A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• Clostridium difficile is a significant nosocomial pathogen causing gastrointestinal infections.
• Cell wall proteins in C. difficile play roles in pathogenesis.
• CwpV is the largest known cell wall protein in C. difficile.

Purpose of the Study:

• To characterize the CwpV cell wall protein in Clostridium difficile.
• To elucidate the mechanism of CwpV phase variable expression.
• To identify the genetic elements involved in CwpV regulation.

Main Methods:

• Analysis of CwpV surface expression and processing.
• Construction of gusA transcriptional reporter fusions to study cwpV expression.
• Investigation of DNA inversion upstream of the cwpV gene.
• Identification of a site-specific recombinase responsible for DNA inversion.

Main Results:

• CwpV is surface-expressed and post-translationally processed similarly to SlpA.
• cwpV expression is phase variable, occurring in approximately 5% of cells.
• Phase variation is mediated by DNA inversion of a 195 bp sequence flanked by inverted repeats.
• A novel phase variation mechanism involving intrinsic terminator formation in the OFF transcript was proposed.
• A C. difficile site-specific recombinase catalyzing the inversion was identified.

Conclusions:

• CwpV is a surface-expressed, phase-variable cell wall protein in Clostridium difficile.
• The identified DNA inversion mechanism represents a novel mode of bacterial phase variation.
• Understanding CwpV regulation provides insights into Clostridium difficile pathogenesis and adaptation.