Improvement in the Growth and α-toxin Production of Clostridium septicum by Magnesium Sulfate.

Clostridium septicum, the anaerobic toxigenic bacterium is the agent that causes dangerous disease in man and animals. There is a lethal toxin of the bacterium namely alpha toxin. The ɑ-toxin has hemolytic, necrotic and lethal activities. Today, Razi Vaccine and Serum Research Institute of Iran produced the C. septicum vaccine in the form of bacterin/toxoid. Because of some problems, the vaccine needs to improve on an industrial scale. The study is going to find an appropriate supplement to improve growth and ɑ-toxin production. Three strains of C. septicum (vaccine, NH1 and NH8 strains) were cultured in the basic vaccine media. Magnesium sulfate, Copper, Ferrous, yeast extract, and trace elements plus vitamins' solution were added to the basic vaccine media in different cultures. The effect of the ingredients on the growth was measured by a spectrophotometer and the α-toxin secretion was assayed by hemolysin test. Growth of the bacterium and α-toxin secretion were increased by Magnesium (80 mg/l) in NH8 and vaccine strains significantly. The black precipitate was difficult to dissolve in magnesium media that must be solved. Trace elements plus vitamins solution mildly influence on NH1strain growth and toxin secretion. Other supplements (Cu, Fe, yeast extract) were not showen any significant changes in the growth and α-toxin production of C. septicum. Overflowing peptone (4%) in the vaccine media, fixes essentials of proteolysis activity, allows the sufficient growth and toxin production without Cu, Fe, and yeast extract. Due to essentially of Mg for growth, extra magnesium was added for improvement of media culture. The study suggests for Magnesium addition in the C. septicum vaccine media during production procedure after precipitation solving problem.

Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

Elevated levels of glycosylphosphatidylinositol (GPI) anchored proteins in plasma from human cancers detected by C. septicum alpha toxin.

The glycosylphosphatidylinositol (GPI) anchor is a glycan and lipid posttranslational modification added to proteins in the endoplasmic reticulum. Certain enzymes within the GPI biosynthetic pathway, particularly the subunits of the GPI transamidase, are elevated in various human cancers. Specific GPI anchored proteins, such as carcinoembryonic antigen and mesothelin, have been described as potential biomarkers for certain cancers; however, the overall levels of GPI anchored proteins present in plasma from cases of human cancers have not been evaluated. We have developed the use of a bacterial toxin known as alpha toxin from Clostridium septicum to detect GPI anchored proteins in vitro. In this study, we use alpha toxin to detect GPI anchored proteins present in plasma from cases of several types of human cancers. Our data indicate that human cancers with previously documented elevations of GPI transamidase subunits show increased alpha toxin binding to plasma from patients with these cancers, indicating increased levels of GPI anchored proteins. Furthermore, our results reveal very low levels of alpha toxin binding to plasma from patients with no malignant disease indicating few GPI anchored proteins are present. These data suggest that GPI anchored proteins present in plasma from these cancers represent biomarkers with potential use for cancer detection.

Necrotizing endometritis and isolation of an alpha-toxin producing strain of Clostridium septicum in a wild sooty mangabey from Côte d'Ivoire.

Few lethal pathogens in wild-living primates have been described, and little is known about infectious diseases of the reproductive tract and their possible impact on health and reproduction. This report describes the pathology and isolation of an alpha-toxin producing strain of Clostridium septicum in a case of necrotizing endometritis in a wild sooty mangabey found dead in a tropical rainforest of West Africa.

Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositol-anchored proteins.

Clostridium septicum alpha-toxin has a unique tryptophan-rich region ((302)NGYSEWDWKWV(312)) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.

[The study on the cloning and expression of alpha toxin gene of clostridium septicum and the immunity of the toxoid].

In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.

Identification of functional domains of Clostridium septicum alpha toxin.

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane beta-hairpin of the prepore oligomer.