Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions...

Area of Science:

• Microbiology
• Infectious Diseases
• Bacterial Pathogenesis

Background:

• Clostridioides difficile infections initiate with spore germination in the small intestine.
• Spore germination requires sensing bile acid germinants and co-germinants (amino acids and divalent cations like Ca2+).
• Previous studies suggested Ca2+ is essential for C. difficile spore germination, based on bulk population assays.

Purpose of the Study:

• To investigate whether both co-germinant signals (amino acids and Ca2+) are required for C. difficile spore germination.
• To overcome limitations of bulk assays for analyzing germination in Ca2+-deficient spores.
• To elucidate the role of Ca2+ and calcium dipicolinic acid (CaDPA) in C. difficile spore germination.

Main Methods:

• Development of a novel automated, time-lapse microscopy-based germination assay.
• Single-spore level analysis of CaDPA mutant spore germination.
• Comparison of germination kinetics between wild-type (WT) and CaDPA mutant spores.

Main Results:

• CaDPA mutant spores can germinate in response to bile acid and amino acid co-germinants alone.
• Higher concentrations of amino acid co-germinants are needed for CaDPA mutant spore germination compared to WT spores.
• CaDPA released by WT spores acts in a feedforward loop to enhance germination of neighboring spores.

Conclusions:

• Ca2+ is dispensable for inducing C. difficile spore germination, indicating parallel signaling pathways for amino acid and Ca2+ co-germinants.
• CaDPA plays a potentiating role in C. difficile spore germination, not an essential initiating role.
• Automated microscopy provides a more accurate method for studying C. difficile spore germination, especially for mutant strains.