Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5-20% of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99% of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens.