Deletion analysis of the Clostridium perfringens enterotoxin

To further our knowledge of the structure-function relationship and mechanism of action of the Clostridium perfringens enterotoxin (CPE), a series of recombinant CPE (rCPE) species containing N- and C-terminal CPE deletion fragments was constructed by recombinant DNA approaches. Each rCPE species was characterized for its ability to complete the first four early steps in the action of CPE, putatively ordered as specific binding, a postbinding physical change to bound CPE, large-complex formation, and induction of alterations in small-molecule membrane permeability. These studies demonstrated that (i) at least 44 amino acids can be removed from the N terminus of CPE without loss of cytotoxicity, (ii) removal of the first 53 amino acids from the N terminus of CPE produces a fragment that appears to be noncytotoxic because it cannot undergo the post-binding physical change step in CPE action, (iii) removal of as few as five amino acids from the C terminus of CPE produces a noncytotoxic fragment lacking receptor binding activity, and (iv) a fragment lacking the first 44 N-terminal amino acids of native CPE formed twice as much large complex and was twice as cytotoxic as native CPE. From these structure-function results, it appears that the minimum-size cytotoxic CPE fragment comprises approximately residues 45 to 319 of native CPE. Results from these deletion fragment studies have also contributed to our understanding of CPE action by (i) independently supporting previous suggestions that binding, the postbinding physical change step, and large-complex formation represent important steps in CPE cytotoxicity and (ii) providing independent evidence confirming the putative sequential order of these early events in CPE action.     

Effect of sulfated compounds on acid sialidase

The inhibitory effects of various sulfated compounds on the activities of sialidases purified from porcine liver and human placenta were investigated. Among the sulfated compounds tested, heparin, dextran sulfate, condroitin sulfates and sulfatide significantly inhibited the 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4-MU-NeuAc) sialidase activities of the two enzyme preparations, but glucose 6-sulfate and glucosamine 6-sulfate did not. Potassium sulfate showed an inhibitory effect only at high concentrations. When the sialidase activities were measured using natural substrates, the sialidase activities for the (alpha2-3) and (alpha2-6) sialyllactoses, and colominic acid, were markedly inhibited by heparin and sulfatide similar to 4-MU-NeuAc, although the fetuin sialidase activity was not significantly influenced by them. The sialidase activity hydrolyzing GM3 was strongly inhibited by heparin, but not by sulfatide.     

Nosocomial diarrhoea in the elderly due to enterotoxigenic Clostridium perfringens

Zinc is an important trace element for immune function. Here, we show that zinc addition in a serum- and lipopolysaccharide-free cell culture system leads to significantly enhanced levels of interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) and to expression of the corresponding mRNA in human peripheral blood mononuclear cells (PBMC). Structurally related divalent cations like cobalt, nickel, and mercury also partially increase monokine secretion but to a much lower and thus insignificant extent. They fail to induce mRNA of TNF-alpha after 3 h of culture. Therefore, monokine induction is a zinc-specific effect influenced by the physicochemical properties of the ion. Confirmation of the unique significance of zinc for immune function provides a better understanding of the mechanisms of specific zinc-mediated immune modulation.     

Effect of antibiotic growth promoters and anticoccidials on growth of Clostridium perfringens in the caeca and on performance of broiler chickens

The purpose of this study was to analyze atomizer performance in the production of respirable spray-dried particles. An ultrasonic nebulizer and a plain-jet airblast atomizer were evaluated in an open cycle, cocurrent spray-drying tower using a 0.5% w/v disodium fluorescein solution. The plain-jet airblast atomizer produced smaller initial droplet sizes (D32 = 4.5-4.8 microns) relative to the ultrasonic nebulizer (D32 = 20-48 microns) over a range of atomizer operating conditions. The airblast atomizer was selected for further analysis in two spray-drying tower configurations: grounded and electrostatically charged. The spray-dried particles produced by the airblast atomizer were of a size range (mass median aerodynamic diameter [MMAD] < 1.6 microns) suitable for inhalation. Significant differences were observed for the grounded and electrostatically charged tower configurations, the latter producing the smaller median particle size at the expense of decreased collection efficiency. The electrostatically charged tower was size selective because of diffusion charging, retaining particles with an aerodynamic diameter (Dae) in the range 1 < Dae < 2 microns. The particle size was reduced with decreasing ambient relative humidity, although a controlled study of this parameter would be required to explicitly define its effects.     

Isolation and properties of the natural and the recombinant sialidase from Clostridium septicum NC 0054714

The natural sialidase of Clostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed by Escherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 degrees C in 60 mM sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having alpha(2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the alpha(2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. alpha(2-3) Linkages are more rapidly hydrolysed than alpha(2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens

The Tet P determinant from the conjugative Clostridium perfringens R plasmid pCW3 two functional overlapping tetracycline resistance genes, tetA(P) and tetB(P). The tetA(P) gene encodes a putative 46-kDa transmembrane protein which mediates active efflux of tetracycline from the cell, while tetB(P) encodes a putative 72.6-kDa protein which has significant similarity to Tet M-like tetracycline resistance proteins (J. Sloan, L.M. McMurry, D. Lyras, S. B. Levy, and J. I. Rood, Mol. Microbiol. 11:403-415, 1994). In the present study, hybridization and PCR analysis of 81 tetracycline-resistant isolates of C. perfringens showed that they all carried the tetA(P) gene. Most of these isolates (93%) carried a second tetracycline resistance gene, with 53% carrying tetB(P) and 40% carrying a tet(M)-like gene. Despite the wide distribution of the tetB(P) and tet(M) genes, no isolate which carried both of these determinants was detected. In isolates that carried both tetA(P) and tetB(P) these genes overlapped, as in pCW3. Isolates carrying this combination of genes originated from diverse geographical locations and environmental sources. The single Clostridium paraputrificum isolate examined carried tetA(P), indicating that this gene is not confined to C.perfringens. However, neither tetA(P) nor tetB(P) was detected in the nine Clostridium difficile isolates tested. Nucleotide sequence analysis of isolates lacking tetB(P) revealed that they contained the tetA408(P) gene, which lacked the codons for the 12 carboxy-terminal amino acids of the TetA(P) protein.     

Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.     

UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.     

Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples

Developing rats between 5 and 44 days of age as well as rats about 2 months old were anesthetized with urethane. Spontaneous activities and muscle potentials to sciatic stimuli were recorded from their exposed medial gastrocnemius muscles using concentric electrodes. Neostigmine of 0.12-0.40 mg/kg was injected into the contralateral muscles. Regardless of age, spontaneous activities were not observed and muscle potentials were evoked by single stimuli primarily in a biphasic wave (main component) before the drug treatment. Developmental differences were revealed in the presence of the drug. 1) Spontaneous activities were detected only in single spikes around postnatal 10 days. Single or double spikes were often found in rats of about two weeks. Burst discharges such as seen in adult rats were observed immediately before weaning. 2) In rats of 2 weeks or so, one or more components were observed obscurely following the main component of muscle potentials, which appeared definitely at the preweaning period. 3) When double shocks with the interval of 2 sec were delivered in the presence of the drug, the second potential was greatly depressed in rats older than two weeks as well as in adult rats. The potential was only slightly reduced in rats around 10 days. Thus, an adult pattern as to impulse transmission was observed immediately before weaning. These alterations would, at least in part, reflect the maturation of acetylcholine receptors at neuromuscular junctions.     

ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity

With increased interest in the pharmacology of cholecystokininA (CCKA) receptors, including their trophic and mitogenic effects, the actions of two new non-peptide CCKA receptor antagonists, PD140548 and SR 27897B, were investigated in a convenient model system, the rat isolated nodose ganglion. CCK (1 nM-1 microM) caused concentration-dependent depolarisations when superfused over the nodose ganglion at 37 degrees C as measured by a silicone grease gap technique, and both CCKA antagonists caused significant rightward shifts in the concentration response curve to CCK. SR 27897B (3 and 10 nM) caused 7.9- and 17.9-fold shifts in the CCK concentration-response curve and the apparent-log KB values for each concentration of antagonist were calculated to be 9.36 and 9.23. Further experiments with PD140548 (30 and 100 nM) yielded shifts of 2.9- and 12.5-fold from which -log KB values were determined to be 7.80 and 8.06. Overall SR 27897B was significantly more efficacious than PD140548. Thus, the isolated nodose ganglion preparation allows a functional assessment of CCKA-mediated responses, with the results indicating that both SR 27897B and PD140548 are efficacious CCKA receptor antagonists.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Characterization and distribution of amylases during vegetative cell growth and sporulation of Clostridium perfringens

Clostridium perfringens produced eight extracellular and two intracellular amylolytic activities when examined by zymograms following polyacrylamide gel electrophoresis under native conditions. The major intracellular amylase was isolated from vegetative cells of C. perfringens. It possessed an estimated molecular mass of 112 kDa. Sulfhydryl and phenol functional groups were essential to its activity. The amylase was endo-acting on starch and also hydrolyzed pullulan. Polyclonal antisera against a purified extracellular amylase did not cross-react with intracellular amylase and the two amylases were biochemically different. The distribution of extracellular amylolytic activities of sporulating cells was different from that of vegetative cells, whereas the distribution of intracellular amylolytic activities remained identical. A significant increase of a particular amylase (A8) occurred in the extracellular fluid during sporulation compared with that during vegetative growth. Regulation of the excretion of amylase(s) may be sporulation and enterotoxingenicity related.     

