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Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the vertebrate gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently determined crystal structure of CspC revealed that its degenerate site residues align closely with the catalytic triad of CspB, so in this study we tested whether the ancestral protease activity of the CspC and CspA pseudoproteases could be “resurrected.” Restoring the catalytic triad to these pseudoproteases failed to resurrect their protease activity, although the mutations differentially affected the stability and function of these pseudoproteases. Degenerate site mutations destabilized CspC and impaired spore germination without impacting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since close homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results imply that bioinformatics predictions of enzyme activity may overlook pseudoenzymes in some cases.

The discovery of specific microbiota metabolite mechanisms has begun to motivate new therapeutic approaches. Inspired by our mechanistic studies of microbiota-derived short chain fatty acid (SCFA) acylation of bacterial virulence factors, here we explored covalent protein acylation therapeutics as potential anti-infectives. For these studies, we focused on acetyl-salicylic acid, aspirin, and discovered that SCFA analogues such as butyryl-salicylic acid showed significantly improved anti-infective activity against Salmonella Typhimurium. Structure-activity studies showed that the ester functionality of butyryl-salicylic acid was crucial and associated with the acylation of key bacterial virulence factors and metabolic enzymes, which are important for Salmonella infection of host cells and bacterial growth. Beyond the Gram-negative bacterial pathogens, butyryl-salicylic acid also showed better antibacterial activity compared to aspirin against Clostridioides difficile, a clinically challenging Gram-positive bacterial pathogen. Notably, coadministration of butyryl-salicylic acid, but not aspirin, effectively attenuated Salmonella pathogenesis in vivo. This study highlights how the analysis of microbiota metabolite mechanisms may inspire the repurposing and development of new anti-infective agents.

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.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection., Author Summary Infection with intracellular pathogens causes a majority of the global infectious disease associated mortality. A number of intracellular pathogens must directly access the host cytosol in order to cause disease; however, non-cytosol adapted bacteria do not survive or replicate upon access to the cytosol. The mechanisms cytosolic pathogens use to adapt to this niche are largely unknown. The model cytosolic bacterial pathogen Listeria monocytogenes contains a single penicillin-binding-protein and serine/threonine associated (PASTA) kinase, PrkA. In other bacteria, PASTA kinases bind cell wall fragments and phosphorylate downstream effectors involved in cell wall synthesis, central metabolism, virulence, cell division, and biofilm formation. We demonstrate that in L. monocytogenes, PrkA is required for cell wall homeostasis, growth under nutrient limiting conditions, survival and replication in host cells, and virulence in vivo. Furthermore, we identify a highly conserved protein of unknown function, YvcK, as a PrkA substrate. We demonstrate that L. monocytogenes YvcK is similarly required for cell wall stress responses, growth on glycerol, cytosolic survival and virulence in vivo. Surprisingly, a phosphomimetic, nonreversible negative charge at the phosphorylation sites on YvcK inactivates functions of the protein related to intracellular survival and virulence, suggesting that the identification of PASTA kinase substrates phosphorylated during infection will be critical to our understanding of this central regulator metabolism, cell wall homeostasis and ultimately virulence.