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Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection.

In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene (act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host-pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT-versus mutant-induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection.

Aeromonas hydrophila↗

Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 A crystal structure reveals the details.

A hybrid between the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin has been described, which exhibits a novel binding specificity to blood group A and B type 2 determinants. In the present investigation, we have determined the crystal structure of this protein hybrid, termed LCTBK, in complex with the blood group A pentasaccharide GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAcbeta, confirming not only the novel binding specificity but also a distinct new oligosaccharide binding site. Binding studies revealed that the new specificity can be ascribed to a single mutation (S4N) introduced into the sequence of Escherichia coli heat-labile enterotoxin. At a resolution of 1.9 A, the new binding site is resolved in excellent detail. Main features include a complex network of water molecules, which is well preserved by the parent toxins, and an unexpectedly modest contribution to binding by the critical residue Asn4, which interacts with the ligand only via a single water molecule.

Asparagine↗

Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats.

Staphylococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB(4) and PGE(2)) and cytokines (TNF-alpha, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 microg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE(2), LTB(4), TNF-alpha, IL-6) and anti-inflammatory mediators (IL-10).

Acute Disease↗

Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast.

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) have been studied intensively as vaccines against diseases caused by those bacteria and as adjuvants for mucosal vaccination. Two major problems interfere with the use of these promising adjuvants: their toxicity and the residual bacterial endotoxins mixed with the desired LT. In this study, subunit B of LT was expressed in Pichia pastoris yeast cells (yrLTB) and the recombinant protein was purified and concentrated by ion-exchange chromatography. The final yield of the recombinant protein was 5-8 mg/l induction medium. The molecule is in pentameric form and binds to GM1 gangliosides. When given orally to chickens, anti-LTB antibodies were produced, exhibiting its ability to cross the digestive system and induce an immune response. The adjuvant activity of yrLTB was proven by fusing it to viral protein 2 (VP2) of infectious bursal disease virus. Birds intramuscularly vaccinated with this molecule exhibit 70-100% protection, in a dose-response-dependent manner. This method eliminated the bacterial endotoxins and enabled the production of large quantities of LTB. Expression in a eukaryotic system allows the production of fusion proteins that require post-translational modifications. This may allow oral vaccination with a protein fused to yrLTB. The approach described in this study will enable the efficient production of a non-toxic, eukaryotically expressed enterotoxin as a vaccine against the toxin itself or as a carrier or adjuvant for foreign vaccine molecules.

Adjuvants, Immunologic↗

Type 2 heat-labile enterotoxin (LT-II)-producing Escherichia coli isolated from ostriches with diarrhea.

The culture supernatant of Escherichia coli, isolated from ostriches with diarrhea in Brazil, caused elongation in Vero cell, rounding in Chinese hamster ovary (CHO) cells and a cytoplasmic vacuolation in ostrich embryo fibroblasts (OEF), but it was not cytotoxic for chicken embryo fibroblasts (CEF). These effects were not neutralized by antiserum to cholera toxin. Polymerase chain reaction assays showed that the ostrich E. coli contained the gene encoding (eltII-A), but not those for type 1 heat-labile enterotoxin (eltA), heat-stable enterotoxins (estA, estB), verocytotoxins (stx-I, stx-II), or cytotoxic necrotizing factors (cnf 1, cnf 2). All isolates belonged to serotype O15:H8. The enteropathogenic relevance of LT-II in ostrich diarrhea remains undetermined.

Animals↗

Primary action of Clostridium perfringens type A enterotoxin on HeLa and Vero cells in the absence of extracellular calcium: rapid and characteristic changes in membrane permeability.

Clostridium perfringens type A enterotoxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca2+ at 37 degrees C. The bound toxin rapidly (within 2 min) caused influx of Na+ and efflux of K+ and Mg2+. Changes in membrane permeability occurred in the absence or presence of extracellular Ca2+ and to the similar extents at 37 degrees C and 4 degrees C, in contrast to the subsequent bleb and balloon formation, which required both extracellular Ca2+ and incubation at 37 degrees C. Substances with molecular weights of over ca. 200 protected the cells from the morphological alterations induced by the toxin, whereas substances with molecular weights of less than ca. 200 did not. The mechanism of the primary action of the enterotoxin is discussed.

Animals↗

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line.

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.

Adrenal Gland Neoplasms↗

Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane.

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.

Clostridium perfringens↗

Rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin B.

