The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

Abstract

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.

Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination

“…coli strains are listed in Supplemental Table S1 in the supplementary material. As previously described [30], the cspC complementation constructs were created using flanking primers, #2189 and 2242 (Supplemental Table S2), in combination with internal primers encoding a given point substitution, ΔcspBA genomic DNA was used as the template. This resulted in cspC complementation constructs carrying 282 bp of the cspBA upstream region in addition to the ΔcspBA sequence and the intergenic region between cspBA and cspC.…”

“…Notably, these bile acid germinants differ from the nutrient germinants sensed by almost all other spore-formers studied to date, and their signal transduction mechanism appears to be unique because C. difficile lacks the transmembrane germinant receptors found in all other spore formers [23][24][25][26]. Instead, the bile acid germinant signal is transduced by members of the clostridial serine protease family known as the Csps [27][28][29][30]. Csps are subtilisin-like serine protease family members [31,32] conserved in many clostridial species [33].…”

“…Instead, they regulate how C. difficile spores sense bile acid germinants as well as cation and amino acid co-germinant signals. C. difficile CspC is thought to directly sense bile acid germinants [28] and integrate signals from the two co-germinant classes [30], while C. difficile CspA may function as the co-germinant receptor [42] and is necessary for CspC to be packaged into mature spores [29]. Thus, C. difficile CspC and CspA both regulate the protease activity of CspB, whose intact catalytic triad is required for proteolytically activating SleC [27].…”

“…The scissor icon marks the autoprocessing sites of the Csp family members that are catalytically active, which separates the prodomain from the subtilase domain. The predicted site that would be cleaved by autoprocessing of the CspC pseudoprotease based on the CspC structure [30] is also shown in red. The YabG cleavage site [42] between CspB and CspA is shown.…”

“…Since we recently showed that the pseudoactive site residues of C. difficile CspC closely align with the catalytic triad of an active CspB protease ( [30], Figure 1C), we asked whether restoring an intact catalytic triad to CspC would be sufficient to convert it into an active protease capable of undergoing autoprocessing like other subtilisin-like serine proteases [32]. Since structural modeling of CspA also predicted close alignment of its pseudoactive site residues with CspB's catalytic triad residues ( Figure 1C), we tested the effect of restoring the catalytic triad to C. difficile CspA to gain insight into the evolution of these 'zombie' proteins from active proteases and the structural requirements for their function in C. difficile.…”