Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.     

Epitope conservation and immunohistochemical localization of the guanylin/stable toxin peptide receptor,guanylyl cyclase C

The heat-stable enterotoxins (ST) are a family of cysteine-rich low-molecular weight peptides produced by pathogenic bacteria, and are one of the major causes of watery diarrhea all over the world. These toxins mediate their action by binding to an intestinal cell surface receptor that is a membrane-associated guanylyl cyclase (GCC). This receptor also serves as the receptor for the recently characterised endogenous ligand, guanylin. We have expressed various domains of the receptor in Escherichia coli and used purified proteins for the generation of both polyclonal and monoclonal antibodies. While polyclonal antibodies were able to partially inhibit ST binding to the native receptor present in the T84 human colonic cell line, GCC:B10 monoclonal antibody did not interfere with ligand binding. Western blot analysis, using membranes prepared from human colonic T84 cells, detected two bands of size 160 and 140 kDa, representing alternately glycosylated forms of the receptor. Using the recombinant proteins, we could map the epitope of GCC:B10 monoclonal antibody to the intracellular domain of the receptor. We used the antibody to localize the receptor throughout the rat intestine, and in the porcine and bonnet monkey colon. We could detect receptor expression in the villus and the crypts of the duodenum, jejunum, ileum, and caecum, and in the crypts of the colon. Receptor expression was observed in cells that had earlier been shown to express cGMP-dependent kinase, but not the cystic fibrosis transmembrane regulator, a known downstream target of cGMP/G-kinase, which suggests that GCC/cGMP could regulate additional cellular signal transduction machinery. J. Cell. Biochem. 66:500–511, 1997. © 1997 Wiley-Liss, Inc.     

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.     

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vβ type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21–32, 93–107 and 202–217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.     

Hemagglutinating activity of the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

Abstract The hemagglutinating activity of the heat-labile enterotoxin (LT_p) isolated from porcine enterotoxigenic Escherichia coli was studied by hemagglutination inhibition. The hemagglutinating activity of LT_p was enhanced 64–512-fold with pronase- and neuraminidase-treated human erythrocytes although both intact human and sheep erythrocytes were not agglutinated by LT_p at the highest concentration used. No enhancement was found in hemagglutination of neuraminidase-treated sheep erythrocytes by LT_p. Hemagglutination of pronase-treated human type A erythrocytes induced by LT_p was inhibited by melibiose and galactose among mono-, di-, and polysaccharides used as inhibitors. Galactose was a slightly better inhibitor than melibiose. These findings suggest that LT_p is a bacterial lectin specific for galactose.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

Apoptosis observed in murine peritoneal macrophages treated with interferon gamma through staphylococcal enterotoxin-dependent cell-mediated cytotoxicity

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.     

Rabbit enterotoxaemia: Purification and preliminary characterisation of a toxin produced by Clostridium spiroforme

Abstract A culture filtrate of Clostridium spiroforme grown in a brain-heart-infusion peptone medium at 37°C for 24 h was concentrated by ultrafiltration, and the toxic fraction separated by ion exchange chromatography. This material was dermonecrotic in depilated guinea pigs, lethal in mice, enterotoxic in rabbit ileal loops and infant mice and cytophathic for HeLa cells. All activity was neutralised by Clostridium perfringens Type E antitoxin. Analysis by size exclusion high-performance liquid chromatography produced a single symmetrical peak corresponding to an M _r of 41 000 to 42 000.     

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE‐like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)‐PCR analysis method established. LA‐PCR products of 13–17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb‐harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq‐harboring SaPIj11 and non‐superantigen‐harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA‐PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.