Identification and characterization of staphylococcal enterotoxin types G and I from Staphylococcus aureus.

Staphylococcal enterotoxins are exotoxins produced by Staphylococcus 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 seg and 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-gamma), 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.

Biochemical and mutational analysis of the histidine residues of staphylococcal enterotoxin A.

The goal of this study was to examine the role of histidine residues in the biological activities of staphylococcal enterotoxin A (SEA). Carboxymethylated SEA was unable to stimulate murine T-cell proliferation but was resistant to monkey stomach lavage fluid degradation, suggesting that native conformation was intact. Site-directed mutagenesis of the histidine residues of SEA was subsequently performed. SEA-H44A (SEA with histidine 44 replaced with alanine), SEA-H44D, SEA-H50A, SEA-H50D, SEA-H114A, SEA-H114D, SEA-H187A, and SEA-H187D retained superantigen and emetic activities, whereas SEA-H225A and SEA-H225D were defective in the ability to stimulate T-cell proliferation. These mutants were unable to compete with SEA for binding to Raji cells, suggesting that the defect in SEA-H225A and SEA-H225D is due to impaired major histocompatibility complex class II binding. SEA-H225D provoked an emetic response in monkeys only if fed at high doses, while SEA-H225A did not provoke an emetic response at low or high doses. In comparison, SEA-H61A and SEA-H61D were defective in emetic activity but not in the ability to stimulate murine T-cell proliferation. Overall, these studies show that the carboxy-terminal histidine at residue position 225 of SEA is important for both the superantigen and emetic activities of this enterotoxin. Histidine 61 appears to be important for emetic activity but not for superantigen activity, consistent with the hypothesis that the two activities are separable in staphylococcal enterotoxins.

Biological activities of staphylococcal enterotoxin type A mutants with N-terminal substitutions.

The purpose of this study was to examine the importance of certain N-terminal amino acid residues of staphylococcal enterotoxin type A (SEA) for biological activity. The results confirm our previous observation that Asn-25, Phe-47, and Leu-48 are important for SEA's emetic and superantigen activities. Substitutions at six other sites (Leu-12, Lys-14, Ser-16, Asp-45, Gln-46, and Thr-51) did not reveal any additional residues required for biological activity. Mutant SEAs with substitutions at 25, 47, or 48 all had decreased T-cell stimulatory activity, with the mutants at position 47 being the most defective. Results of a competition assay for binding to the major histocompatibility complex (MHC) class II-expressing cell line Raji suggested that the decreased superantigen activities of the mutants with substitutions at positions 47 and 48 are due to poor interactions with MHC class II molecules, whereas the defects of the mutants at position 25 are a consequence of faulty interactions with T-cell receptors. With respect to emetic activity in rhesus monkeys, the mutants at position 25 or 48 exhibited decreased but significant activity. Interestingly, the two mutants at position 47 had different emetic activities; SEA-F47G was nonemetic when administered intragastrically at 500 micrograms per animal, whereas SEA-F47S was emetic at this dosage. Since the mutants at position 47 were equally defective for superantigen activity, this further supports our previous suggestion of an incomplete correlation between SEA's emetic and superantigen activities.

In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus.

The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70.

Promoter analysis of the staphylococcal enterotoxin A gene.

The promoter region of the staphylococcal enterotoxin A (SEA) gene (sea) of Staphylococcus aureus was localized by primer extension analysis in conjunction with in vitro mutagenesis. The 5'-end of sea mRNA was located 86 base pairs upstream of the translational initiation codon. A DNA region with good agreement with canonical promoter sequences was observed beginning 8 base pairs upstream of the apparent transcriptional start site. Analysis of a series of progressive deletions of upstream DNA revealed that no DNA upstream of the putative -35 region was required for transcription of sea (determined by primer extension analysis) or for SEA production as detected by Western immunoblot analysis. Deletion mutants extending into the -35 region or mutants containing nucleotide substitutions in the -10 region both showed dramatic reductions in SEA production and transcription of sea. Analysis of a deletion mutant in which 59 base pairs between the transcriptional and translational start sites were deleted revealed slightly increased levels of SEA production.

