Toxic shock syndrome (TSS) is a complex of generalized symptoms caused by a local staphylococcal infection, and a circulating toxin is thought to be involved. Indeed, nearly 100% of TSS isolates produce an exoprotein, TSSE, that is thought to have an aetiological role on the basis of positive animal tests (refs 1,2 and F. Quimby, personal communication) and human serological data. Although the precise role of TSSE in TSS remains unclear (E. Kass, personal communication), no other staphylococcal factor has been implicated. Our preliminary studies of the genetics of TSSE production failed to demonstrate plasmid or phage involvement or linkage with known chromosomal genes (ref. 4 and B.N.K. et al., unpublished data); however, Schutzer et al. have found that most TSS strains harbour prophages with common plating characteristics and suggest that the toxin(s) involved in TSS are transmitted by lysogenic conversion. We show here that TSSE is not demonstrably transferred by lysogeny; moreover, we have cloned the gene and found that the cloned product is serologically and biologically indistinguishable from the native protein, and that the TSSE determinant is associated with a larger DNA segment that is absent or rearranged in TSSE- strains.

The gene for staphylococcal enterotoxin A (entA), in two wild-type strains, is carried by related temperate bacteriophages. Hybridization analysis of DNA from entA-converting phage PS42-D and its bacterial host suggests that this phage integrates into the bacterial chromosome by circularization and reciprocal crossover (the Campbell model) and that the entA gene is located near the phage attachment site. DNA from three of eight staphylococcal strains that did not produce enterotoxin A and seven wild-type enterotoxin A-producing (EntA+) strains had extensive homology to the entA-converting phage PS42-D DNA, although there was a high degree of restriction-fragment length polymorphisms. At least one EntA+ strain did not produce detectable viable phage after induction. These data indicate that a polymorphic family of Staphylococcus aureus phages (some of which may be defective) can carry the entA gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and Cl, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and Cl hybridized well to DNA isolated from EntA', EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.

Staphylococcal enterotoxins are exotoxins produced byStaphylococcus aureus that possess emetic and superantigenic properties. Prior to this research there were six characterized enterotoxins, staphylococcal enterotoxin types A to E and H (referred to as SEA to SEE and SEH). Two new staphylococcal enterotoxin genes have been identified and designated segand sei (staphylococcal enterotoxin types G and I, respectively). seg and sei consist of 777 and 729 nucleotides, respectively, encoding precursor proteins of 258 (SEG) and 242 (SEI) deduced amino acids. SEG and SEI have typical bacterial signal sequences that are cleaved to form toxins with 233 (SEG) and 218 (SEI, predicted) amino acids, corresponding to mature proteins of 27,043 Da (SEG) and 24,928 Da (SEI). Biological activities for SEG and SEI were determined with recombinant S. aureus strains. SEG and SEI elicited emetic responses in rhesus monkeys upon nasogastric administration and stimulated murine T-cell proliferation with the concomitant production of interleukin 2 (IL-2) and gamma interferon (IFN-γ), as measured by cytokine enzyme-linked immunoassays. SEG and SEI are related to other enterotoxins of S. aureus and to streptococcal pyrogenic exotoxin A (SpeA) and streptococcal superantigen (SSA) of Streptococcus pyogenes. Phylogenetic analysis and comparisons of amino acid and nucleotide sequence identities were performed on related staphylococcal and streptococcal protein toxins to group SEG and SEI among the characterized toxins. SEG is most similar to SpeA, SEB, SEC, and SSA (38 to 42% amino acid identity), while SEI is most similar to SEA, SEE, and SED (26 to 28% amino acid identity). Polyclonal antiserum was generated against purified histidine-tagged SEG and SEI (HisSEG and HisSEI). Immunoblot analysis of the enterotoxins, toxic-shock syndrome toxin 1, and SpeA with antiserum prepared against HisSEG and HisSEI revealed that SEG shares some epitopes with SEC1 while SEI does not.

The effect of alkaline pH on expression of the accessory gene regulator (agr) in Staphylococcus aureus was examined. agr, a global regulator, affects the expression of numerous exoproteins, including a-hemolysin, toxic shock syndrome toxin 1, protein A, and staphylococcal enterotoxins types B, C, and D. agr contains two major, divergent transcripts, designated RNAII and RNAHI. In this study, the level of RNAIII was used to monitor agr expression because this transcript and/or its protein product(s) appears to be responsible for altering target gene expression. S. aureus FRI1230 and its Agr-derivative were examined in a fermentor system which allowed batch cultures to be maintained at a constant pH. FRI1230 cultures were grown at pH 6.5, 7.0, 7.5, and 8.0. Northern (RNA blot) analysis of samples revealed that maximal agr expression occurred at pH 7.0, with virtually no RNAI observed at pH 8.0. The effect of alkaline pH on an agr target gene, sec, was also evaluated. sec expression was reduced at alkaline pH in strain FRI1230 (Agr+) but not in its Agr-derivative, indicating that an intact agr allele is required for the pH effect on sec. Examination of batch cultures under conditions of nonmaintained pH gave results that were also consistent with a role for alkaline pH in repressing agr expression.The Staphylococcus aureus accessory gene regulator (agr) was identified initially as a locus which, when inactivated, results in altered production of several exoproteins and cell surface-associated proteins (7,25). An Agr+ strain produces more ax-hemolysin, ,-hemolysin, toxic shock syndrome toxin 1 (TSST-1), and staphylococcal enterotoxin types B, C, and D (SEB, SEC, and SED, respectively) and less coagulase and protein A (2,3,12,13,25,27) than its Agrderivative. The ability of agr to function as both an activator and a repressor suggests the existence of two mechanisms for agr-mediated regulation.Nucleotide and mutational analysis of the agr locus revealed that it is a divergent operon containing two major transcripts that are referred to as RNAII and RNAIII (3.5 and 0.5 kb in length, respectively [1,15,16,20,23,24]).

