|Abstract or Summary
- Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these toxins. Although a small percentage of C. perfringens isolates (mostly belonging to type A) produce C. perfringens enterotoxin (CPE), these are very important human gastrointestinal (GI) pathogens, causing C. perfringens type A food poisoning (FP) and nonfood-borne GI diseases (NFBGID). Due to its anaerobic nature and the ability to form extremely resistant spores found ubiquitously in the environment, to cause the wide array of C. perfringens-associated diseases (CPAD), these C. perfringens spores must germinate, release the nascent cell, grow and produce their toxins. Therefore, germination of C. perfringens spores is the initial and perhaps most important step for the progression of diseases in animals and humans. Although extensive research has been conducted on the mechanism of spore germination of Bacillus species, very few studies of spore germination have been conducted in Clostridium species mainly due to the lack of molecular genetic tools. Genomic comparisons reveal significant differences in the backbone of the germination apparatus between Bacillus and Clostridium species. Consequently, a detail understanding of the molecular mechanism of germination of C. perfringens spores is essential for the development of novel preventive strategies for CPAD as well as diseases caused by other pathogenic Clostridium species.
The first focus of this work was to identify and characterize the germinants and the receptors involved in C. perfringens spore germination. Result from these studies found differential germination requirements between spores of FP and NFBGID isolates in that: (i) while a mixture of L-asparagine and KCl was a good germinant for spores of FP and NFBGID isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in FP isolates only; and ii) L-alanine and L-valine induced significant germination of spores of NFBGID but not FP isolates. In contrast to B. subtilis, C. perfrinegns genomes sequenced to date possess no tricistronic gerA-like operon, but has a monocistronic gerAA that is far from a gerK locus. The gerK locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream and in the opposite orientation to gerKA-gerKC. Consequently, through the construction of mutations into strain SM101, a C. perfringens FP isolate, the role of gerAA, gerKA-gerKC and gerKB genes in C. perfringens spore germination were investigated. Results indicated that KCl, L-asparagine and Ca-DPA required GerKA and/or GerKC receptors, while GerAA and GerKB played an auxiliary role in germination. Lack of GerKA and/or GerKC, and GerKB significantly reduced spores colony forming efficiency, indicating a role in spore viability.
The fact that C. perfringens spores lacking the main germinant receptor(s) proteins, GerKA and/or GerKC, are still able to germinate albeit poorly compared to wild-type, and that C. perfringens spores germinate with K+ ions alone, raises the hypothesis that GrmA-like antiporters might also play some role in C. perfringens spore germination. Two putative GrmA-like antiporters (i.e., GerO and GerQ) are encoded in the genome of all C. perfringens sequenced to date. This study shows that gerO and gerQ genes are expressed uniquely during sporulation and the mother cell compartment of the sporulating cell. Complementation studies of K+ uptake and Na+ sensitive E. coli mutants indicate that while GerO is capable of translocating K+ and Na+, GerQ is only capable of translocating, to a small extent, Na+ ions. Spores lacking GerO had defective germination in rich medium, KCl, L-asparagine, and Ca-DPA, but not with dodecylamine, defect that might be prior to DPA release during germination. In contrast, loss of GerQ had a much smaller effect on spore germination. Two adjacent Asp residues, important in ion transcloation of the E. coli Na+/H+ antiporter NhaA were also present in GerO, but not GerQ, and replacement of these residues for Asn reduced the protein’s ability to complement gerO spores. Although results from this study indicate that putative antiporters have some role on C. perfringens spore germination, it is unclear whether their role is direct or during spore formation.
C. perfringens type A FP spores are capable of germinating with K+ ions, an intrinsic mineral of meats commonly associated with FP. Inorganic phosphate (Pi) is also intrinsically found in meat products. Consequently, we hypothesized that FP spores are capable of germinating in presence of Pi. Results from this study show that spores of the majority of FP, but not NFBGID isolates, are able to germinate in presence of Pi. Pi-induced germination of FP spores is primarily through the GerKA and/or GerKC protein, while GerAA and to a much lesser extent, GerKB, play auxiliary roles. The putative Na+/K+-H+ antiporter, GerO, is also required for normal Pi-induced germination. These results suggest that the differential germination phenotypes between spores of FP and NFGID isolates is tightly regulated by their adaptation to different environmental niches.
A second focus of this work was to investigate the mechanism of signal transduction between the germinant receptors and the downstream effectors. In B. subtilis, the SpoVA proteins have been associated with Ca-DPA uptake and subsequent release during sporulation and germination, respectively. In addition, Ca-DPA acts as a signal molecule for cortex hydrolysis during B. subtilis spore germination, activating the cortex lytic enzyme (CLE) CwlJ. Results from this study show that in contrast to B. subtilis spoVA mutants, where spores lyse quickly during purification, C. perfringens spoVA spores were stable and germinated similarly as wild-type spores. These results suggest major differences in the regulation of the germination pathway between C. perfringens and B. subtilis, and suggest that activation of CLEs in C. perfringens might be through a different pathway than the Ca-DPA pathway of B. subtilis.
A third focus of this work was to investigate the in vivo role of the CLE involved in peptidoglycan (PG) spore cortex hydrolysis during C. perfringens spore germination. Two C. perfringens CLEs (i.e., SleC and SleM) degrade PG spore cortex hydrolysis in vitro, however, due to lack of genetic tools, their in vivo role in spore germination remains unclear. Results from this study show that C. perfringens sleC spores released their DPA slower than wild-type and were not able to germinate with nutrients and non-nutrient germinants. In contrast, sleM spores germinated similar as wild type in presence of nutrient and non-nutrient germinants, indicating that while SleC is essential for cortex hydrolysis and viability of C. perfringens spores, SleM although can degrade cortex PG in vitro, is not essential.
A fourth focus of this work was to investigate the in vivo role of the Csp proteases in the initiation of cortex hydrolysis. In vitro work has shown that Csp proteins process the inactive proSleC into the mature enzyme, SleC. However, the in vivo role of the Csp proteins has not been established. In this study, spores a C. perfringens cspB mutant exhibited significantly less viability than wild-type spores, and were unable to germinate with either rich medium or Ca-DPA. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis. Results from this study indicate that CspB is essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. In contrast to B. subtilis, Ca-DPA did not activate the CLEs during spore germination present in cspB spores supporting previous results that Ca-DPA acts trough the GerKA and/or GerKC receptor.
A final focus of this work was to develop a strategy to inactivate C. perfringens spores in meat products. C. perfringens spores posses high heat and pressure resistance, however, they loss their resistance properties during early stages of germination. In contrast to B. subtilis spores, germination of C. perfringens spores could not be triggered with low pressures. However, they germinated efficiently when heat activated in presence of L-asparagine and KCl at temperatures lethal for vegetative cells, and these germinated spores were efficiently inactivated by subsequent treatment with pressure assisted thermal processing (586 MPa at 73ºC for 10 min). This study shows the feasibility of a novel strategy to inactivate C. perfringens spores in meat products formulated with germinants.
Collectively, the present study contributes to the understanding of the mechanism of spore germination in the pathogenic bacterium C. perfringens, and developed an alternative and novel strategy to inactivate C. perfringens spores in meat products.