Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin ( cpe ) gene (C- cpe ), while C. perfringens -associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P- cpe ). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C- cpe and P- cpe isolates in that (i) while a mixture of l -asparagine and KCl was a good germinant for spores of C- cpe and P- cpe isolates, KCl and, to a lesser extent, l -asparagine triggered spore germination in C- cpe isolates only; and (ii) l -alanine or l -valine induced significant germination of spores of P- cpe but not C- cpe isolates. Spores of a gerK mutant of a C- cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with l -asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca ²⁺ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C- cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C- cpe and P- cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold, and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involve their production of a variant small acid-soluble protein-4 that binds more tightly to spore DNA than to the small acid-soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the United States. During this foodborne disease, C. perfringens is ingested with food and then, by using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines.

Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two Clostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of C. perfringens spores. C. perfringens sleC spores did not germinate completely with nutrients, KCl, or a 1:1 chelate of Ca ²⁺ and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleC spores was 10 ³ -fold lower than that of wild-type spores. However, sleC spores incubated with various germinants released most of their DPA, although slower than wild-type or sleM spores, and DPA release from sleC sleM spores was very slow. In contrast, germination and viability of sleM spores were similar to that of wild-type spores, although sleC sleM spores had 10 ⁵ -fold-lower viability. These results allow the following conclusions about C. perfringens spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca ²⁺ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca ²⁺ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l -asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca ²⁺ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10 ³ - to 10 ⁴ -fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

Clostridium difficile is the causative agent of the majority of antibiotic associated diarrhoea cases. C. difficile spores are recognized as the persistent and infectious morphotype as well as the vehicle of transmission of CDI. However, there is a lack of knowledge on how C. difficile spores interact with the host’s epithelial surfaces. In this context, we have characterized the ability of C. difficile spores to adhere to human Caco-2 cells. Despite the similarities in spore-surface hydrophobicity between spores of C. difficile and Clostridium perfringens (another enteric pathogen that also sporulates in the gut), spores of C. difficile adhere better to Caco-2 cells. Adherence to Caco-2 cells was significantly reduced when C. difficile spores were treated with trypsin. Sonication of C. difficile spores altered the ultrastructure of the outermost exosporium-like structure, releasing two protein species of ~40 kDa and significantly reduced spore hydrophobicity and adherence to Caco-2 cells. Using a trifunctional cross-linker, we were able to co-immunoprecipitate four protein species from the surface of Caco-2 cells. In conclusion, this study provides evidence that C. difficile spores adhere to human intestinal enterocyte-like cells through spore- and enterocytic-surface-specific ligand(s) and/or receptor(s).

Previous work has suggested that a group of α/β-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding α/β-type SASP in this organism. We have now used antisense RNA to decrease levels of α/β-type SASP in C. perfringens spores by ∼90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of α/β-type SASP in the resistance of C. perfringens spores.