Sensitive, rapid and reproducible detection of staphyloccocal enterotoxin B (SEB) in a range of different biological matrices was achieved using the ORIGEN((R)) Immunoassay System (Igen, Inc). The homologous immunoassay format consisted of a double antibody sandwich in which a biotinylated capture antibody, pre-bound to streptavidin-coated paramagnetic beads, was used to bind antigen from test samples. A detector antibody, labeled with ruthenium (II) tris-bipyridal chelate, was added and, when bound to the bead immunocomplex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the ORIGEN analyzer. The sensitivity of this assay was 1 pg of enterotoxin per ml of serum, urine, tissue, or buffer and was highly reproducible. Concentration curves generated from SEB standards produced consistently wide linear ranges (0.1-100 ng/ml), making quantitation possible with only two dilutions of sample (undiluted and 1:1000). The assay used 50 microl of sample per test and required a 30 min incubation period in addition to a 1 min per tube reading time (50 tubes maximum). This assay was significantly better in terms of sensitivity, linear range, and assay time than the standard microplate enzyme-linked immunosorbent assay and should permit early SEB detection in clinical samples, food, and environmental samples.

Animals↗

Diarrhoea associated with heat-stable enterotoxin-producing strains of Escherichia coli.

Five patients who developed acute watery diarrhoea while travelling in Mexico in October, 1974, were found to have enterotoxigenic Escherichia coli in their stool which produced heat-stable enterotoxin (S.T.) without producing heat-labile enterotoxin (L.T.). These S.T.-only E. coli, which have previously been described as causing diseases in animals, must now be regarded as pathogenic for humans as well.

Adult↗

Cholera-like enterotoxin produced by Campylobacter jejuni. Characterisation and clinical significance.

The presence and clinical significance of enterotoxins produced by Campylobacter jejuni were investigated. The supernatant of a prototype virulent strain grown in supplemented medium induced intraluminal fluid secretion in rat ileal loop but not in rabbit ileal loop or the infant mouse assay. It induced elongation and increased intracellular cyclic AMP levels in Chinese hamster ovary cells. Toxin activity was blocked by cholera antitoxin and was destroyed by heat and high or low pH; its molecular weight is in the range 10(4)-10(5) daltons. Toxin production was detected in 24 of 32 C jejuni strains from patients with diarrhoea and 1 of 6 from carriers. Antibody response to autologous C jejuni somatic antigen was investigated in 19 subjects for whom serial serum specimens were available. A fourfold rise was observed in all 10 patients with enterotoxigenic C jejuni diarrhoea, in 1 of 3 patients with non-enterotoxigenic C jejuni, and in none of the symptomless carriers of non-enterotoxigenic strains. These findings demonstrate that C jejuni produces an enterotoxin that may be important in pathogenesis of diarrhoea.

Animals↗

Intramolecular epitope spreading induced by staphylococcal enterotoxin superantigen reactivation of experimental allergic encephalomyelitis.

The staphylococcal enterotoxin superantigens are among the most potent T cell stimulators known. They have been shown to alter the course of disease in experimental allergic encephalomyelitis, an animal model for multiple sclerosis (MS). We have previously demonstrated that two of the staphylococcal enterotoxins, SEA and SEB, are able to reactivate paralysis in PL/J mice which had been immunized with myelin basic protein (MBP) and resolved an initial episode of paralysis. In PL/J mice, Ac1-11 is the dominant encephalitogenic determinant of MBP. We hypothesized that superantigen reactivation of experimental allergic encephalomyelitis (EAE) may result in the spreading of T cell specificities for other epitopes of MBP. PL/J mice which had resolved an initial episode of EAE were treated with SEA and developed a second episode of paralysis. At the onset of symptoms, mice were sacrificed and splenocytes were stimulated in vitro with a panel of MBP peptides. EAE reactivation by SEA resulted in the spreading of T cell specificites to residues 100 to 120 of MBP. While intramolecular spreading did occur, spreading to other antigens did not, as evidenced by the lack of response to a proteolipid protein (PLP) peptide and heat shock protein 60 (hsp 60). To further characterize the epitope MBP 100-120, PL/J mice were immunized with MBP 100-120. No initial development of disease was observed. However, administration of SEA 2 weeks after MBP 100-120 immunization resulted in the onset of paralysis. In addition to a proliferative response to MBP 100-120, these mice also exhibited a proliferative response to the flanking MBP peptides 81-100 and 120-140. Thus, SEA is able to induce intramolecular epitope spreading in PL/J mice after reactivation of EAE.

Animals↗

Analytical chromatography for recovery of small amounts of staphylococcal enterotoxins from food.

