Phosphorylation and functionality of CdtR in Clostridium difficile

Bilverstone, T.W.; Minton, N.P. and Kuehne, S.A. (2018). Phosphorylation and functionality of CdtR in Clostridium difficile. In: The 14th Biennial Congress of the Anaerobe Society of the Americas, 09-12 Jul 2018, Las Vegas, Nevada, USA..

Abstract

The aim of this study was to identify the phosphorylation site of the virulence regulator CdtR in Clostridium difficile R20291, and establish the functionality of its PCR-ribotype 078 homolog.

We generated dephosphomimetic and potentially-phosphomimetic CdtR-encoding variants in which our predicted phosphoAsp residue had been substituted with Ala and Glu respectively. We also amplified cdtR from the archetypal ribotype 078 strain M120, containing 9 non-synonymous mutations including a premature truncation, and integrated all three cdtR- variants at the pyrE locus in R20291 PaLoc cdtR.

The Asp-Glu CdtR-expressing strain mirrored the cdtR-null parental strain, in which CdtA was reduced to levels undetectable by Western blot. Conversely, the Asp-Glu CdtR phosphomimic was at least partially functional, as CdtA was detectable for this mutant, although the level of production was considerably lower than its wild-type counterpart. M120-derrived CdtR was again comparable to the cdtR-null parental indicating its lack of functionality. To ensure that this observation stemmed from the mutations within the coding region and not the polymorphic promoter region, we tested the capability for E. coli to tolerate chloramphenicol using R20291 and M120-derrived cdtR promoters to drive CatP expression. Our data indicated that both constructs drove constitutive expression. Finally, we amplified cdtR from seven ribotype 027 and five ribotype 078 clinical isolates from our culture collection and sequenced their open reading frames. Nucleotide sequences were 100% identical to their archetypal strains within each ribotype with a 4.4% divergence between ribotypes.

Our preliminary data provide valuable insights into the activation of functional CdtR and the non-functionality of the ribotype 078 homolog.

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