Coat and enterotoxin-related proteins in Clostridium perfringens spores

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.     

Activation and injury of Clostridium perfringens spores by alcohols

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Activation and injury of Clostridium perfringens spores by alcohols.

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.     

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores.

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.     

Extraction of Clostridium perfringens spores from bottom sediment samples.

Two extraction-separation procedures were developed and evaluated for use in conjunction with the mCP membrane filter method for the enumeration of Clostridium perfringens spores in bottom sediments. In the more facile of the two procedures, a distilled-water suspension of the sediment sample is pulse sonicated for 10 s and allowed to settle. Portions of the supernatant are then removed for membrane filtration. This procedure is recommended for general use. The more complicated procedure is recommended for situations in which the presence of high levels of toxic materials is suspected or in which relatively low spore densities are present in fine silts. In this procedure, sonication is followed by a distilled water wash. The centrifuged sediment is resuspended in distilled water and mixed with the components of a two-phase separation system (50% polyethylene glycol in distilled water and 25% sucrose in 3 M phosphate buffer [pH 7.1]). After equilibration of the system and low-speed centrifugation, the top phase and interphase are removed, mixed, and membrane filtered. The recoveries of C. perfringens spores by the two procedures, when used in conjunction with the mCP method, were comparable to each other and significantly greater than those by the British most-probable-number method. It was estimated that more than 85% of the spores were recovered by the procedures. The precision of the sonicate-and-settle-mCP procedure was markedly better than that obtained theoretically by the most-probable-number method and approached that theoretically attributable to counting an average of 85 colonies on each of two plates.     

The effect of hydrogen peroxide on spores of Clostridium perfringens

Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H₂O₂ in the presence of Cu²⁺ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H₂O₂ involves formation of OH by Cu²⁺-catalysed decomposition of the peroxide. DTT-treated spores took up Cu²⁺ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu²⁺.     

Sensitization of Clostridium perfringens spores to heat by gamma radiation.

Spores of Clostridium perfringens, type A, were given separate or sequential treatments of gamma radiation (0 to 0.7 Mrad) and/or high temperature (93 to 103 degrees C). Prior heating, sufficient to inactivate 40 to 99% of the viable spores, had no effect on the subsequent radiation inactivation rate. Prior irradiation had a sensitizing effect on subsequently heated spores. The degree of sensitization to heat, as measured by thermal inactivation rate, increased with increased radiation pretreatment dose.     

Sensitization of Clostridium perfringens spores to heat by gamma radiation.

Spores of Clostridium perfringens, type A, were given separate or sequential treatments of gamma radiation (0 to 0.7 Mrad) and/or high temperature (93 to 103 degrees C). Prior heating, sufficient to inactivate 40 to 99% of the viable spores, had no effect on the subsequent radiation inactivation rate. Prior irradiation had a sensitizing effect on subsequently heated spores. The degree of sensitization to heat, as measured by thermal inactivation rate, increased with increased radiation pretreatment dose.     

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T_lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT_release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T_lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T_lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T_lag and ΔT_release values in dodecylamine germination that does not utilize GRs. Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.     

Purification and properties of spore-lytic enzymes from Clostridium perfringens type A spores

Spores of Clostridium perfringens contain at least two spore-lytic enzymes active in hydrolysing cortical peptidoglycan. One enzyme has been purified 1800-fold and has a molecular weight of 17 400 determined from chromatography on Sephadex G-75. Two protein bands were apparent after SDS-PAGE. The isolated enzyme was investigated for response to temperature, pH, ionic strength and enzyme inhibitors, and for mode of action. A second enzyme activity, differing from the first in apparent molecular weight (29 800) as determined by gel exclusion chromatography, and also in its pH optimum and activity on cortical substrate, was also isolated, although not purified to the same extent.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of Clostridium perfringens

A precursor of the spore-lytic enzyme of Clostridium perfringens was extracted with alkali from dormant spores of the organism. The enzyme precursor was activated by incubating it with germinated spores which had been treated with alkali. The activation was greatly enhanced by the addition of 3-phosphoglycerate, suggesting that the conversion of precursor to active enzyme depends on endogenous energy-producing metabolism during germination.     

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores

The mechanism(s) of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms by base-exchange technique and these spores were heated at 95°C. Of the four ionic forms, i.e. Ca²⁺, Na⁺, H⁺ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.