Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate

The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969 and SleCSM101 by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969 was processed into mature SleCF4969 in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of ?-asparagine and KCl, or with a 1:1 chelate of Ca2+ and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.     

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.     

Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.

The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.     

The N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores

A spore cortex-lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. After cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present results demonstrated that the cleaved N-terminal prepeptide remained associated with C35. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide-C35 complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide-C35 complex, and the full length of the N-terminal presequence was needed to fulfil its role. Although the C-terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.     

Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM.

Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM. Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores. Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species. The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5. It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss. The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed.     

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.     

A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme.

A gene (sleB) encoding a 24-kDa germination-specific spore cortex-lytic enzyme, probably an N-acetylmuramyl-L-alanine amidase, was cloned from Bacillus cereus, and its nucleotide sequence was determined. It was indicated that the enzyme is produced as a 259-residue protein with a signal sequence of 32 residues and is present in dormant spores in its active form. Sulfhydryl reagents inactivated the enzyme, but mutation of a single cysteine of the protein, Cys-258, to Gly did not cause complete inactivation of the enzyme, suggesting that the residue does not function as the catalytic center of enzyme.     

Clostridium perfringens alpha-toxin: characterization and mode of action

Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C. perfringens. The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin. Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin. To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.     

Subcellular localization of a germiantion-specific cortex-lytic enzyme, SleB, of Bacilli during sporulation

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.     

Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin

Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.     

Multilocus enzyme typing of human and animal strains of Clostridium perfringens

Abstract The stability of plasmids introduced in Lactobacillus lactis IL1403 was followed in continuous cultures. Four strains with or without pIL205 and pIL252 or pIL253 (low or high copy number) were studied. pIL205 was remarkably stable even after 180 generations, suggesting the presence of a partitioning system which is still unidentified. In contrast, pIL252 and pIL253 were unstable, in the presence as well as in the absence of pIL205. The multicopy plasmid pIL253 was nevertheless more stable than pIL252 (90% loss after 180 and 60 generations, respectively). The instability was not related to an incompatibility between pIL205 and pIL252 (or pIL253). The segregational instability of pIL252 and pIL253 plasmids could be explained by the loss of the resolvase gene during their construction.