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The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor., Author summary The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

SUMMARYMany viruses globally shut off host gene expression to inhibit activation of cell-intrinsic antiviral responses. However, host shutoff is not indiscriminate, since viral proteins and host proteins required for viral replication are still synthesized during shutoff. The molecular determinants of target selectivity in host shutoff remain incompletely understood. Here, we report that the influenza A virus shutoff factor PA-X usurps RNA splicing to selectively target host RNAs for destruction. PA-X preferentially degrades spliced mRNAs, both transcriptome-wide and in reporter assays. Moreover, proximity-labeling proteomics revealed that PA-X interacts with cellular proteins involved in RNA splicing. The interaction with splicing contributes to target discrimination and is unique among viral host shutoff nucleases. This novel mechanism sheds light on the specificity of viral control of host gene expression and may provide opportunities for development of new host-targeted antivirals.