Figure 1.

Structure of the spore and life cycle.

a. The robust properties of the spore are due to its multi-layered structure, with each layer contributing to the overall resilience [25]. The dense core is dehydrated due to the presence of up to 25% Ca-DPA, and the DNA is bound to and protected by the small acid-soluble proteins. Surrounding the core is an extremely impermeable inner membrane and the germ (primordial) cell wall. This thin layer of peptidoglycan has the same composition as in vegetative cells – and will become the nascent cell wall during germination. Around the germ cell wall is a much thicker layer of peptidoglycan, the cortex. Within the cortex peptidoglycan approximately 25% of the N-acetylmuramic acid moieties are modified to muramic-δ-lactam, and there are few crosslinks between adjacent N-acetylmuramic acid-N-acetylglucosamine polymers [26]. This results in a much more flexible peptidoglycan structure, with a distinct chemical signature that allows specific degradation during germination, without risk of compromising the germ cell wall. The cortex is surrounded by a second membrane, derived from the mother cell during engulfment, and then finally, the protein coat. The coat is a lamellar structure consisting of a large number of often highly crosslinked proteins. The outermost layers of the coat in C. difficile appear less organized, with an amorphous structure, and vary in thickness. This layer has been described as exosporium but does not appear to have the same loose-attachment and hexameric organization seen in other spore-formers [27]. Some of these structures can be seen in the transmission electron micrograph of a negative-stained, thin-sectioned spore on the right. b. When conditions are favorable, a C. difficile cell will normally be divided by binary fission. However, when environment conditions are less than ideal, most likely due to nutrient limitation, the cell can enter the sporulation pathway instead. Upon initiation of sporulation, the cell first undergoes asymmetric septation, producing the mother cell compartment and smaller forespore. A copy of the genome is transferred into the nascent spore, and the forespore is then engulfed by the mother cell - a phagocytosis-like event that results in an immature spore, bounded by two membranes, free in the cytoplasm of the mother cell. The spore then undergoes a maturation process whereby the DNA is compacted by the small acid-soluble proteins, Ca-DPA is synthesized, the core is dehydrated, and cortex and protein coats are synthesized. The final mature spore is released by lysis of the mother cell.