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Inhibition of Clostridium botulinum by strains of Clostridium perfringens isolated from soil.

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.

Antibiosis↗

Effect of Clostridium toxoids, especially of Clostridium perfringens toxoid, on mouse transplanted tumors.

The antitumor activities of toxoids of Clostridium perfringens, C. nouyi, C. septicum, and C. tetani were tested against sarcoma-180 and Ehrlich carcinoma. Among them, C. perfringens toxoid showed a high antitumor activity against the growth of the implanted sarcoma-180 ascites form. The results of the inhibiting effect of C. perfringens toxoid on Nakahara-Tokuzen-Fukuoka (NTF) reticulum cell sarcoma and methylcholanthrene-induced fibrosarcoma were also described.

Animals↗

Effect of okadaic acid on the cytotoxic activity of Clostridium difficile toxin B and Clostridium sordellii toxin L.

Clostridium difficile toxin B and Clostridium sordellii toxin L, which are immunologically related toxins, possess a cytotoxic activity inducing depolymerization of microfilaments and cellular retraction of cell bodies that are different for toxin B- and toxin-L-treated cells. The biological mechanisms responsible for these effects are unknown, but a previous study revealed that both toxins induce modification of phosphorylation of cellular proteins extracted from toxin B- and toxin L-treated cells without changes in protein kinase C activity or cAMP concentration. In the present study, we have investigated the effect of okadaic acid, an inhibitor of protein phosphatases, on the cytotoxic activity of toxins B and L in MacCoy cells. Firstly, we reveal by cytotoxic assay and staining of F-actin that okadaic acid (1 microM or higher) induces depolymerization of microfilaments and cellular morphological modifications which are similar to that of cells treated with toxin L. Secondly, we show that 1 microM okadaic acid potentials the cytotoxic activity of toxin L but not of toxin B. These observations suggest that the cytotoxic mechanisms induced by okadaic acid and toxin treatment are partly common, indicating that an increase in phosphorylation favors the cytotoxicity of toxin L. Since okadaic acid had no influence on the cytotoxicity of toxin B, we suggest that toxin B and L, alter the cells by different cellular biological mechanisms.

Actins↗

Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.

Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.

Journal Article↗

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin.

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.

ADP Ribose Transferases↗

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of beta-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60 degrees C and produced 9 U of beta-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.

Journal Article↗

Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum).

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.

Journal Article↗

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum").

Clostridium beijerinckii ("Clostridium butylicum") NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.

Journal Article↗

Variability of Clostridium difficile surface proteins and specific serum antibody response in patients with Clostridium difficile-associated disease.

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors, such as surface proteins. Clostridium difficile surface proteins have been identified as (i) adhesins (the flagellar cap protein FliD; the flagellin FliC; and the cell wall protein Cwp 66 with a two domain-structure [Cw 66 N-terminal and Cwp 66 C-terminal domains]) and (ii) protease (the Cwp 84 protein). To address the roles of these proteins in the pathogenesis of Clostridium difficile and to identify vaccine antigen candidates, we analyzed the variability of the proteins and their immunogenicities in 17 patients with C. difficile-associated disease. PCR-restriction fragment length polymorphism analysis of amplified gene products revealed interstrain homogeneity with fliC and fliD, in contrast to cwp 66 genes. Immunoblot analysis showed that FliC and FliD were detected in the majority of isolates. The N-terminal domain of Cwp 66 and Cwp 84 were present in all strains tested, in contrast to the Cwp 66 C-terminal domain, the expression of which was heterogeneous. The 17 sera from the corresponding patients were analyzed by enzyme-linked immunosorbent assay to detect antibodies directed against these proteins. Many patients developed antibodies to FliC, FliD, Cwp 84, and the Cwp 66 C-terminal domain, but not to the Cwp 66 N-terminal domain. In conclusion, this study confirms the expression of these surface proteins of C. difficile during the course of the disease. In addition, the FliC, FliD, and Cwp 84 proteins appeared to be good potential vaccine candidates.

Animals↗

Serum antibody response to Clostridium difficile toxins in patients with Clostridium difficile diarrhoea.

Consecutive serum samples from 61 patients with Clostridium difficile diarrhoea were investigated for antibody response to C. difficile toxins A and B in an indirect enzyme immunoassay (ELISA) and in a neutralization assay against C. difficile cytotoxin. Sera from 64 blood donors, elderly healthy females and patients with other known intestinal enteropathogens served as controls. An immune response was detected by ELISA in approximately half of the patients with C. difficile diarrhoea. The specificity of the ELISA was 94% or 97%, depending on the control material used. Furthermore, a correlation was found between clinical recovery without relapse of C. difficile diarrhoea and high IgG titers to toxin B in the ELISA, and/or appearance of neutralizing antibodies. It is concluded that the ELISA for detection of serum antibodies to C. difficile toxins may be of diagnostic value in combination with the conventional tissue culture assay for cytotoxin in stool. High ELISA IgG titres to toxin B and/or the appearance of neutralizing antibodies may also be a positive prognostic sign in patients with C. difficile diarrhoea.

Adolescent↗

Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755).

Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E). Here we have determined the nucleotide sequences of the BoNT/E genes of these two C. butyricum strains and from C. botulinum E strain Beluga. We show that the sequences of the BoNT/E genes from the two C. butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C. botulinum. Our data suggest a transfer of the BoNT/E gene from C. botulinum to the originally nontoxigenic C. butyricum strains.

Amino Acid Sequence↗

Similarity in nucleotide sequence of the gene encoding nontoxic component of botulinum toxin produced by toxigenic Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike.

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C. butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.

Amino Acid Sequence↗

Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

Amino Acid Sequence↗

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.

Clostridium↗

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗

Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5).

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same "global behavior" was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH2 flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD+ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.

Clostridium acetobutylicum↗

Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E.

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.

Bacterial Toxins↗