Phage-associated differences in staphylococcal enterotoxin A gene (sea) expression correlate with sea allele class.

Staphylococcus aureus strains which produced either high or low levels of staphylococcal enterotoxin A (SEA) with a minimal eightfold difference between the two groups were identified. For FRI100 and FRI281A (prototypes for each group), strain differences in the expression of the SEA-encoding gene (sea) were found to occur at the level of sea mRNA concentration, and part of the difference in expression was associated with the sea-containing phages. Southern blot analysis revealed that this phage-associated difference was not due to differences in the copy number of sea. Nucleotide sequence analysis of sea from FRI281A revealed a new allele of sea, with the majority of the sequence differences occurring in the upstream promoter region. Although a strict correlation was observed between the level of SEA production and sea allele class for several strains, the sequence differences observed in the upstream region were not sufficient in themselves to alter the expression level of sea.

Mutations in the promoter spacer region and early transcribed region increase expression of staphylococcal enterotoxin A.

The mechanism leading to increased production of staphylococcal enterotoxin type A (SEA) in mutant Staphylococcus aureus FRI722 compared within its wild-type parent strain, FRI100, was examined. Sequence analysis revealed two mutations in the upstream promoter region of FRI722 at nucleotides -28 and +3 with respect to the transcriptional initiation site at An sea translational fusion of the upstream region of FRI722 to the structural gene from FRI100 showed an increase in sea expression by Northern (RNA) analysis and in SEA production by Western (immunoblot) analysis. To independently evaluate the effect of each mutation, site-directed mutagenesis was done and revealed that each mutation was responsible for an increase in SEA production.

Lack of complete correlation between emetic and T-cell-stimulatory activities of staphylococcal enterotoxins.

This study examined the emetic activity of several staphylococcal enterotoxin type A and B (SEA and SEB, respectively) mutants that had either one or two amino acid residue substitutions. New sea gene mutations were constructed by site-directed mutagenesis; gene products were obtained with glycine residues at position 25, 47, 48, 81, 85, or 86 of mature SEA. Culture supernatants from Staphylococcus aureus RN4220, or derivatives containing either sea or a sea mutation, were analyzed for the ability to stimulate proliferation of murine splenocytes, as determined by incorporation of [3H]thymidine. Culture supernatants containing SEA-N25G (a SEA mutant with a substitution of glycine for the asparagine residue at position 25), SEA-F47G, or SEA-L48G did not stimulate T-cell proliferation, unlike supernatants containing the other substitution mutants. Purified preparations of SEA-N25G had weak activity and those of SEA-F47G and SEA-L48G had essentially no activity in the T-cell proliferation assay. All mutants except SEA-V85G, which was degraded by monkey stomach lavage fluid in vitro, were tested for emetic activity. SEA-C106A and two SEB mutants, SEB-D9N/N23D and SEB-F44S (previously referred to as BR-257 and BR-358, respectively), whose construction and altered immunological properties have been reported previously, were also tested in the emetic assay. Each mutant was initially administered intragastrically at doses of 75 to 100 micrograms per animal; if none of the animals responded, the dose was increased four-to fivefold. SEA-F47G, SEA-C106A, and SEB-D9N/N23D were the only mutants that did not induce vomiting at either dose tested; these three mutants had reduced immunological activity. However, there was not a perfect correlation between immunological and emetic activities; SEA-L48G and SEB-F44S retained emetic activity, although they had essentially no T-cell-stimulatory activity. These studies suggest that these two activities can be dissociated.

Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation.

Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to SEA appeared to be dependent on the conformation of SEA.

High sodium chloride concentrations inhibit staphylococcal enterotoxin C gene (sec) expression at the level of sec mRNA.

