Structure of streptococcal pyrogenic exotoxin A reveals a novel metal cluster.

The streptococcal pyrogenic toxins A, B, and C (SPEA, SPEB, and SPEC) are responsible for the fever, rash, and other toxicities associated with scarlet fever and streptococcal toxic shock syndrome. This role, together with the ubiquity of diseases caused by Streptococcus pyogenes, have prompted structural analyses of SPEA by several groups. Papageorgiou et al. (1999) have recently reported the structure of SPEA crystallized in the absence of zinc. Zinc has been shown to be important in the ability of some staphylococcal and streptococcal toxins to stimulate proliferation of CD4+ T-cells. Since cadmium is more electron dense than zinc and typically binds interchangeably, we grew crystals in the presence of 10 mM CdCl2. Crystals have been obtained in three space groups, and the structure in the P2(1)2(1)2(1) crystal form has been refined to 1.9 A resolution. The structural analysis revealed an identical tetramer as well as a novel tetrahedral cluster of cadmium in all three crystal forms on a disulfide loop encompassing residues 87-98. No cadmium was bound at the site homologous to the zinc site in staphylococcal enterotoxins C (SECs) despite the high structural homology between SPEA and SECs. Subsequent soaking of crystals grown in the presence of cadmium in 10 mM ZnCl2 showed that zinc binds in this site (indicating it can discriminate between zinc and cadmium ions) using the three ligands (Asp77, His106, and His110) homologous to the SECs plus a fourth ligand (Glu33).

Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome.

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.

Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals.

Staphylococcus aureus and Streptococcus pyogenes express pyrogenic toxin superantigens (PTSAgs) that are associated with toxic shock syndrome (TSS) and staphylococcal food poisoning (SFP). Most PTSAgs cause TSS in deep-tissue infections, whereas only TSS toxin 1 (TSST-1) is associated with menstrual, vaginal TSS. In contrast, SFP has been linked only with staphylococcal enterotoxins (SEs). Because of the differential abilities of PTSAgs to cause systemic or localized symptoms in a site-dependent manner, the present study was undertaken to assess the toxins' abilities to cross mucosal barriers. The activity of three PTSAgs when delivered orally, vaginally, or intravenously to rabbits and orally to monkeys was investigated. TSST-1 induced shock via all three routes in rabbits. Although active when administered intravenously, SEC1 and streptococcal pyrogenic exotoxin A (SPEA) did not cause symptoms when administered orally or vaginally. Only SEC1 induced emesis in the monkey feeding assay. TSST-1, albeit less stable than SEC1 and SPEA to pepsin, induced diarrhea in monkeys. Our results may explain the unique association of TSST-1 with menstrual TSS and why SPEA is only rarely associated with TSS after pharyngitis, despite being highly associated with TSS after subcutaneous infections. Finally, our studies indicate that enterotoxicity in SFP is not the result of superantigenicity.

Purification of streptococcal pyrogenic exotoxin a.

Group A streptococci secrete a variety of molecules, many of which are recognized as virulence factors important in the establishment of streptococcal infections. Among these extracellular products is streptococcal pyrogenic exotoxin A (SPE A, scarlet fever toxin A, erythrogenic toxin A) (1). Other SPEs include toxin serotypes B and C (1), streptococcal superantigen (SSA) (2), and SPE F (mitogenic factor) (3,4). Combinations of these toxins are believed to be important in streptococcal toxic shock syndrome. The latter two molecules will not be discussed in this chapter, but methods utilized to purify SPEs A-C also may be used to purify SSA and SPE F.

Analysis of toxicity of streptococcal pyrogenic exotoxin A mutants.

Streptococcal pyrogenic exotoxin A (SPE A) is secreted by some strains of Streptococcus pyogenes and is strongly associated with streptococcal toxic shock syndrome (STSS), a severe and often fatal illness. SPE A possesses a number of biological properties, some of which are shared with a group of exotoxins of streptococcal and staphylococcal origins, the pyrogenic toxin superantigens (PTSAgs). SPE A's most extensively studied property is superantigenicity. Superantigenic activation of T cells and monocytes stimulates the release of cytokines such as tumor necrosis factors alpha and beta, interleukin 1, and gamma interferon. These endogenous mediators are considered to be the primary cause of capillary leak, hypotension, and shock, the most severe manifestations of STSS. However, several studies have suggested that other properties of SPE A, such as ability to greatly enhance host susceptibility to endotoxin and ability to interact directly with endothelial cells, may play substantial roles in the syndrome. In this work we generated single- and double-site mutations of SPE A at residues K16, N20, C87, C90, C98, K157, S195, N20/C98, and N20/K157. The mutant SPE A's were analyzed in vivo for their lethal activity and in vitro for their superantigenic ability. Our results indicate that SPE A's ability to induce lethality and endotoxin enhancement does not require superantigenicity, and conversely superantigenicity does not necessarily lead to lethality. Thus, these properties and their relative contributions to the onset of hypotension and shock may be separable. Furthermore, evidence is presented that certain mutant toxins may be suitable for use as vaccine toxoids.

The major role of Spo0A in genetic competence is to downregulate abrB, an essential competence gene.

We show that the major role for Spo0A in the development of genetic competence is to downregulate expression of abrB. AbrB is both a negative regulator and a positive regulator of competence. The negative effects are exerted at multiple points in competence regulation. A regulatory mechanism that is independent of mecA and abrB operates on comK expression.

ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA.

ComA is a response regulator protein of Bacillus subtilis which is required for the transcription of several genes which are involved in late-growth expression and in responses to environmental stress. Among these genes are degQ, gsiA, and srfA. The last is an operon needed for the development of genetic competence, surfactin production, and normal sporulation. We show here that partially purified ComA protein, isolated from an overproducing Escherichia coli strain, is phosphorylated in vitro by incubation with acetyl phosphate and that ComA could bind specifically to a DNA fragment containing the promoter of srfA and associated sequences. The binding affinity is enhanced when ComA is phosphorylated. DNase I protection analysis identified two protected sites located upstream from the srfA promoter. The presence of DNase I-hypersensitive bonds induced by ComA binding which are located between the protected sequences is consistent with a model for ComA action involving the bending of DNA.

Growth medium-independent genetic competence mutants of Bacillus subtilis.

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.

Suppression of early competence mutations in Bacillus subtilis by mec mutations.

Although competence normally develops only in glucose-minimal salts media, mecA and mecB mutations permit the expression of competence and of late competence genes in complex media as well (D. Dubnau and M. Roggiani, J. Bacteriol. 172:4048-4055, 1990). The expression of late competence genes is dependent on the products of the regulatory genes comA, comB, comP, sin, abrB, spo0H, and spo0A. We show here that this list must be extended to include degU, csh-293, and spo0K. mecA and -B mutations bypass most of these requirements, making the expression of late competence genes and of competence itself independent of all of these regulatory genes, with the exceptions of spo0A and spo0K (in the case of mecB). The expression of late competence genes in mec mutants that are deficient for each of the bypassed regulatory functions is still under growth stage-specific regulation. The implications of these findings are discussed, and a provisional scheme for the flow of information during the development of competence is proposed.