OBJECTIVE: To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. METHOD: SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. RESULT: The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. CONCLUSION The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.
Cell Line, Tumor ; Enterotoxins ; genetics ; Gene Expression ; Genetic Vectors ; Humans ; Plasmids ; Superantigens ; genetics ; Transfection

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

OBJECTIVETo investigate superantigen gene profiles of group A Streptococcus (GAS) isolated in Beijing pediatric patients in 2014, and to explore the the relationship between superantigen gene profiles with emm types, and GAS infections with diseases.

METHODSA total of 259 GAS strains were isolated from pediatric patients clinically who diagnosed with scarlet fever and pharyngitis from 36 hospitals in Beijing from May to July, 2014.The Superantigens genes of strains were performed by Real-time PCR (speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, ssa). PCR amplification of GAS strain M protein N gene segments were carried ort; products after sequencing comparison were analyzed to determine the GAS types of emm. The differences in distributions of superantigen genes and emm types of GAS isolates were compared between subgroups.

RESULTSAmong the 259 GAS strains, the detection rates of 13 superantigens were as the following: speA 48.6% (126), speB 99.2% (257), speC 99.2% (257), speF 98.8% (256), speG 98.5% (255), speH 43.6% (113), speI 46.3% (120), speJ 49.0% (127), smeZ 99.2% (257) and ssa 98.5% (255), respectively, however, speK, speL, and speM were not found. Eleven superantigen gene profiles in all were observed (A-K). The percentage of emm1 strains harbored spe A and speJ were 94.2% (113/120), 95.0% (114/120), respectively, which were significantly higher than those of emm12 strains (5.6% (7/124), 5.6% (7/124), respectively; χ(2) = 191.20, 194.80, P < 0.001). The percentage of emm12 strains harbored speH and speI were 83.9% (104/124), 88.7% (110/124), respectively, which were significantly higher than those of emm1 strains (3.3% (4/120), 4.2% (5/120), respectively; χ(2) = 160.30, 174.90, P < 0.001).The superantigen genotypes of GAS strains and emm types, which were isolated from scarlet fever and pharyngitis cases, were not significant different (P > 0.05).

CONCLUSIONThe GSA strains isolated in Beijing pediatric patients in 2014, the relevance ratio of speB, speC, speF, smeZ, speG, ssa were higher than others, while speK, speL, and speM were no detected in any GAS strains. The superantigen genes appeared to be associated with the emm type. Furthermore, emm type distribution and superantigen genes were not different between scalet fever and pharyngitis.

Antigens, Bacterial ; genetics ; Beijing ; Child ; Genotype ; Humans ; Real-Time Polymerase Chain Reaction ; Streptococcal Infections ; microbiology ; Streptococcus pyogenes ; genetics ; immunology ; Superantigens ; genetics

BACKGROUND: Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). METHODS: A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. RESULTS: The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. CONCLUSIONS: Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.
Bacterial Toxins/*genetics ; Chromosomes, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins/*genetics ; Humans ; Methicillin Resistance/*genetics ; Phylogeny ; Staphylococcal Infections/epidemiology/microbiology ; Staphylococcus aureus/drug effects/*genetics/isolation & purification ; Superantigens/*genetics

BACKGROUND: Various virulence factors and superantigens are encoded by mobile genetic elements. The relationship between clonal background and virulence factors differs in different geographic regions. We compared the distribution and relationship of spa types and virulence genes among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from a tertiary hospital in 2000-01 and 2007-08. METHODS: In 2000-01 and 2007-08, 94 MRSA strains were collected from 3 intensive care units at a Korean tertiary hospital. We performed spa typing and multiplex PCR for 19 superantigen genes. RESULTS: Relatively frequent spa types were t037 (40.5%), t002, t601, and t2138 in 2000-01, and t2460 (43.9%), t002, t037, t601, t324, and t2139 in 2007-08. We identified 4 novel spa types, 2 of which were designated as t5076 and t5079. Superantigen profiles were closely linked to spa types. For example, sea, sek, and seq superantigen genes were mainly detected in t037 strains. CONCLUSIONS: Major spa types differed depending on study periods, and the distribution of superantigen genes correlated with spa type.
Bacterial Typing Techniques ; DNA, Bacterial/chemistry ; Genotype ; Humans ; Intensive Care Units/statistics & numerical data ; Methicillin-Resistant Staphylococcus aureus/genetics/*isolation & purification/pathogenicity ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Staphylococcal Infections/microbiology ; Superantigens/genetics ; Virulence/genetics ; Virulence Factors/*genetics

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.
Animals ; Enterotoxins ; genetics ; immunology ; Female ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Receptors, Antigen, T-Cell ; immunology ; Superantigens ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo analyse the relationship between superantigens produced by Staphylococcus aureus and the mRNA expression of T-cell receptor V beta region (TCR Vbeta), and to investigate the possible role of Staphylococcal superantigens in the pathogenesis of nasal polyps.