Expression of the staphylococcal enterotoxin type C gene (sec) is regulated in response to both high NaCl concentrations (osmolarity) and the accessory gene regulator (agr). agr is a global regulator that alters the expression of many genes in Staphylococcus aureus. In this report, we have demonstrated that osmoregulation of sec occurs at the level of mRNA independently of an intact agr allele. Northern (RNA) and Western blot (immunoblot) analyses of samples from cultures grown in low- (0 M NaCl) and high-osmotic-strength (1.2 M NaCl) media revealed that the low-osmotic-strength culture contained approximately 16-fold more SEC and sec mRNA than the high-osmotic-strength culture. sec expression in high-osmotic-strength medium was enhanced when osmoprotective compounds were added. Osmoregulation of sec expression in Agr- strains was also examined; SEC and sec mRNA levels decreased in response to high osmolarity in a manner similar to that seen in the Agr+ strains.

Staphylococcal enterotoxin A gene (sea) expression is not affected by the accessory gene regulator (agr).

The goal of this work was to determine whether staphylococcal enterotoxin type A gene (sea) expression is regulated by an accessory gene regulator (agr). The Tn551 insertionally inactivated agr allele of Staphylococcus aureus ISP546 was transferred to three Sea+ S. aureus strains. Each of the Agr- strains produced as much staphylococcal enterotoxin A (SEA) as its parent strain. These results suggest that sea expression is regulated differently from that of seb, sec, and sed, which previously have been shown to require a functional agr system for maximal expression.

Dual roles for class II major histocompatibility complex molecules in staphylococcal enterotoxin-induced cytokine production and in vivo toxicity.

The staphylococcal enterotoxins (SE) specifically bind to class II major histocompatibility complex (MHC) proteins, resulting in activation of monocytes and T cells. The SE cause weight loss in mice, which is dependent on T-cell stimulation and tumor necrosis factor alpha (TNF-alpha) production. Here we use a mutant of staphylococcal enterotoxin A that binds class II MHC molecules and activates monocytes but not T cells to evaluate the relative contributions of monocyte- and T-cell-stimulatory activities to in vivo toxicity. The mutant toxin did not cause weight loss in B10. BR mice but did stimulate monocyte TNF-alpha production in vitro, as did the wild-type toxin. Addition of a supernatant from toxin-activated T cells enhanced monocyte-stimulatory activity of both mutant and wild-type toxins fivefold. The effect of the supernatant could be mimicked by recombinant gamma interferon (IFN-gamma) and was inhibited by antibody to IFN-gamma. These results suggest that toxin-induced monocyte TNF-alpha production is upregulated by IFN-gamma, which likely represents the T-cell requirement in SE-mediated weight loss. Our studies thus implicate two distinct class II MHC-dependent signaling pathways for SE, the first involving direct signal transduction through class II MHC molecules mediated by either mutant or wild-type toxin and the second requiring T-cell stimulation by toxin-class II MHC complexes with consequent production of IFN-gamma. We suggest that both pathways are required for optimal monocyte TNF-alpha production in vitro and SE-induced toxicity in vivo.

Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus.

The effect of glucose on accessory gene regulator (agr) expression in Staphylococcus aureus was examined. agr is a global regulator that affects the expression of numerous genes, including those for some factors implicated in virulence, such as toxic shock syndrome toxin 1, alpha-hemolysin, and protein A. The agr locus determines two divergent transcripts, designated RNAII and RNAIII. RNAII contains four open reading frames (agrABCD), and RNAIII encodes delta-hemolysin. The mechanisms responsible for agr-mediated regulation are not well understood, but it appears that the RNAIII transcript plays a central role in the regulation of a number of target genes, including those for alpha-hemolysin (hla), beta-hemolysin (hlb), protein A (spa), and staphylococcal enterotoxin B (seb+). In this study, S. aureus cultures were grown either in a shake flask system with a complex medium or in a fermentor system with a completely defined medium in which the pH and glucose concentration were maintained. Northern (RNA) blot analysis revealed that a dramatic reduction in agr expression was apparent only when the cultures contained glucose and when the pH was 5.5 or was not maintained. The effect of glucose on two agr target genes, sec+ and hla, was also studied. Glucose-containing cultures produced less sec+ and hla mRNAs at maintained pH (6.5). In addition, the glucose effect on sec+ and hla was enhanced under conditions that inhibited agr expression (i.e., pH 5.5 or a nonmaintained pH).

