Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation

Abstract

Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. difficile. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Using genetic analyses, we determined the functions of these engulfment-related proteins along with the putative endopeptidase, SpoIIQ, during C. difficile sporulation. While SpoIID, SpoIIP and SpoIIQ were critical for engulfment, loss of SpoIIM minimally impacted C. difficile spore formation. Interestingly, a small percentage of ∆spoIID and ∆spoIIQ cells generated heat-resistant spores through the actions of SpoIIQ and SpoIID, respectively. Loss of SpoIID and SpoIIQ also led to unique morphological phenotypes: asymmetric engulfment and forespore distortions, respectively. Catalytic mutant complementation analyses revealed that these phenotypes depend on the enzymatic activities of SpoIIP and SpoIID, respectively. Lastly, engulfment mutants mislocalized polymerized coat even though the basement layer coat proteins, SpoIVA and SipL, remained associated with the forespore. Collectively, these findings advance our understanding of several stages during infectious C. difficile spore assembly.

Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment

“…Work presented here and by Ribis et al . ( 2018 ) shows that organization of the engulfment machinery in C. difficile differs from that proposed in the model organism B. subtilis .…”

“…Work presented here and by A. Ribis et al . () shows that organization of the engulfment machinery in C. difficile differs from that proposed in the model organism B. subtilis . We propose that a DP machinery, interlinked with the Q:AH channel, provides the driving force for engulfment, with SpoIIM being largely dispensable.…”

“…Importantly, data from Ribis et al . () shows that SpoIIP expression is regulated by the forespore‐specific σ F regulator and is expected to be present in the forespore membrane. Conversely, SpoIID is anchored to the mother cell membrane, with expression under the mother cell‐specific regulator σ E , strengthening the idea that a SpoIID‐SpoIIP (DP) direct interaction across the intersporangial space could provide a simpler PG degradation machinery.…”

“…Combining this data with the observations by Ribis et al . (), the engulfment machinery in C. difficile would be anchored to the forespore membrane by SpoIIP and SpoIIQ and to the mother cell membrane by SpoIID and SpoIIIAH, with SpoIIM playing an ancillary, redundant role (Fig. ).…”

“…Instead, SpoIID seems to interact with SpoIIM and SpoIIP directly. Importantly, data from Ribis et al . ( 2018 ) shows that SpoIIP expression is regulated by the forespore-specific σ F regulator and is expected to be present in the forespore membrane.…”