Staphylococcal enterotoxins cause the intoxication staphylococcal food poisoning syndrome (3). The enterotoxins are classified by serological criteria into five major groups (A to E, referred to as SEA to SEE, respectively); SEC can be further subdivided into SEC1, SEC2, and SEC3 (3,29). Cross-reactivity has been observed between SEA and SEE and between SEB and the SECs (9,18,19,40).The protein sequences of SEA, SEB, and SEC1 and nucleotide sequences of their genes (entA, entB, and entC1, respectively) have been determined (6,7,11,12,16,33). Comparison of the amino acid sequences of SEA, SEB, and SEC1 has demonstrated that these proteins are not only related to each other but also to type A streptococcal pyrogenic exotoxin (SPE A) (6,7,14,16,44). SPE A, like the enterotoxins, is mitogenic and pyrogenic and enhances endotoxic shock (1, 3).The genes for entA and SPE A (speA) are encoded by lysogenic phages in Staphylococcus aureus and Streptococcus pyogenes, respectively (5,15,43). The relationship between these phages is not known. The entB and entC, genes are linked to the same plasmid in at least one Staphylococcus aureus strain (2). Another S. aureus strain has an entD-encoding plasmid (K. W. Bayles, and J. J. Iandolo, Abstr. Annu. Meet. Am. Soc. Microbiol., 1987, B187, p. 56).Previously, we have shown (6) that DNA isolated from SEE-producing (EntE+) S. aureus strains has homology with the entA gene. In this report, cloning and nucleotide sequence determinations of the homologous DNA demonstrated that it is the entE gene. The entE gene has 84% nucleotide sequence homology with the entA gene. Comparison of amino acid sequence data obtained from nucleotide and peptide analyses suggest that SEE is synthesized as a precursor of 257 amino acid residues (molecular weight; 29,358) and is processed to yield a mature form of 230 amino acid residues (molecular weight, 26,425). Amino acid sequence comparisons also agreed with the relationships suggested by serological data (9,18,19,40). SEE is more closely related to SEA than it is to SEB, SEC1, or SPE A. * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmids, phages, and culture conditions. S. aureus MJB265 was strain RN450 that was lysogenized with the entA-converting phage FRI337-1 (5 ) contained pBR322, pMJB9, and pMJB38, respectively. pMJB9 is a pBR322 derivative that contains a 2.5-kilobase-pair (kb) HindIII fragment insert encoding a functional entA gene. pMJB38 is a pBR322 derivative that contains a 624-base-pair fragment (designated A-624) that contains only entA structural gene sequences. E. coli strains were routinely grown in LB (21) containing 100 p,g of ampicillin per ml.The propagation of E. coli phage M13 derivatives (24, 27, 42) and E. coli JF626, which was used for M13 phage propagation (obtained from Jeff Felton), has been described previously (35).DNA isolation procedures. Genomic DNA was isolated from S. aureus protoplasts (37). Staphylococcal DNA probes for hybridization reactions were obtained as follows. Phage FRI337-1 was prop...

We used three related strains of Staphylococcus aureus to determine whether capsule size influenced bacterial virulence. Strain SA1 mucoid elaborated a large capsule demonstrable by transmission electron microscopy (TEM). Nonmucoid isolates were derived from strain SA1 mucoid by Tn551 insertional mutagenesis. By TEM, strain JL24 produced a "microcapsule," whereas strain JL25 was unencapsulated. Strain SA1 mucoid had a 50% lethal dose for mice greater than 3,000-fold lower than that of strains JL24 and JL25. Quantitative cultures of blood and kidney from animals challenged intravenously revealed that strain SA1 mucoid was cleared less readily from the bloodstream and kidneys than the nonmucoid mutants. In an in vitro assay, only strain SA1 mucoid demonstrated antibody-dependent, complement-mediated opsonophagocytosis by human leukocytes. Strains JL24 and JL25 were opsonized for phagocytosis by complement alone. Thus a highly encapsulated strain of S. aureus was more virulent in mice than two related nonmucoid strains. The microencapsulated mutant was not more virulent than the unencapsulated mutant.

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