Regulation of spore germination by an intrinsically disordered protein

The clinically significant pathogen Clostridioides difficile initiates infection when its metabolically dormant spores germinate in the gut. To initiate germination, C. difficile utilizes three soluble clostridial-specific proteases (Csp; CspA, CspB, and CspC) to transduce germinant signals and activate the SleC lytic enzyme, which degrades the spore cortex and allows for germination. The stable incorporation of Csp proteins in spores is controlled by the gel-forming protein GerG. Deletion of gerG results in a >100-fold germination defect likely due to the decreased levels of Csp proteins. While GerG is a critical regulator for C. difficile germination, the mechanism by which it controls Csp levels in mature spores remains unclear. Structural modeling of GerG predicts that it consists of a central -helical region that is flanked by long intrinsically disordered regions (IDRs). The β-helical region shares some structural similarities with bactofilins, a ubiquitous family of cytoskeletal proteins that spontaneously polymerizes into filaments and recruits proteins to this scaffold, while IDRs frequently bind multiple protein clients to regulate diverse cellular processes. My preliminary data indicate that the central β-helical region of GerG mediates spontaneous filament formation in vitro, but this region does not affect Csp levels in spores or spore germination. Interestingly, I have shown that mutations of highly conserved, negatively charged residues in GerG’s C-terminal IDR (C-IDR) decrease Csp levels and disrupt spore germination. In contrast, mutation of conserved tyrosine residues in GerG’s N-terminal IDR (N-IDR) does not impact Csp levels, although the mutant spores are more sensitive to germinant. These data suggest that GerG’s N-IDR regulates germinant sensitivity by modulating the functions of the Csp germination proteins, while the C-IDR is critical for Csp protein incorporation into spores. Thus, GerG regulates multiple aspects of C. difficile spore germination. Based on these observations, I hypothesize that GerG directly binds Csp proteins through its IDRs and recruits them to specific sites in C. difficile spores. I will test these hypotheses by identifying key functional regions within GerG that regulate Csp incorporation into spores and spore germination (Aim 1). I will also identify the binding partners of GerG through an unbiased approach and test whether GerG interacts, directly or indirectly, with the Csp proteins (Aim 2). Finally, I will determine the impact of GerG on the localization of the Csp proteins in C. difficile and that of binding partners identified in Aim 2 (Aim 3). By elucidating the mechanisms used by GerG to control the levels of essential germination regulators in C. difficile spores, my dissertation project will advance our understanding of the critical process of C. difficile spore germination. It will also provide broadly useful insights into how IDRs in prokaryotes regulate diverse cellular processes. Project Number: 1F31AI197433-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Abigail Rivera Seda | Institution: TUFTS UNIVERSITY BOSTON, BOSTON, MA | Award Amount: $46,302 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F07A-W (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31AI19743301