title: Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT, 05405, USA.
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Kevorkian
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT, 05405, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Shirley
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT, 05405, USA. Electronic address: aimee.shen@uvm.edu.
authors.forename: Aimee
authors.initials: A
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013171:Spores, Bacterial / Q000235:genetics / Q000378:metabolism / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C092058:CspA protein, Clostridium; C102917:CspC protein, Clostridium; C077188:cold-shock protein CspB, Bacteria; D010447:Peptide Hydrolases
keywords: Cap family protease; Clostridium difficile; Pseudoprotease; Spore germination