Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.Group A streptococcus (GAS) causes many diseases in humans, ranging in severity from milder infections such as pharyngitis, simple cellulitis, erysipelas, and scarlet fever to life-threatening necrotizing fasciitis, septicemia, and toxic shock syndrome. One of the many potentially important virulent factors produced by this organism is streptococcal pyrogenic exotoxin B (SpeB). As a potent cysteine proteinase, SpeB cleaves multiple streptococcal virulence factors, including M protein (3), as well as many host factors controlling inflammation (18,20).The gene for SpeB (speB) is chromosomally located on every GAS strain studied and consists of a 1,196-base pair (bp) open reading frame yielding a 371-amino-acid polypeptide with a predicted molecular weight of 40,000 (16). SpeB is secreted strictly in the late log/early stationary phase of growth as a proteinase precursor that must be proteolytically cleaved to the mature active form having a calculated molecular mass of approximately 28 kDa. SpeB is also found on the surfaces of the bacteria and possesses glycoprotein and laminin binding activities (19). While all strains of GAS are endowed with the gene for SpeB, not all strains produce the toxin in vitro, and even among strains that do, the quantity produced varies greatly from strain to strain (6,15,16,22,31). Other environmental factors, such as acidic pH, concentration of NaCl, the availability of nutrients, the presence of kanamycin, etc., also affect speB expression (7, 9, 39).Current knowledge regarding SpeB's transcriptional regulation and maturation is derived from many labs around the world. At the transcriptional level, rgg (also known as ropB) positively regulates SpeB expression and production (5), as does the global regulator mga (35). In addition, inactivation of both oligopeptide and dipeptide transport systems diminished speB mRNA levels (33, 34). At the posttranscriptiona...

A gene unique to Streptococcus pyogenes, called vfr, that negatively regulates speB, an important extracellular proteinase, has been identified. Disruption of vfr markedly increased SpeB production in a clinical strain of S. pyogenes and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive S. pyogenes infections.Group A streptococcus (GAS) causes many human diseases ranging in severity from milder infections such as pharyngitis to life-threatening necrotizing fasciitis/myonecrosis and streptococcal toxic shock syndrome (reviewed in reference 3). Among the myriad of virulence factors involved in pathogenesis is streptococcal pyrogenic exotoxin B (SpeB), an extracellular cysteine protease that facilitates bacterial survival and dissemination by degrading critical protein components of the host immune system and by modification of bacterial surface proteins (reviewed in reference 29).We have recently demonstrated that a protease maturation protein gene, prsA, is located immediately downstream of speB-spi (11), that speB-spi and prsA are cotranscribed, and that functional SpeB activity depends on PrsA production (20). In addition, we systematically elucidated the complex transcriptional unit of the polycistronic speB operon (20). Adding to previous reports of two speB transcripts (11,19,21), we demonstrated four clearly defined mRNA species hybridizing to the speB-specific probe. The unique characteristics of this operon include two speB promoters separated by 560 bp, two transcriptional terminators separated by the gene prsA, and an independent prsA promoter (20).Regulation of SpeB gene expression is complex and not fully understood. Neely and Caparon have shown that RopB, an Rgg family member, binds to the speB promoter and is required for speB expression (21). Other transcriptional regulators, including CsrR (also known as CovR) (9), LacD.1 (15), Pel/SagA (14), and Mga (18, 26), have also been implicated in speB regulation; however, direct DNA binding of these regulators to the speB promoter region has not been demonstrated. Finally, some investigators have speculated that additional unknown factors together with RopB control speB gene expression (17).Transposon mutatgenesis of wild-type GAS. To further investigate speB regulation, the Tn917 plasmid derivative, pTV1-OK (8), was transformed into a clinical isolate of M-type 3 GAS (strain 88-003) by electroporation. This strain was isolated from a fatal case of streptococcal toxic shock syndrome with necrotizing fasciitis/myonecrosis and was characterized in a previous report (31). Growth of the transformed bacteria, first at 30°C and then at 42°C (8), followed by selection of erythromycin-resistant colonies, yielded mutants with Tn917 transposed to the host chromosome, thereby creating a Tn917-mutagenized library of GAS 88-003. This library was screened for SpeB protease activity by a milk agar plate hydrolysis assay (20). SpeB activity in those with visibly altered protease activity was quantitated ...