# Regulation of Spore Germination in Clostridioides difficile

> **NIH NIH R01** · TUFTS UNIVERSITY BOSTON · 2024 · $333,752

## Abstract

Germination is essential for many spore-forming bacteria to initiate disease. Spores of the nosocomial pathogen
Clostridioides difficile induce germination when they sense bile acid (germinants) combined with amino acids
and/or Ca2+ (co-germinants). C. difficile senses these small molecules using a mechanism that is distinct from
previously studied spore-formers because it lacks the transmembrane germinant receptors encoded by all other
spore-formers. Instead, C. difficile uses two soluble proteins of the clostridial serine protease family, CspC and
CspA, to sense germinant and co-germinant signals. While most Csps are active proteases, C. difficile CspC
and CspA are pseudoproteases due to mutations in their catalytic triads. Despite their lack of catalytic activity,
the CspC and CspA pseudoproteases control the activity of a related protease, CspB, during germination. While
CspC and CspA were identified as the likely bile acid germinant and co-germinant receptors, respectively,
through genetic screens, we unexpectedly discovered in the past funding period that CspC not only senses bile
acid germinant but also co-germinant signals. These findings raise the question as to how CspC and CspA
integrate signals from such different small molecules, especially since we lack biochemical evidence that either
of these proteins binds any of these signaling molecules.
 We recently gained biochemical insight into this question by demonstrating that CspC and CspA directly
interact. We also determined that CspA forms a homodimer, in contrast with CspC, by solving CspA’s crystal
structure. Intriguingly, when we model CspC onto our unpublished structure of CspA, CspC residues that control
the sensitivity of C. difficile spores to germinants and/or co-germinants cluster to the predicted CspC:CspA
heterodimerization interface. These data lead us to hypothesize that germinant and co-germinant signals alter
the equilibrium between CspC:CspA heterodimers and CspA homodimers. Since CspA is also needed for CspC
to be stably incorporated into spores, we propose that a partner-swap mechanism regulates C. difficile spore
germination. According to this model, CspC:CspA heterodimerization allows CspC to be stably incorporated into
dormant spores. The inactive heterodimer is disrupted upon germinant and co-germinant addition, which triggers
germination potentially by promoting CspA homodimerization. We will test this model by evaluating the functional
significance of CspC:CspA heterodimerization and CspA homodimerization (Aim 1) and the impact of germinants
and co-germinants on these protein:protein interactions (Aim 2). We will also determine the role of CspA’s
unstructured prodomain in regulating the equilibrium between CspA homodimers and CspC:CspA heterodimers
in the presence of germinant and co-germinant signals (Aim 3). Collectively, these analyses will provide atomic-
level insight into how C. difficile spores transduce germinant and co-germinant signals to induce germ...

## Key facts

- **NIH application ID:** 10896312
- **Project number:** 5R01GM108684-08
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Aimee Shen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $333,752
- **Award type:** 5
- **Project period:** 2014-07-10 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10896312

## Citation

> US National Institutes of Health, RePORTER application 10896312, Regulation of Spore Germination in Clostridioides difficile (5R01GM108684-08). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10896312. Licensed CC0.

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