Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

Abstract

The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C. difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C. difficile spore germination.

The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

“…Our results support the first model whereby CspB, CspA, and CspC are in a germinosome complex – similar to the germinosome complex found in B. subtilis [53]. Recent studies on GerS have shown that GerS likely does not form a complex with other germination proteins [52, 54]. Rather, it is required to generate cortex-specific modifications in the spore and thus, important for germination of the spore because the SleC cortex lytic enzyme depends on cortex-specific modifications to degrade the cortex layer efficiently [54].…”

“…Recent studies on GerS have shown that GerS likely does not form a complex with other germination proteins [52, 54]. Rather, it is required to generate cortex-specific modifications in the spore and thus, important for germination of the spore because the SleC cortex lytic enzyme depends on cortex-specific modifications to degrade the cortex layer efficiently [54]. Based upon prior work from our lab, proSleC is unlikely to be part of this complex because SleC is three to four times more abundant than CspB or CspC, depending upon the strain analyzed [33].…”

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

“…Sporulation was induced in 70:30 agar plates for 2 to 3 days as previously described ( 25 , 53 ). C. difficile sporulating cells were washed 5 to 6 times in ice-cold water, incubated overnight at 4°C, and treated with DNase I (New England BioLabs) at 37°C for 60 minutes.…”

Lipoproteins in Gram-Positive Bacteria: Abundance, Function, Fitness

“…In group of rod shape bacteria producing endospores, Lpp genes are also involved in the spore germination process for example GerA/D in Bacillus subtilis or GerS in Clostridium difficile (Pelczar and Setlow, 2008;Li et al, 2014;Diaz et al, 2018). In fact, a Bacillus anthracis lgt mutant lacking the lipid moiety germinated inefficiently; in line, the spore displayed attenuated virulence in the mouse infection model (Okugawa et al, 2012).…”