The Sialidases of Clostridium perfringens Type D Strain CN3718 Differ in Their Properties and Sensitivities to Inhibitors

Clostridium perfringens causes histotoxic infections and diseases originating in animal or human intestines. A prolific toxin producer, this bacterium also produces numerous enzymes, including sialidases, that may facilitate infection. C. perfringens type D strain CN3718 carries genes encoding three sialidases, including two large secreted sialidases (named NanI and NanJ) and one small sialidase (named NanH) that has an intracellular location in log-phase cultures but is present in supernatants of death phase cultures. Using isogenic mutants of CN3718 that are capable of expressing only NanJ, NanI, or NanH, the current study characterized the properties and activities of each sialidase. The optimal temperature determined for NanJ or NanH enzymatic activity was 37°C or 43°C, respectively, while NanI activity increased until temperature reached 48°C. NanI activity was also the most resistant against higher temperatures. All three sialidases showed optimal activities at pH 5.5. Compared to NanJ or NanH, NanI contributed most to the sialidase activity in CN3718 culture supernatants, regardless of the substrate sialic acid linkage; NanI also released the most sialic acid from Caco-2 cells. Only NanI activity was enhanced by trypsin pretreatment and then only for substrates with an α-2,3- or α-2,6-sialic acid linkage. NanJ and NanI activities were more sensitive than NanH activity to two sialidase inhibitors (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B). The activities of the three sialidases were affected differently by several metal ions. These results indicated that each C. perfringens sialidase has distinct properties, which may allow these enzymes to play different roles depending upon environmental conditions.     

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin

Abstract A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay ( r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining ( r = 0.995).     

Neurological disorders produced by Clostridium perfringens type D epsilon toxin

Although the epsilon toxin of Clostridium perfringens type D produces disease in many species of domestic livestock, neurological disturbance is more common and better studied in sheep. High levels of circulating toxin, especially in lambs, cause cerebral microvascular endothelial damage with disruption of the blood-brain barrier leading to a severe, diffuse vasogenic oedema and an acute or peracute clinical course to death. With lower toxin levels, or in partially immune sheep, a focal, bilaterally symmetrical encephalomalacia sometimes occurs in selectively vulnerable brain regions after a more protracted clinical course, but the pathogenesis is uncertain.     

The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin

The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium perfringens is described. All five monoclonal antibodies located three proteins in a trypsinized preparation of epsilon prototoxin. These proteins were located at 37.6 kDal, 35.6 kDal and 33.7 kDal by Western blots. Two of the monoclonal antibodies, M26/2 and M27/12, neutralized epsilon toxin in the mouse lethality assay. Four of the five monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay procedures.     

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation.     

Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia

Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia.     

Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D

The sequence of the epsilon toxin gene of Clostridium perfringens type D was determined and compared with that of the previously reported type B sequence. It showed two nucleotide changes in the open reading frame, giving rise to one amino acid substitution. The promoter sequences were not homologous, and different putative -35 and -10 regions have been identified in each. The sequence information was used to develop PCR primers which were specific for the epsilon toxin gene. The utility of this system for identifying type B or D strains of C. perfringens was demonstrated.     

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.     

In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.     

Effects of Clostridium perfringens alpha and epsilon toxins in the bovine gut

Clostridium perfringens alpha and epsilon toxins produce enterotoxaemia in sheep and goats. However, the information regarding the pathophysiology of alpha and epsilon toxins in the bovine intestine is still scanty. In this study, intestinal loops were performed in the ileum and colon of three one-week-old Holstein and two four-week-old crossbreed calves. Laparotomy was performed in all calves under anaesthesia and four loops -three cm long- were performed in the small and large intestines. For both intestines, loops were inoculated with alpha or epsilon toxins. Tissue samples from all loops were obtained and processed for routine histology and for transmission electron microscopy. Congestion was observed in toxin treated loops. Fluid accumulation in the gut lumen was prominent in all treated loops, but in epsilon treated ones the mucous was also haemorrhagic. The histology revealed large amount of exfoliated epithelial cells in the lumen of alpha toxin treated loops and severe haemorrhage was observed in the lamina propria of epsilon toxin treated colonic loops. Despite some necrotic exfoliated enterocytes, no ultraestructural changes were observed in alpha toxin treated loops, though with epsilon toxin the loops exhibited dilation of the intercellular space in the mucosa of both, small and large intestines. These observations indicate that both, alpha and epsilon toxins can alter the intestinal barrier, in calves and are pathogenic for this species.     

Pathogenesis of Hobbs' heat-sensitive spore forming Clostridium perfringens type A strain.

Food poisoning in man due to heat-sensitive strains of Cl. perfringens type A appeared to be mediated through enterotoxin synthesized in vivo during sporulation. A minimum of 2.0 X 10(5) vegetative cells suspended in sporulation medium was sufficient to elicit gut-loop response in rabbits. The functional disturbance in the gut as well as the structural changes were progressive and proportional to the size of the inoculum up to a dose limit of 2.0 X 10(7) vegetative cells and beyond this the changes remained steady.     

Functional and structural characterization of soluble recombinant epsilon toxin of Clostridium perfringens D, causative agent of enterotoxaemia

Clostridium perfringens types B and D are responsible for enterotoxaemia, one of the major causes of cattle mortality and is therefore of great economic concern. The epsilon toxin produced by the organism is the major antigenic determinant and has been directly implicated for the disease causation. In the present paper, we evaluated the biological activity of the recombinant epsilon toxin (rEtx) produced as soluble protein in Escherichia coli. The rEtx was purified to near homogeneity by a one-step anion-exchange chromatography. The immunological identity of purified rEtx was confirmed by Western blotting using a monoclonal antibody against the native toxin. The rEtx formed heptamer in the Madin–Darby canine kidney (MDCK) cells and synaptosomal membrane of mouse brain and was cytotoxic to the MDCK cells with a CT₅₀ of 30 ng/ml. The rEtx was highly stable and its thermostability profile related well with its biological activity. The rEtx was purified in large amounts and exhibited all the properties of native toxin and therefore can be used for the development of vaccine against the pathogen.     

Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract

The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract.     

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.     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.