Search results for "Enterotoxin"

Nucleotide sequence of Clostridium difficile toxin A.

1990

Cloning and Characterization of Overlapping DNA Fragments of the Toxin A Gene of Clostridium difficile

1989

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of vari…

Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile

1995

A library of total Clostridium novyi DNA was established and screened for the alpha-toxin gene (tcn alpha) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcn alpha fragments were isolated and subsequently the total sequence of tcn alpha was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M(r) 250,166 and pI 5.9. The N-terminal alpha-toxin (Tcn alpha) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it co…

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

1992

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…

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

1996

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…

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

2009

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of …

Staphylococcal food poisoning case and molecular analysis of toxin genes in Staphylococcus aureus strains isolated from food in Sicily, Italy.

2014

A case of staphylococcal food poisoning was observed in two individuals of the same family after consumption of primosale, a semiripened sheep cheese produced in Sicily. Staphylococcus aureus isolated from the cheese produced enterotoxin C (SEC) and carried both the enterotoxin C (sec) and the toxic shock syndrome toxin (tsst-1) gene. Following this case, an extensive survey was conducted on 971 food samples (raw milk, cheese, meat, and food preparations). S. aureus was detected in 102 of 971 food samples, from all types of food with the exception of ricotta cheese. The tsst-1 gene was present in 42% of the strains, either alone or in combination with other toxin genes. The enterotoxin C ge…

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques.

2005

Abstract The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus , 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease ( nuc ) and enterotoxin ( sea to see ) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T…

Concomitant loss of conformation and superantigenic activity of staphylococcal enterotoxin B deletion mutant proteins.

1993

The T-cell-stimulating activity of staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases. To investigate the immunologically active domains of the SEB molecule, we have produced truncated fragments of recombinant SEB by C-terminal and N-terminal deletions. The fragments were expressed as fusion proteins with protein A, including a cleavage site to remove the protein A part. Mutant proteins were tested for the ability to stimulate human resting T cells and SEB-reactive T-cell clones. Deletion of only 9 amino acids from the C terminus leads to complete loss of T-cell-stimulating activity. Removing further amino acids from the SEB mole…

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

1997

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…