- Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that can produce as many as 17 different toxins and are responsible to cause a wide array of gastrointestinal (GI) and histotoxic diseases in humans and animals. As individual strains produce a subset of these toxins, C. perfringens strains can be classified into five toxinotypes (A-E). C. perfringens type A strains that produce C. perfringens enterotoxin (CPE) cause food-poisoning (FP) and non-food borne (NFB) diseases in humans and domestic animals which account for a substantial amount of economic loss every year in the United States. To cause a wide variety of diseases in different hosts, C. perfringens possesses several unique characteristics; i) this bacterium is anaerobic and can survive in many conditions that are not regularly exposed to the air or has limited oxygen concentration, such as soil, sewage, GI tract of humans and animals, ii) it can form spores that are resistant to heat and other environmental stresses, iii) by sensing favorable conditions, spores of C. perfringens can rapidly transform into vegetative cells through a process called germination when conditions are favorable, and iv) it can produce variety of toxins in different conditions. Considering these unique features regarding C. perfringens survival in the environment and pathogenicity towards different hosts, it is important to identify genes and proteins that are involved in C. perfringens sporulation and germination process. Also, identification of potential germinant molecules is important to effectively kill spores with minimal effort. The studies conducted for this dissertation were focused to identify and characterize several putative germination and sporulation specific proteins as well as characterize germinant molecules for C. perfringens isolates from various sources.
The initial focus of this dissertation was to investigate the role of two serine proteases of Csp family proteins, CspA and CspC, in C. perfringens spore germination. Previously, it has been shown that another serine protease CspB is involved in the processing of pro-SleC into mature, active SleC. SleC is a cortex lytic enzyme that is involved in the spore cortex hydrolysis in C. perfringens spore germination. Our current study demonstrated that i) cspA and cspC are transcribed as a bicistronic operon during the sporulation and the location of the transcripts are present in mother cell only, ii) both CspA and CspC have role in spore germination, as spores of double cspA-cspC and single cspC mutants exhibited very low extent of germination with different nutrient and non-nutrient germinants than wild-type and complemented strains, iii) cspA and cspC spores were defective in outgrowth and colony formation in nutrient rich media, iv) CspA and CspC are involved in spore cortex hydrolysis by processing of pro-SleC into active SleC, and finally v) CspA and CspC do not activate and regulate the levels of CspB.
The second focus of this dissertation was to identify and characterize several putative sporulation proteins in C. perfringens FP strain SM101. Nine genes (ylzA, ymxH, spoIIM, ytxC, ylxY, ytaF, yyaC1, yyaC2 and bkdR) were identified based on the homology with respective genes in Bacillus subtilis. Among the selected genes, promoters of seven genes (ymxH, spoIIM, ytxC, ytaF, yyaC1, yyaC2 and bkdR) were expressed at different time points during sporulation as determined by β-glucuronidase assay. By using group-II intron based TargeTron technique, null mutations were inserted in seven genes (ymxH, spoIIM, ytxC, ytaF, yyaC1, yyaC2 and bkdR) and analyzed for spore formation and germination properties. Results from this study revealed that, ii) SpoIIM plays very crucial role in spore formation as no spores were produced by spoIIM mutant strain, ii) YmxH, YtxC, YtaF, YyaC1 and BkdR have minor role in spore formation as C. perfringens ymxH, ytxC, ytaF, yyaC1 and bkdR strains showed significantly reduced spore formation compared to the wild-type strain, and iv) YyaC2 and YtxC have roles in spore germination as lower germination rate and decreased DPA release compared to wild-type was observed with yyaC2 and ytxC mutant spores.
The final focus of this dissertation was to identify the germinants for the spores of
C. perfringens strains isolated from diseased animals and investigate the presence and expression of major germination genes in these animal isolates. Spores of C. perfringens animal isolates (horse isolates 106902 and 106903, pig isolates JGS1071 and JGS1807, dog isolates 294442 and 294443, and poultry isolates JGS4122 and JGS4125) were tested with different nutrient and non-nutrient germinants. Spores of C. perfringens animal isolates showed very low germination with different nutrient, non-nutrient germinants, rich media and cell culture media compared to human FP isolate SM101. When spores of four strains (106903, JGS1807, 294442, and JGS4122) of animal isolates were tested with all amino acids at pH 7.0 and pH 6.0, the germination rate varied among strains and was not consistent. However, most of the strains germinated better at pH 6.0 than at pH 7.0 with all amino acids tested. Germination assay with L-cysteine and L-lysine at different pHs (5.0 to 9.0) and different concentrations (1 mM to 200 mM) revealed that except strain JGS4122, all other strains did germinate better with higher concentrations of L-cysteine and L-lysine at pH range of 5.5-7.0. qRT-PCR analyses with RNA extracted from 8 h sporulation culture showed that level of expressions of gerKA, gerKB, gerKC and gerAA were higher in animal isolates compared to SM101. However, the transcript levels of cspA, cspB, cspC and sleC were lower in animal isolates. This finding was further confirmed by the quantitative Western blot analyses as the levels of both CspB and SleC was low in C. perfringens animal isolates than in human isolates. Together this finding suggests that C. perfringens animal isolates may have low activity in cortex hydrolysis that results in the lower spore germination.
Collectively, our studies contribute towards understanding the mechanism of sporulation and germination in pathogenic bacterium C. perfringens by 1) dissecting the role of two new Csp proteases in C. perfringens spore germination; 2) identifying and characterizing new sporulation and germination genes in the pathogenic bacterium C. perfringens, and 2) characterizing germination of spores of C. perfringens isolated from diseased animals.