Differential Effects of 'Resurrecting' Cap 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 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.