The ClosTron: A universal gene knock-out system for the genus Clostridium

Heap, John; Pennington, Oliver; Cartman, Stephen T.; Carter, Glen P.; Minton, Nigel P.

doi:10.1016/j.mimet.2007.05.021

Cited by 595 publications

(656 citation statements)

References 33 publications

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“…Mutants were constructed from the parental strain C. difficile 630erm 8 using the ClosTron system 5,6 . The retargeted plasmids pMTL007C-E2::Cdi-tcdA-1584s, 7 pMTL007C-E2::Cdi-tcdB-1511a and for the double pMTL007S-C7::Cdi-tcdA-1584s…”

Section: Methods Summarymentioning

confidence: 99%

“…The C. difficile single mutant strains A -B + and A + B -were made using ClosTron technology as described previously 5,6 . The A -B -double mutant strain was made from the A + B -mutant using a catP-based ClosTron using the 'pseudo-suicide' vector principle as described elsewhere 25 .…”

Section: Molecular Biology Techniquesmentioning

confidence: 99%

“…To verify the correct insertions, primers used for tcdA were: Cdi-tcdA-F2 (5´-TCAATTGACAGAACAAGAAATAAATAGTCTATGGAGC-3´) and EBS universal 5 , and Cdi-tcdA-R2 (5´-TACCCCATTGTCTTCAGAAAGAGATCCACC-3´) and ErmRAM-R (5´-ACGCGTGCGACTCATAGAATTATTTCCTCCCG-3´); and for tcdB were: Cdi-tcdB-F1 (5´-TGATAGTATAATGGCTGAAGCTAATGCAGATAATGG-3´) and ErmRAM-R, and Cdi-tcdB-R1 (5´-CTTGCATCGTCAAATGACCATAAGCTAGCC-3´) and EBS universal.…”

Section: Molecular Biology Techniquesmentioning

confidence: 99%

“…The intron sequence itself encompasses an erythromycin resistance determinant which permits selective isolation of mutants. Furthermore, it has been shown experimentally that the insertions are completely stable, meaning that inactivation of a gene is permanent 5 .…”

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confidence: 99%

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The role of toxin A and toxin B in Clostridium difficile infection

Kuehne

Cartman

Heap

et al. 2010

Nature

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759

638

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*These authors contributed equally to this work.Clostridium difficile infection is the leading cause of healthcare associated diarrhoea in Europe and North America 1, 2 . During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have suggested that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A, or there is prior damage to the gut mucosa 3 . However, a recent study suggested that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent 4 . This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knock-out system 5, 6 to permanently inactivate the toxin genes, we found that C. difficile producing either one or both toxins displayed cytotoxic activity in vitro, which translated directly into virulence in vivo.Furthermore, by constructing the first ever double mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue considering both toxins in the development of diagnostic tests and effective counter-measures against C. difficile.2 Toxin A and toxin B both catalyse the glucosylation, and hence inactivation, of Rho-GTPases; small regulatory proteins of the eukaryotic actin cell cytoskeleton. This leads to disorganisation of the cell cytoskeleton and cell death 7 . The toxin genes, tcdA and tcdB, are situated on the C. difficile chromosome in a 19.6 kilobase pathogenicity locus (PaLoc), along with the three accessory genes, tcdC, tcdR and tcdE (Fig. 1a). To address the individual importance of toxin A and toxin B, we used the ClosTron gene knock-out system 6 to inactivate the toxin genes of C. difficile. This system inactivates genes by inserting an intron into the protein-encoding DNA sequence of a gene, thus resulting in a truncated and non-functional protein. The intron sequence itself encompasses an erythromycin resistance determinant which permits selective isolation of mutants. Furthermore, it has been shown experimentally that the insertions are completely stable, meaning that inactivation of a gene is permanent 5 .Using the ClosTron system, we targeted insertions to tcdA and tcdB at nucleotide positions 1584 and 1511, respectively (Fig. 1a). In both cases, this placed the intron within DNA sequence encoding the toxin catalytic domain. Three separate isogenic The genotype of each toxin mutant was characterised by PCR and DNA sequence analysis to confirm the exact location of each intron insertion made (data not shown).Southern blot analysis of EcoRV-digested genomic DNA samples, using an intron-3 specific probe, confirmed that the A -B + and A + B -mutants ...

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Section: Methods Summarymentioning

confidence: 99%

Section: Molecular Biology Techniquesmentioning

confidence: 99%

Section: Molecular Biology Techniquesmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

The role of toxin A and toxin B in Clostridium difficile infection

Kuehne

Cartman

Heap

et al. 2010

Nature

Self Cite

759

638

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show abstract

“…Attempts have been made to improve the butanol concentration up to 17.8 g/L by genetically manipulating the wild-type clostridial species [12]. Nevertheless, genetically modified bacteria are usually unstable due to plasmid excision [14], leading to the deterioration of butanol-producing capability within batches of experiments. Hence, the search for novel and enhanced wild-type microbes with improved butanol tolerance is of great necessity for industrial applications [15].…”

Section: Introductionmentioning

confidence: 99%