METHODSSinonasal mucus and polyp/mucosa tissue were obtained from patients with chronic rhinosinusitis (22 patients with bilateral nasal polyps, 15 without nasal polyps) and 12 normal subjects as comparative negative controls. Mucus specimens were assayed by enzyme-linked immunosorbent assay (ELISA) for Staphylococcal exotoxins,and analyzed for the expression of TCR Vbeta genes using the technique of reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSThe percentages of Staphylococcus exotoxins in nasal polyps were 54.54% (21/22) for chronic rhinosinusitis with nasal polyp (CRSwNP) subjects. There were no positive results in the CRSsNP or control groups. The expressional intensity of Vbeta3 (10.02), Vbeta14 (3.54), Vbeta15 (2.39), Vbeta17 (3.48), and Vbeta20 (2.94) was increased significantly for Staphylococcal exotoxin B (SEB) positive subjects (P < 0.05). Vbeta2 (13.8) and Vbeta6. 1-3 (6.53) were significantly highly expressed for toxic shock syndrome toxin-1 (TSTf-1) positive subjects in CRSwNP group (P < 0.05). There were no dominantly used Vbeta fragments in ELISA- negative specimens. In the group of chronic rhinosinusitis without nasal polyp (CRSsNP), most of TCR Vbeta gene subfamilies demonstrated a trend toward higher expressional levels compared with those of normal controls, although there was no statistical difference (P > 0.05).

CONCLUSIONSThere was relationship between Staphylococcal superantigens and the excursion of TCR Vbeta gene spectra in nasal polyp, and superantigens possibly play an important role in the pathogenesis of CRSwNP.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Genes, T-Cell Receptor beta ; Humans ; Male ; Middle Aged ; Nasal Polyps ; genetics ; immunology ; Receptors, Antigen, T-Cell ; genetics ; Sinusitis ; genetics ; immunology ; Staphylococcus aureus ; immunology ; Superantigens ; immunology ; Young Adult

It has been well established that bacterial superantigens lead to the induction and aggravation of chronic inflammatory skin diseases. We investigated the clinical significance of serum specific immunoglobulin E (lgE) to the staphylococcal superantigens staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), and toxic shock syndrome toxin (TSST)-1 in patients with chronic urticaria (CU), focusing on the differences in these prevalences between aspirin-intolerant CU (AICU) and aspirin-tolerant CU (ATCU) patients. Aspirin sensitivity was confirmed by oral aspirin provocation test. There were 66 patients AICU and 117 patients ATCU in the study. Serum IgE antibodies specific for SEA, SEB, and TSST-1 were measured by the ImmunoCAP test and the patients were compared with 93 normal controls (NC). The prevalences of serum specific IgE to staphylococcal superantigens were significantly higher in CU than in NC patients (IgE to SEA, 13.7% vs. 5.4%; IgE to SEB, 12.0% vs. 4.3%; IgE to TSST-1, 18.0% vs. 6.5%; p<0.05, respectively). The patients with specific IgE to SEA, SEB, and TSST-1 had higher serum total IgE levels and higher rates of atopy. Significant associations were noted between the prevalence of specific IgE to SEA and SEB and the HLA DQB1*0609 and DRB1*1302 alleles in the AICU group. We confirmed that a sub-population of patients with CU possesses serum IgE antibodies to SEA, SEB, and TSST- 1. Particularly, the IgE immune response to TSST-1 is associated with aspirin sensitivity in CU patients.
Adolescent ; Adult ; Aged ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Antigens/*chemistry ; Aspirin/pharmacology ; Bacterial Toxins/metabolism ; Chronic Disease ; Enterotoxins/metabolism ; Female ; Humans ; Immunoglobulin E/*chemistry/metabolism ; Male ; Middle Aged ; Phenotype ; Staphylococcus/*genetics/immunology ; Superantigens/metabolism ; Urticaria/*immunology

OBJECTIVETo develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect.

METHODSThe pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined.

RESULTSThe laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged.

CONCLUSIONA new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

Animals ; Cancer Vaccines ; therapeutic use ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Enterotoxins ; immunology ; Gene Expression ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Superantigens ; immunology ; Transfection

Clinical application of staphylococcal enterotoxin C2 (SEC2) was restricted during the cure of malignant tumor due to its side-effects. The aim of this study was to obtain SEC2 mutant, preserving the important functional sites responsible for the T-cell stimulatory activities but removing the sites responsible for emetic activity, through truncation of SEC2. It would efficiently solve the question of SEC2 side-effect. According to the results of methyl thiazol tetrazolium (MTT) assay in vitro, novel truncated SEC2 mutant (NSM) efficiently stimulated T-cell proliferation and inhibited the growth of such tumor cells as human colorectal cancer cells (Cx-1) and human breast cancer cells (MCF-7) in vitro. Activities of T cell stimulating and anti-tumor of NSM were similar to those of SEC2. According to results of animal experiments, the mutant no longer induced emetic response even if the dose was a 10-fold excess of the amount of SEC2 required. And also, NSM obviously inhibited the tumor growth in tumor-bearing mice. Therefore, we obtained novel truncated staphylococcal enterotoxin C2 mutant, which could efficiently inhibit the growth of tumor cells. It will become novel anti-tumor agents with the lowest side-effects and best treatment effects in clinic.
Animals ; Antineoplastic Agents ; adverse effects ; pharmacology ; Breast Neoplasms ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; immunology ; pathology ; Enterotoxins ; genetics ; immunology ; Humans ; Mice ; Mutant Proteins ; immunology ; Staphylococcus aureus ; immunology ; Superantigens ; immunology ; T-Lymphocytes ; immunology ; Vomiting ; prevention & control