Graduate Thesis Or Dissertation
 

New Insights into the Molecular Mechanism of Clostridium perfringens Spore Germination

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  • C. perfringens is a spore-forming, gram-positive, anaerobic pathogenic bacterium capable of causing a wide variety of diseases in both humans and animals. However, the two most common illnesses in humans are C. perfringens type A food poisoning (FP) and non-food-borne (NFB) gastrointestinal (GI) illnesses. Interestingly, these two major diseases are caused only by C. perfringens Type A isolates that are able to produce the C. perfringens enterotoxin (CPE). Importantly, the CPE-encoding gene (cpe) can be found in different places in FP and NFB isolates. C. perfringens FP isolates carry the cpe gene on the chromosome, while NFB isolates causing GI illnesses (i.e., sporadic diarrhea and antibiotic-associated diarrhea) carry a plasmid-borne copy of the cpe. C. perfringens spores are highly resistant and can survive in the environment for decades. When spores are in a favorable environment, they can initiate germination and return to active growth to cause disease. Spore germination is an early step and essential stage in the progression of C. perfringens infection in humans and animals. Recent findings have identified the germinants of spores of C. perfringens FP and NFB isolates. A variety of germinants can initiate the process, including nutrients, amino acids, cationic surfactants, and enzymes termed germinant. Further understanding of the germination of Clostridium species is needed, since Bacillus species spore germination has been studied well. In the first study, we demonstrated the individual role of each of these germinant receptors (GRs) in C. perfringens spore germination. To reach the goal, we constructed mutant strains carrying single mutations in gerKA or gerKC, and double mutant gerKB gerAA, and characterized the germination phenotypes of their spores in the presence of nutrient and non-nutrient germinants. Through Western blot analyses, the precise location of GerKC protein in spores was also determined. This study offers the following findings: (i) Spores of gerKC mutant did not germinate with KCl, L-asparagine, a mixture of asparagine and KCl, and NaPi at pH 6.0. (ii) The gerKC spores germinated poorly compared to wild-type and other GRs mutant spores with the non-nutrient germinants dodecylamine and a 1:1 chelate of Ca²⁺ and dipicolinic acid. (iii) The germination defect in gerKC spores was restored by complementing the gerKC mutant with wild-type gerK operon, indicating that GerKC is essential for germination of C. perfringens spores. (iv) GerKC is essential for the release of DPA from the spore's core during germination with KCl and dodecylamin. (v) GerKC is also essential for spore’s viability; Finally, (vi) GerKC localizes in the spore’s inner membrane. A second study of this work investigated the precise location for the Csp proteases CspB and cortex lytic enzymes SleC in spores of C. perfringens FP strain SM101. It was shown that CspB and pro-SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. In addition, quantitative Western blot analyses demonstrated that there are approximately 2,000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore. The third study was to identify and characterize the germinants and receptors involved in C. perfringens NFB strain F4969. Results from these studies indicate that NFB strain F4969 germinates with AK in a cooperative manner, while FP strain SM101 mixture of L-Asparagine and KCl (AK) components are capable of triggering spore germination through independent pathways. Spores of gerKA-KC, gerKA, gerKC, gerAA, and gerKB knock-out mutants indicate that (i) germination of FP and NFB spores differs significantly in rich media and several defined germinants; (ii) L-asn and KCl induced germination in NFP F4969 spores cooperatively, while either germinant alone triggers germination of FP spores; (iii) GerKC and GerAA proteins are required for normal germination of NFB spores with AK and L-cys;(iv) The colony-forming ability of gerKC or gerKA-KC spores was significantly lower than that of wild-type spores; although the ability of gerAA spores to outgrow was significantly affected, they gave rise to similar titers as wild-type spores. Together, this dissertation study will help further understanding of the mechanism of spore germination of C. perfringens and the insights into the roles of spore germination for both FP and NFB CPE-producing C. perfringens isolates.
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