Inhibition of emetic and superantigenic activities of staphylococcal enterotoxin A by synthetic peptides

Staphylococcus aureus is a major human pathogen producing different types of toxins. Enterotoxin A (SEA) is the most common type among clinical and food-related strains. The aim of the present study was to estimate functional regions of SEA that are responsible for emetic and superantigenic activities using synthetic peptides. A series of 13 synthetic peptides corresponding to specific regions of SEA were synthesized, and the effect of these peptides on superantigenic activity of SEA including interferon γ (IFN-γ) production in mouse spleen cells, SEA-induced lethal shock in mice, spleen cell proliferation in house musk shrew, and emetic activity in shrews were assessed. Pre-treatment of spleen cells with synthetic peptides corresponding to the regions 21–40, 35–50, 81–100, or 161–180 of SEA significantly inhibited SEA-induced IFN-γ production and cell proliferation. These peptides also inhibited SEA-induced lethal shock. Interestingly, peptides corresponding to regions 21–40, 35–50 and 81–100 significantly inhibited SEA-induced emesis in house musk shrews, but region 161–180 did not. These findings indicated that regions 21–50 and 81–100 of SEA are important for both superantigenic and emetic activities of SEA molecule while region 161–180 is involved in superantigenic activity but not emetic activity of SEA. These regions could be important targets for therapeutic intervention against SEA exposure.     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Lowering of bactericidal activity of mouse peritoneal macrophages under combined use of staphylococcal enterotoxin type A and endotoxin

The present study investigated the effect of staphylococcal enterotoxin type A (SEA) and endotoxin Serratia marcescens (LPS) on the phagocytosis and killing of Staphylococcus aureus by mouse peritoneal macrophages. Two hours after enterotoxin intraperitoneal injection phagocytic and bactericidal activity were depressed. 24 hours later there was increased functional activity of macrophages by SEA and LPS, apart. But when two toxins were administered together (LPS four hours later enterotoxin) marked inhibition of bacterial killing was observed. When peritoneal macrophages were treated in vitro for 24 hours with the same toxins they were also markedly suppressed in bactericidal activity.     

Transport and processing of staphylococcal enterotoxin A

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.     

Guanidination and nitroguanidination of staphylococcal enterotoxin B

Guanidination of the free amino groups of staphylococcal enterotoxin B with 3,5-dimethyl-1-guanylpyrazole converted 31-32 of 33 epsilon-amino groups and 30% of the N-terminal residue. This product, although markedly reduced in solubility, suffered no gross change in conformation and retained full biological activity. A derivative prepared by reaction with O-methylisourea with only one lysyl residue unaltered lost most of its emetic activity. Nitroguanidination with 3,5-dimethyl-1-nitroguanylpyrazole converted up to 28 of the epsilon-amino groups and essentially all of the N-terminus. This material was greatly reduced in ability to produce emesis and like the O-methylisourea prepared guanidinated enterotoxin, gave only a line of partial identity in double diffusion. The loss of activity is attributed to unfolding and it is concluded that the free amino groups of enterotoxin B do not critically participate in either its antigenic determinants or its active center for emesis.     

Immunosuppression induced by staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) is a potent mitogen for both human and murine T lymphocytes. We report here studies which demonstrate that a suppressor cell population, capable of suppressing the primary immune response of normal syngeneic mouse splenocytes to heterologous sheep erythrocytes (SRBC), is activated by SEB. Enterotoxin concentrations ranging from 0.05 to 5.0 Mg ml^?1 are capable of activating this suppressor cell population, and significant suppression can be detected with relatively small numbers of SEB-primed spleen cells (SEB-PSC) in culture. Elimination of macrophages before or after priming splenocytes with SEB does not reduce the suppression of plaque-forming cell (PFC) responses when SEB-PSC are added to normal cells in Mishell-Dutton cultures. Treatment of cells with anti-Thy-1 serum plus complement, after priming with SEB, effectively eliminates the activity of the suppressor cell population. Enrichment for T cells before priming with SEB results in greater suppression of PFC responses than do SEB-PSC generated in cultures of nonfractionated splenocytes. Activation of suppressor cells with SEB in vitro appears to occur through the induction of the T-cell subpopulation expressing the Lyt-1^?,2⁺,3⁺ cell surface phenotype, since selective depletion of this T-cell subpopulation with monoclonal rat anti-mouse Lyt-2 antisera after priming cells with SEB virtually eliminates the suppressor activity.