Phosphorylation and functionality of CdtR in Clostridium difficile.

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• Clostridium difficile toxin production is regulated by the CdtR two-component system.
• The signal transduction pathway for CdtR activation remains largely uncharacterized.

Purpose of the Study:

• To elucidate the CdtR signal transduction pathway and the role of phosphorylation.
• To investigate CdtR function and regulation in different Clostridium difficile ribotypes (027 and 078).

Main Methods:

• Generated Clostridium difficile R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants (Asp61Ala, Asp61Glu).
• Assessed the ability of CdtR variants to restore CDT production.
• Analyzed CdtR coding and promoter regions in clinical isolates of ribotypes 027 and 078.

Main Results:

• Phosphorylation of Asp61 is essential for CdtR activation; Asp61Ala mutant was non-functional, while Asp61Glu showed partial activity (38-52% of wild-type).
• PCR ribotype 078 CdtR was non-functional due to coding gene polymorphisms, but its promoter exhibited enhanced activity compared to ribotype 027.
• Conserved cdtR nucleotide sequences were confirmed in clinical isolates of both ribotypes.

Conclusions:

• CdtR activation is dependent on phosphorylation of Asp61.
• Polymorphisms in CdtR influence its function and regulation differently across Clostridium difficile ribotypes, affecting toxin production.