile toxins.

Large clostridial cytotoxins — a family of glycosyltransferases modifying small GTP-binding proteins

Some Clostridium species produce AB x -type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases.

A detailed analysis of the 8130-bp open reading frame (ORF) of gene toxA and of an upstream ORF designated utxA, indicates the presence of a transcription terminator stem-loop for toxA, promoter sequences, and Shine-Dalgarno boxes for toxA and utxA. No transcription terminator between toxA and utxA is suggested by the sequence. ToxA contains two domains, one-third (C-terminal) with a repetitive structure and the residual two-thirds with no repetitions. The 2499-bp sequence encoding the repetitive structure is composed of nine groups of different short repetitive oligodeoxyribonucleotides (SRONs). A combination of these SRONs codes for five groups of combined repetitive oligopeptides (CROPs)…

Comparative sequence analysis of the Clostridium difficile toxins A and B.

The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison…

Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B.

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open r…

Nucleotide sequence of Clostridium difficile toxin A.

Definition of the single integration site of the pathogenicity locus in Clostridium difficile.

We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in pol…

Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases.

The homologous C-terminal repeats of Clostridium difficile toxins (ToxA and ToxB) and streptococcal glucosyltransferases appear to mediate protein-carbohydrate interactions at cellular binding sites with sugar moieties as substrates. A consensus sequence of 134 repeating units from gram-positive bacteria indicates that these repeats have a modular design with (i) a stretch of aromatic amino acids proposed to be involved in the primary carbohydrate-protein interaction, (ii) an amplification of this interaction by repetition of the respective sequences, and (iii) a second domain, not characterized, that is responsible for carbohydrate specificity.