Production, purification and identification of the staphylococcal enterotoxins

Tranter, Howard S.; Brehm, Rossalyn D.

doi:10.1111/j.1365-2672.1990.tb01803.x

Cited by 10 publications

(13 citation statements)

References 100 publications

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“…Because RDA has been used to purify several staphylococcal enterotoxNovel Superantigenfrom Streptococcus pyogenes 711 ins (23,24), we thought this material might be useful in identifying novel S. pyogenes superantigens. Concentrated culture supernatants from strain Weller were chromatographed on a RDA column, the column was eluted with a phosphate step gradient, and fractions were tested for the presence of a class II-dependent T cell mitogen (7).…”

Section: Resultsmentioning

confidence: 99%

“…The use ofRDA chromatography in our purification protocol may have provided an essential advantage in separating SSA from SPE-A and SPE-C. RDA has been shown to be an effective means of purifying virtually all staphylococcal enterotoxins (23,24). Given the striking resemblance ofthe NH2 terminus of SSA to SEB and SEC1,3, it is not surprising that SSA was also amenable to RDA purification.…”

Section: Discussionmentioning

confidence: 99%

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A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Mollick¹,

Miller²,

Musser³

et al. 1993

J. Clin. Invest.

150

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Streptococcus pyogenes (group A Streptococcus) has reemerged in recent years as a cause of severe human disease. Because extracellular products are involved in streptococcal pathogenesis, we explored the possibility that a disease isolate expresses an uncharacterized superantigen. We screened culture supernatants for superantigen activity with a major histocompatibility complex class II-dependent T cell proliferation assay. Initial fractionation with red dye A chromatography indicated production of a class II-dependent T cell mitogen by a toxic shock-like syndrome (TSLS) strain. The amino terminus of the purified streptococcal superantigen was more homologous to the amino termini of staphylococcal enterotoxins B, C1, and C3 (SEB, SEC,, and SEC3), than to those of pyrogenic exotoxins A, B, C or other streptococcal toxins. The molecule, designated SSA, had the same pattern of class II isotype usage as SEB in T cell proliferation assays. However, it differed in its pattern of human T cell activation, as measured by quantitative polymerase chain reaction with Va-specific primers. SSA activated human T cells that express V#1, 3, 15 with a minor increase of V#5.2-bearing cells, whereas SEB activated V,63, 12, 15, and 17-bearing T cells. Immunoblot analysis of 75 disease isolates from several localities detected SSA production only in group A streptococci, and found that SSA is apparently confined to only three clonal lineages as defined by multilocus enzyme electrophoresis typing. Isolates of one of these lineages, (electrophoretic type 2) are strongly associated with TSLS. The data identify SSA as a novel streptococcal superantigen that appears to be more related structurally to staphylococcal enterotoxins than to streptococcal exotoxins. Because abundant SSA production is apparently confined to only three streptococcal clonal lineages, the data also suggest that the SSA gene has only recently been acquired by S. pyogenes. (J. Clin.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Mollick¹,

Miller²,

Musser³

et al. 1993

J. Clin. Invest.

150

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“…Conventionally, molecular sieving and ion exchange chromatography have been used either independently or in tandem for purification of Staphylococcal enterotoxins (Tranter and Brehm, 1990;Coffman et al 2002;Dainiak et al 2005). Isoelectric focusing has been proved useful for purification of small amounts of staphylococcal enterotoxins but on a preparative scale the procedure has generally been used in combination with ion exchange chromatography and gel filtration (Tranter and Brehm, 1990).…”

Section: Staphylococcal Food Poisoning (Sfp) Is Caused By the Membersmentioning

confidence: 99%

“…This effect is more severe in case of intoxication involving SEB which is also a listed biological warfare (BW) agent (Franz et al 1997; Baker et al 2002;Pettersson and Forsberg, 2002; Boles et al 2003). Therefore, it is of utmost importance to develop immunological detection system for SEB, the success of which is dependent on the purity of SEB used for generating anti SEB antibodies.Conventionally, molecular sieving and ion exchange chromatography have been used either independently or in tandem for purification of Staphylococcal enterotoxins (Tranter and Brehm, 1990;Coffman et al 2002;Dainiak et al 2005). Isoelectric focusing has been proved useful for purification of small amounts of staphylococcal enterotoxins but on a preparative scale the procedure has generally been used in combination with ion exchange chromatography and gel filtration (Tranter and Brehm, 1990).…”

mentioning

confidence: 99%

“…Therefore, SEB should be available in highly pure form. Purification of SEB is tedious and time consuming by conventional protein purification methods like size exclusion and ion-exchange chromatography (Tranter and Brehm, 1990;Coffman et al 2002;Dainiak et al 2005). An alternative approach to achieve high level of protein purity is heterologous expression of desired gene into a suitable vector containing a fusion partner that can be used as affinity tag for single step purification (Nilsson et al 1997;Constans, 2002;Mukherjee et al 2003).…”

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confidence: 99%

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Heterologous expression of staphylococcal enterotoxin B (seb) gene for antibody production

Kamboj¹,

Nema²,

Pandey³

et al. 2006

Electron. J. Biotechnol.

