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

Abstract

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 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 solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to ‘resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases.

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

“…It is likely that PrkC phosphorylates additional targets to promote germination, although these are yet to be defined. Additionally, Clostridia possess Csp-type receptors indirectly connected to the OsM via lipoproteins and coupled to the cortex lytic enzyme SleC, which is activated by cleavage upon recognition of germinant ( Fimlaid et al, 2015a ; Bhattacharjee et al, 2016 ; Kochan et al, 2018 ; Donnelly et al, 2020 ). Organisms such as C. difficile lack Ger-type receptors altogether and instead rely on Csp proteins, such as CspC ( Bhattacharjee et al, 2016 ).…”

Imaging Clostridioides difficile Spore Germination and Germination Proteins

“…In the majority of the studied endospore-forming organisms, spores detect germinants via Ger-type receptors embedded in the inner spore membrane (46). C. difficile does not encode Ger-type germinant receptors and, instead, uses the CspA and CspC proteins as germinant receptors (47)(48)(49)(50)(51). Prior to the work done in C. difficile, CspA, CspB, and CspC proteins were characterized in Clostridium perfringens, and found to be subtilisin-like serine proteases.…”

“…Unlike what is found in C. perfringens, C. difficile cspB and cspA are translationally fused (CspBA), and cspC is encoded downstream from the cspBA gene. Interestingly, both CspA and CspC have lost their catalytic triad and, therefore, are pseudoproteases (47,48).…”

Single-spore germination analyses reveal that calcium released duringClostridioides difficilegermination functions in a feed-forward loop

“…Upon sensing germinants and co-germinants, CspC and CspA activate the CspB protease through an unknown mechanism (11, 13). CspA, CspB, and CspC are all members of the subtilisin-like serine protease family, but only CspB is catalytically active (26, 30). Activated CspB proteolytically removes an inhibitory propeptide from the cortex lytic enzyme, SleC (26), which then degrades the cortex, a protective layer of modified peptidoglycan critical for maintaining the metabolic dormancy of spores.…”