Sample preparation is an important element in the detection of toxins in food samples. In this work, a simple analytical sample preparation method for recovery of small amount of staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin A (SEA) in food samples was developed. Cation exchanger carboxymethylcellulose (CM) was used for small-scale batch chromatography isolation of SEB from infant formula and from mushrooms spiked with SEB. The resulting materials were analyzed for SEB by Western immunoblotting. Nearly all of the extraneous substances in the sample were removed by this procedure with no significant loss of the toxin. Using this method, even small amounts of SE (0.75 ng/g) can be recovered and immunologically analyzed by Western blotting or by ELISA with a very low background. Because this method is effective, rapid, simple and inexpensive, it has the potential to be a general method for the preparation of samples used for analysis of SEs.

Agaricales↗

Development and use of PCR primers for the investigation of C1, C2 and C3 enterotoxin types of Staphylococcus aureus strains isolated from food-borne outbreaks.

Staphylococcus aureus is a major food-borne pathogen in many countries. Enterotoxins produced by S. aureus strains include staphylococcal enterotoxins (SEs) A, B, C, D, E and G, H, I, etc. For SEC, in addition to the three major SEC subtypes, i.e., SEC1, C2 and C3, other molecular variants may exist. Although the detection methods and the distribution of SEA, B, C, D, E types of S. aureus in staphylococcal infections or food-borne outbreaks have been well documented, the differentiation method and the distribution of SEC subtypes in staphylococcal infections are rarely reported. In this study, four polymerase chain reaction (PCR) primers used in pairs (ENTC1/ENTCR, ENTC2/ENTCR and ENTC3/ENTCR) for the specific detection of SEC1, C2 and C3 genes of S. aureus strains were developed. When 39 SEC S. aureus strains isolated from fecal samples of randomly selected diarrheal patients associated with food-borne outbreaks in central Taiwan in 6 years (1995-2000) were analyzed, it was found that the major SEC subtypes for these S. aureus strains were SEC2 and C3.

DNA Primers↗

Staphylococcal enterotoxin genes of classical and new types detected by PCR in France.

The PCR detection of the genes coding for the newly described staphylococcal enterotoxins (SE) G, H, I and J was carried out for 332 foodborne staphylococci, isolated from a variety of foods in France. The frequency of the Staphylococcus aureus strains harboring these genes was found to be very high (57%) and greater than that of the strains harboring "classical" SE genes as previously established. If one takes into account the newly described SE genes, in addition to the classical SE genes, the percentage of foodborne enterotoxigenic S. aureus strains doubles. The S. aureus biovars that were rarely or never enterotoxigenic (i.e., the poultry and bovine biovars) frequently become more potentially toxigenic, if taking into account the seg. seh, sei and sej genes. Furthermore, this work indicated a systematic association between the seg and sei genes and their high incidence among the S. aureus strains, which suggests that these two SE's could be an important phylogenetic link among the staphylococcal enterotoxins.

DNA, Bacterial↗

MPN-PCR-quantification method for staphylococcal enterotoxin c1 gene from fresh cheese.

PCR detection methods have been extensively used in diagnostic microbiology. However, a lack of a simple and reliable method for quantification of the PCR products has partly hindered the use of PCR in routine food laboratories. The quantification of PCR products can be done by combining the principles of MPN statistics and PCR technique. We have developed a simple and sensitive MPN-PCR assay for detection and enumeration of enterotoxin C producing Staphylococcus aureus NCTC 10655 from fresh cheese. By amplifying single copy chromosomal enterotoxin C gene fragment, we could detect as little as 20 cfu/g. By Moran's test, most of the DNA dilution series appeared to fulfill the basic mathematical assumptions of ordinary MPN methods. The analysis with MPN-PCR took one day to perform compared with three days analysis time with plate counting. This MPN-PCR method can be readily applied with different primer systems without extensive development work.

Cheese↗

Amino acid requirements for the growth and enterotoxin production by Staphylococcus aureus in chemically defined media.

The production of enterotoxins A, B, and C by five strains of Staphylococcus aureus was studied in a series of defined media, each differing from the complete defined medium in the lack of one amino acid. Valine was required for growth; arginine and cystine for growth and enterotoxin production. Omission of individual amino acid affected in different ways the yields of the toxins produced.

Amino Acids↗

A survey of human serum samples for antibody against Clostridium perfringens type A enterotoxin in humans in Korea.

Serum samples from humans in Chongju City, Korea, were tested for the presence of Clostridium perfringens enterotoxin antibody by enzyme linked immunosorbent assay (ELISA). About half of the 1054 samples tested had high ELISA readings, which could be the result of exposure to enterotoxin produced by C. perfringens. The high prevalence of the antibody among the Korean population suggests that foodborne illnesses caused by C. perfringens may be common in Korea even though the occurrence has not been monitored.

Adolescent↗