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Staphylococcal food poisoning (SFP) is caused by the members of superantigen family called staphylococcal enterotoxins (SEs). About 20 different types of SEs are produced by Staphylococcus aureus out of which type A (SEA), B (SEB), C (SEC) and D (SED) are commonly implicated in SFP.Among these, SEB is the most potent toxin and has also gained the status of biological warfare (BW) agent. Therefore, detection of SEB is of utmost importance. Any immunological detection system for SEB requires specific and sensitive antibodies which inturn depends on the purity of the SEB. In the present investigation, seb gene of S. aureus was cloned and expressed in E. coli along with biotin as fusion partner to facilitate the purification process. The yield of purified recombinant SEB was 13.1 mg/L of culture broth. Biotin tag from the biotinylated toxin was removed by protease cleavage, and both biotinylated and non-biotinylated toxin types were used for raising hyperimmune antiserum. Antisera were also specific for SEB amongst different kinds of food poisoning agents tested by indirect plate ELISA and western blot analysis. The quality of the antisera raised in this study was found superior to the commercially available antiserum. The investigation suggests that construction of recombinant staphylococcal enterotoxin B is a good alternative for production of pure enterotoxin to be used in antibody generation.Staphylococcal food poisoning (SFP), a form of enteritis, is intoxication rather than a disease resulting from ingestion of food contaminated with preformed staphylococcal enterotoxins (Bergdoll et al. 1974). Symptoms of SFP usually occur within 1-6 hrs after the food intake and are characterized by nausea, vomiting, abdominal cramps and diarrhoea. These symptoms usually subside in 1-3 days but the patient remains sick for 7-10 days due to the result of toxic shock (Jett et al. 1994; Do Carmo et al. 2004). Toxic shock causing SEs are single chain polypeptides having a molecular weight ranging from 27-29 kDa. SEs can withstand boiling temperature for several minutes, extremes of pH (3-11) and protease digestion by gastric enzymes (Soriano et al. 2002). Twenty different types of SEs, viz., SEA through SEE, SEG through SER and SEU have already been discovered, however, only a few of the toxin serotypes are frequently associated with food poisoning outbreaks (Martin et al. 2004;Fernández et al. 2006). Toxin serotypes frequently associated with food poisoning outbreaks are SEA, SEB, SEC and SED (Balaban and Rasooly, 2000;Smyth et al. 2005).Staphylococcal enterotoxins are 'Superantigens' and are able to bind to the major histocompatability complex (MHC) class II antigen, causing massive T cell stimulation coupled with the release of cytokines. The release of cytokines results in stimulation of neuro-receptors in the intestinal tract, and triggers vomiting center in the brain (Komisar et al. 1994;Kaempfer et al. 2002;Hemalatha et al. 2004). This effect is more severe in case of intoxication involving SEB which is also a listed biol...

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Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System

et al. 2011

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Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning throughout the world. Staphylococcal enterotoxin B (SEB) is one of the most potent and a listed biological warfare agent. Therefore, its quick, accurate and sensitive detection is of paramount importance. But availability of sensitive and specific antibodies against SEB is the major bottleneck in the development of an immunodetection system. Therefore, in the present study seb gene was cloned and expressed in a heterologous host resulting in a yield of 92 mg pure toxin per litre of culture broth after Ni-NTA affinity purification. Antibodies raised against the recombinant toxin did not cross react with related enterotoxins and organisms that can gain access in the food. Further, a sandwich ELISA was developed to detect SEB after extraction from artificially spiked food samples like milk, orange juice, skim milk and khoya. The sandwich ELISA was able to detect SEB in the range of 0.25 to 0.49 ng/ml or g of food. The detection system developed in the present study is at least as specific and sensitive as other commercially available kits which use monoclonal antibodies.

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Production, purification and identification of the staphylococcal enterotoxins

Cited by 10 publications

References 100 publications

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Heterologous expression of staphylococcal enterotoxin B (seb) gene for antibody production

Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System

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