Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination

Abstract

causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the operon. To overcome these limitations, we have used-based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δ Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that and mutants spontaneously germinate more frequently in 630Δ than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of 630Δ spores can spontaneously germinate, in contrast with spores. We also show that clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA. is a leading cause of health care-associated infections. Initiation of infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Differential Effects of “Resurrecting” Csp Pseudoproteases duringClostridioides difficileSpore Germination

“…C. difficile strain construction was performed using 630Δ erm Δ cspC Δ pyrE [31] and 630Δ erm Δ pyrE -Δ cspBA as the parental strains via pyrE -based allele-coupled exchange (ACE [34]). This system allows for single-copy complementation of the Δ cspC and Δ cspBA parental mutants, respectively, from an ectopic locus.…”

“…This resulted in cspC complementation constructs carrying 282 bp of the cspBA upstream region in addition to the Δ cspBA sequence and the intergenic region between cspBA and cspC . This extended construct was required to produce wild-type levels of CspC when expressing the constructs in the pyrE locus [31, 34]. For example, the T170H mutation was constructed using primer pair #2189 and 2355 to amplify a 5’ cspC complementation construct fragment encoding the T170H mutation at the 3’ end, while primer pair #2354 and 2242 were used to amplify a 3’ cspC complementation construct encoding the T170H mutation at the 5’ end.…”

“…Similarly, for cspBA complementation constructs, each construct was designed with 126 bp of the Δ cspC sequence downstream of cspBA in order to fully complement the Δ cspBA mutant as previously described [31]. All primers used for strain construction are listed in Table S3 .…”

“…To clone the construct encoding the cspBA prodomain trans-complementation construct, primer pair #2189 and 951 was used to amplify the 5’ fragment, and primer pair #950 and 2242 were used to amplify the 3’ fragment. In both cases, Δ cspC genomic DNA was used as a template as described previously [31]. The resulting two fragments were joined together using PCR SOE with primer pair #2189 and 2242, and the PCR SOE product was cloned into pMTL-YN1C digested with NotI and XhoI using Gibson assembly.…”

“…While CspA, CspB, and CspC are all active in Clostridium perfringens [28], C. difficile CspC and CspA carry substitutions in their catalytic triad that render them pseudoproteases [23, 24, 31]. CspC is thought to directly sense bile acid germinants [24] and integrate signals from cation and amino acid cogerminants that potentiate germination in the presence of bile acid germinants [32].…”