Development of an ELISA assay for Clostridium perfringens phospholipase C (alpha toxin)

A new method for the assay of Clostridium perfringens alpha toxin (phospholipase C) is described using a sandwich ELISA. This assay has been shown to be quantitative, to have a high specificity for the toxin and is capable of detecting purified Clostridium perfringens phospholipase C at concentrations of as little as 0.005 units/ml in cooked meat culture medium.     

Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI from Chlamydomonas eugametos was shown to cleave the circular chromosomes of all Clostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins, beta, epsilon, and tau, in addition to the enterotoxin and lambda-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins, theta and mu, were missing.     

Protection of goats against experimental enterotoxaemia by vaccination with Clostridium perfringens type D epsilon toxoid

Enterotoxaemia in goats is mainly characterized by enterocolitis, and it has been suggested that the poor efficacy of commercial vaccines in preventing the disease is due to the local action of Clostridium perfringens toxin/s within the intestine, where circulating antibodies might not exert their action. Five goat kids were vaccinated with an incomplete Freund's adjuvant C perfringens type D epsilon toxoid vaccine on three occasions at three-week intervals, four similar kids were vaccinated with a commercial enterotoxaemia vaccine at the same times, and five other unvaccinated kids were used as controls. All the animals were challenged intraduodenally, one week after the last vaccination, with C perfringens type D filtered culture supernatant. At the time of challenge, the level of epsilon toxin antibodies in the serum of the Freund's adjuvant-vaccinated kids ranged between 2.45 and 230 iu/ml, while the kids that received the commercial vaccine had levels between 0.22 and 1.52 iu/ml. No clinical or postmortem changes were observed in the kids that received the Freund's adjuvant-vaccine. Three of the four kids that received the commercial vaccine developed mild, pasty diarrhoea, with a slight reddening of the colonic mucosa being observed postmortem. All the unvaccinated kids developed severe diarrhoea, respiratory distress and central nervous system signs, and were killed humanely between six and 24 hours after challenge. The postmortem changes consisted of pseudomembranous colitis, lung oedema and perivascular oedema of the brain. Moderate to high serum levels of anti-epsilon antibody appeared to protect the goats against both the systemic and the intestinal effects of C perfringens type D toxins.     

Enzyme linked immunosorbent assay for potency testing of vaccines containing Clostridium perfringens type D epsilon-toxoid

Patients with hyperprolactinemia often present with emotional difficulties. These occasionally persist even after successful treatment. Insight into the roots of their diseased state makes a difference in the handling of all cases, but becomes crucial in the not-so-rare situations in which the normalization of hormonal levels is not followed by a feeling of cure. This chapter attempts to provide details, discuss and situate in context the following blocks of pertinent information: (1) prolactin acts upon the central nervous system and variations in its concentrations do affect mood, emotions and behavior; (2) most actions of prolactin are directed to metabolical and behavioral adaptation to pregnancy and the care of the young; (3) even in the absence of pregnancy prolactin secretion responds to environmental stimuli under specific conditions. Whether adaptive, as in the case of surrogate maternity, or pathological, as in the case of pseudopregnancy, prolactin responds to a perceived need to take care of a child; (4) the facts that the clinical onset of prolactinomas often follows life-events and that these tumors occur preferentially in women brought up under specific conditions suggest the possibility that psychological factors may predispose to prolactinomas; (5) dealing with individual cases requires the perception that the relations between prolactin, emotions and feelings are circular, i.e., prolactin affects the brain and mood but, on the other hand, personality traits and environmental factors may stimulate the secretion of prolactin and may play a role in the genesis of the disease.     

Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ

The 6.3 kb Clostridium perfringens transposon Tn4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RSA site, and an RSA-like sequence has been identified upstream of the tnpZ gene. In Escherichia coli, in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RSA site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens. Site-directed mutagenesis of two bases within the RSA site resulted in a significant reduction in mobilization frequency, demonstrating that the RSA site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn4451 is the first mobilizable but non-self-transmissible transposon to be identified from a gram-positive bacterium.     

Gastric mucormycosis in a sika deer (Cervus nippon) associated with proliferation of Clostridium perfringens

Seven sika deer (Cervus nippon) died in a park where 30 deer were kept. One adult female deer died suddenly was necropsied. Severe hemorrhages were noted beneath the serous membranes of the forestomach and abomasum. Hyphal proliferation with neutrophil infiltration was observed in the mucous membranes of the stomaches, and the hyphae showed characteristics of order Mucorales. Catarrhal enteritis with hemorrhages was also observed. A large number of Clostridium perfringens was isolated from the contents of the abomasum and small intestine. The case was diagnosed as gastric mucormycosis associated with proliferation of Clostridium perfringens. The incidence occurred during breeding season and incorrect management was considered to be a predisposing factor for the infection.