Graduate Thesis Or Dissertation
 

The Signaling Role of Divalent Cations in Clostridium perfringens Spore Germination

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  • Clostridium perfringens is an anaerobic, gram-positive, rod-shaped, spore-forming bacterium that leads to a broad range of diseases in humans and animals [23, 27, 50]. Among seven C. perfringens types (type A-G), type F is known to be the most common bacteria that is responsible for causing human food-borne disease outbreaks worldwide [23, 27, 50]. This pathogen becomes a problem for human health because of its ability to form metabolically dormant spores that can tolerate environmental stresses such as radiation, pH, osmotic stress, desiccation, and temperature [62, 63, 73, 84]. As a result, spore resistance causes a broad range of harmful effects including food-poisoning (FP), food spoilage, and gastrointestinal diseases [50, 80]. However, in order to have these negative effects, dormant spores must undergo germination to become metabolically active cells [36, 50, 59]. Therefore, the germination process starts when the spores sense germinants through specific receptors located in the spore’s inner membrane [4, 28, 61, 86]. The germination process can be started through a different type of germinants including, cationic surfactants, amino acids, and enzymes [30, 66, 67, 83, 91]. The previous study has shown that exogenous dipicolinic acid (DPA) chelated with calcium (Ca2+) (Ca-DPA) can significantly enhance spore germination in C. perfringens [20, 60, 66, 83]. However, it is unclear whether Ca2+ or DPA alone is needed to enhance spore germination. Therefore, in the current study, we aimed to evaluate the possible role of Ca2+ and other divalent cations present in the spore’s core (Mn+2 and Mg+2) in germination of C. perfringens spores. To accomplish this, our study consists of three parts. The first part of this study evaluates the role of Ca2+ and DPA in the spore germination process. We found that Ca2+, but not DPA, is sufficient to trigger spore germination in C. perfringens FP isolates. All tested calcium salts (calcium-chloride, calcium-carbonate, or calcium-nitrate) induced germination of spores of C. perfringens FP isolates, indicating that exogenous Ca2+ ion is significant for spore germination. The second part of this study evaluates whether spore-specific divalent cations (Mn2+, and Mg+2) can induce spore germination. Our result suggested that all spore core-specific divalent cations (Ca2+, Mn2+ and Mg+2) contribute to spore germination in C. perfringens FP isolates with slight variations in the percentage of germination. In contrast, non-core-specific divalent cation Zn+2 did not induce germination of spores of C. perfringens FP isolates. The third part of this study evaluates whether the exogenous or endogenous spore-specific divalent cations (Ca2+, Mn2+ and Mg2+) are needed to induce C. perfringens spore germination. Our results indicated that endogenous Ca2+ and Mg2+ are not necessary to initiate the spore germination process. While exogenous and endogenous Mn+2 are needed to enhance spore germination. In conclusion, our results indicated that spore-specific divalent cations play a signaling role in C. perfringens (FP) spore germination. Further germination assay on spores of germinant receptor mutants and cortex-lytic enzyme mutants in the presence of spore-specific divalent cations should clarify the possible mechanism of divalent cation mediated spore germination. In addition, further experiments in food products is needed to evaluate whether this specific concentration and pH of divalent cations can also trigger the spore germination in food products.
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