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...

Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene.

Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, and amputation remains the single best treatment. Previous studies have demonstrated that tissue destruction follows C. perfringens phospholipase C (PLC)-induced, platelet gpIIbIIIa-mediated formation of occlusive intravascular platelet/ leukocyte aggregates. In this study, the intracellular signaling events leading to activation of gpIIbIIIa by PLC were investigated. PLC activated surface expressed gpIIbIIIa and mobilized gpIIbIIIa from internal stores. Chelation of intracellular calcium or inhibition of store-operated calcium entry each blocked PLC-induced activation of gpIIbIIIa, whereas inhibition of protein kinase C was without effect. Thus, PLC initiates an "inside-out" signaling cascade that begins with depletion of internal calcium stores, is sustained by an influx of calcium through store-sensitive channels, and culminates in the functional activation of gpIIbIIIa. These findings suggest that calcium-channel blockade and strategies targeting gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with gas gangrene.Clostridium perfringens type A is the most common organism isolated from patients with trauma-induced gas gangrene [1]. Initial growth of the organism occurs within the devitalized anaerobic milieu. However, the invasion of healthy, living tissue rapidly ensues, with margins of tissue destruction often advancing several

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.