Construction and Characterisation of CDR20291 ΔPaLoc Model Strains for the Study of Binary Toxin Regulation in Clostridium difficile.

Bilverstone, Terry; Kinsmore, Natasha; Minton, Nigel and Kuehne, Sarah (2016). Construction and Characterisation of CDR20291 ΔPaLoc Model Strains for the Study of Binary Toxin Regulation in Clostridium difficile. In: Microbiology Society Annual Conference 2016, 21-24 Mar 2016, Liverpool, UK..

Abstract

Clostridium difficile is a Gram-positive, spore forming, anaerobic bacterium and is the leading cause of hospital-associated diarrhoea in the developed world. The two large monoglycosylating toxins A and B have long been recognised as the major virulence factors of C. difficile infection (CDI). The genes encoding these toxins reside on a conserved locus referred to as the PaLoc. So-called hyprevirulent or epidemic strains of C. difficile produce a third toxin in addition to A and B, the binary toxin, or CDT, from a different chromosomal locus. Initially the relevance of binary toxin to CDI was unknown but recent advances suggest that CDT plays a major role in C. difficile virulence.

The Kuehne group seeks to further investigate the relevance of CDT to C. difficile virulence, and to unravel the molecular mechanisms regulating the production of binary toxin. In order to accomplish this effectively, it is necessary to be able to quantify binary toxin production without interference from the main virulence factors, Toxin A and B. This is best achieved by the construction of PaLoc-minus mutants of a hypervirulent strain such as CDR20291.

In this study we have deleted the entire 18.42Kb PaLoc by two-step allelic exchange in CDR20291 ΔpyrE and CDR20291 ΔpyrEΔcdtR backgrounds. Supernatants collected from cultured mutants contained no detectable toxin A/B as determined by ELISA and did not induce cell-rounding in the Vero cell culture model without prior CDT activation.

To our knowledge, this is the first published description of a clean in-frame ΔPaLoc mutant of C. difficile.

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