# Pseudoprotease-mediated regulation of germinant sensing in Clostridioides difficile

> **NIH NIH F31** · TUFTS UNIVERSITY BOSTON · 2024 · $45,162

## Abstract

ABSTRACT
Clostridioides difficile infections begin when its metabolically dormant spores encounter germinants in the
vertebrate gut and initiate the process of germination. This process is essential for C. difficile to produce the
vegetative cells that will release toxins and cause disease. Interestingly, distinct mechanisms control C. difficile
germinant signaling relative to most spore-forming bacteria. First, C. difficile spore germination requires two
distinct signals: a bile acid germinant signal must be combined with amino acid and/or Ca2+ co-germinant signals.
Second, C. difficile lacks the transmembrane germinant receptors conserved in almost all spore-forming
receptors and instead uses two soluble pseudoproteases, CspA and CspC, to sense co-germinants and
germinants, respectively. CspA and CspC then go on to activate the CspB protease, which in turn activates a
lytic enzyme responsible for degrading the protective cortex layer of the spore. While CspA and CspC clearly
play key roles in regulating the initiation of germination, precisely how they integrate germinant and co-germinant
signals is poorly understood. We recently discovered that CspA forms a homodimer while CspC and CspA form
a stable heterodimer which is preferred over the CspA homodimer. We solved the crystal structure of the CspA
homodimer and the CspA:CspC heterodimer and identified residues that are predicted to regulate these
interactions. Given that we previously showed that CspA is required for CspC to be stably incorporated into
mature spores, we propose that the CspA:CspC heterodimer is the inactive signaling complex found in dormant
spores and that germinants and co-germinants destabilize the heterodimer. Disruption of the CspA:CspC
heterodimer likely promotes CspA homodimerization and frees CspC to bind and activate CspB, which activates
downstream germination events. In this proposal, I will test this “Partner-Swap” model by identifying residues
that mediate CspA homodimer and CspA:CspC heterodimer binding and determine the impact of mutating these
residues on C. difficile germination. Using a combination of biochemical, genetic, and cytological analyses, I will
also determine the effect of germinants and co-germinants on the stability of the CspA:CspC heterodimer.
Collectively, these analyses will reveal how these critical proteins regulate the initiation of C. difficile germination
and advance our understanding of pseudoenzymes and their responsibility in the regulation of key cellular
processes.

## Key facts

- **NIH application ID:** 10997790
- **Project number:** 1F31AI186517-01
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Morgan McNellis
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $45,162
- **Award type:** 1
- **Project period:** 2024-08-01 → 2028-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10997790

## Citation

> US National Institutes of Health, RePORTER application 10997790, Pseudoprotease-mediated regulation of germinant sensing in Clostridioides difficile (1F31AI186517-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10997790. Licensed CC0.

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