Alkaline pH decreases expression of the accessory gene regulator (agr) in Staphylococcus aureus.

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 alpha-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 RNAIII. 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 RNAIII 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 carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.

Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay.

Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose.

The effects of the accessory gene regulator (agr) and glucose on staphylococcal enterotoxin type C (SEC) gene (sec+) expression were examined. For the agr studies, a Tn551 insertionally inactivated agr was transferred into two different sec+ Staphylococcus aureus strains. Western blot (immunoblot) analysis showed that each of the sec+ Agr- derivatives produced less extracellular SEC than their Agr+ parent strains. Analysis of Northern (RNA) blots was consistent with at least part of the agr effect being at the level of steady-state sec+ mRNA. We examined the glucose effect on sec+ expression by utilizing both a fermentor system with a completely defined amino acid-containing medium in which the pH of the medium was maintained at 6.5 and a shake flask system with a complex medium in which the pH was allowed to fluctuate during bacterial growth. In both systems, samples from the cultures containing glucose had less extracellular SEC and less steady-state sec+ mRNA compared with the control cultures which lacked glucose. An intact agr was not required for the glucose effect on sec+ expression; MJB407, an Agr- sec+ strain, produced more SEC and had more steady-state sec+ mRNA when grown in medium that lacked glucose compared with medium that contained glucose.

Identification of a bacteriophage containing a silent staphylococcal variant enterotoxin gene (sezA+).

A variant enterotoxin gene, referred to as sezA+, has been identified. Staphylococcus aureus FRI1106, a staphylococcal enterotoxin type D producer (Sed+), contained HindIII fragments of 3.8 and 9.4 kilobase pairs (kbp) that hybridized in Southern blot analysis to a probe containing only staphylococcal enterotoxin type A structural gene sequences. Presumably, probe A-624 hybridized to the 9.4-kbp HindIII fragment because of the sequence homology between sea+ and sed+. This 9.4-kbp HindIII fragment, which was part of a staphylococcal plasmid, was isolated and ligated into an Escherichia coli plasmid vector; Sed+ E. coli recombinant clones were isolated. The 3.8-kbp HindIII fragment was shown to be part of a viable lysogenic bacteriophage, and it contained sezA+. This sezA(+)-containing fragment was cloned into E. coli, and its DNA sequence was determined. Examination of the nucleotide sequence revealed a 771-bp region that contained an open reading frame with 85 and 77% nucleotide and derived amino acid sequence identifies with sea+ and staphylococcal enterotoxin type A, respectively. This open reading frame has 83 to 50% nucleotide sequence identities with the other types of staphylococcal enterotoxin genes. sezA+ was shown to be transcribed into stable mRNA. However, the sezA+ mRNA was not translated into an enterotoxinlike protein because it lacks an appropriate translation initiation codon.

Nucleotide sequence of the type C3 staphylococcal enterotoxin gene suggests that intergenic recombination causes antigenic variation.

The nucleotide sequence of the structural gene for staphylococcal enterotoxin type C3 (entC3) was determined. This gene contains 798-base-pair open reading frame that encodes a protein of 266 amino acid residues. Sequence analysis suggests that staphylococcal enterotoxin type C3 is synthesized in a precursor form that is processed to yield a mature extracellular form of 238 amino acid residues (molecular weight, 27,438). The entC3 gene is closely related to the gene for staphylococcal enterotoxin type C1, with 98% nucleotide sequence identity. Sequence comparisons between the entC3, entC1, and entB genes suggest that an ancestral entC1-like gene was formed by recombination between the entC3 and entB genes.

Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region.

A 456 basepair HindIII fragment that encoded a portion of the type A botulinum neurotoxin gene was cloned into Escherichia coli using a plasmid vector. DNA sequence analysis revealed that this botulinum DNA insert encoded an open reading frame of 35 amino acid residues of which 34 corresponded to the N-terminal residues of botulinum neurotoxin type A.