Clostridium difficile is a spore-forming obligate anaerobe that is a leading 
cause of healthcare-associated infections. C. difficile infections begin when 
its metabolically dormant spores germinate in the gut of susceptible 
individuals. Binding of bile salt germinants to the Csp family pseudoprotease 
CspC triggers a proteolytic signaling cascade consisting of the Csp family 
protease CspB and the cortex hydrolase SleC. Conserved across many of the 
Clostridia, Csp proteases are subtilisin-like serine proteases that activate 
pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the 
protective cortex layer, allowing spores to resume metabolism and growth. This 
signaling pathway, however, is differentially regulated in C. difficile, since 
CspC functions both as a germinant receptor and regulator of CspB activity. CspB 
is also produced as a fusion to a catalytically inactive CspA domain that 
subsequently undergoes interdomain processing during spore formation. In this 
study, we investigated the role of the CspA pseudoprotease domain in regulating 
C. difficile spore germination. Mutational analyses revealed that the CspA 
domain controls CspC germinant receptor levels in mature spores and is required 
for optimal spore germination, particularly when CspA is fused to the CspB 
protease. During spore formation, the YabG protease separates these domains, 
although YabG itself is dispensable for germination. Bioinformatic analyses of 
Csp family members suggest that the CspC-regulated signaling pathway 
characterized in C. difficile is conserved in related Peptostreptococcaceae 
family members but not in the Clostridiaceae or Lachnospiraceae. Our results 
indicate that pseudoproteases play critical roles in regulating C. difficile 
spore germination and highlight that diverse mechanisms control spore 
germination in the Clostridia.