Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia

^* To whom correspondence should be addressed. Email: Julian.Rood{at}med.monash.edu.au.

	Abstract

Clostridium difficile binary toxin is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, designated here as cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR⁺ and cdtR^- strains of C. difficile it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68 bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.