Furthermore, the cell death percentage of the sagB mutant-infected phagocytes is lower than that of the wild type GAS-infected ones 19. Our previous study indicates that mutation of sagB attenuates GAS-induced production of proinflammatory cytokines and mice death. SagB serves as a dehydrogenase which cooperates with cyclodehydratase to catalyze heterocycle formation and maturation of streptolysin S with cytolytic activity 18. SLS is encoded by the streptococcal sagA gene and posttranslationally modified by heterocycle-forming synthetase proteins SagB, SagC and SagD to produce a mature pore-forming streptolysin S 5, 17. GSK-3β plays an essential role in regulating inflammatory responses, in which it may either enhance or inhibit the inflammatory response, depending on different pathogens, virulence factors, and the type of the infected cells 16. The activity of GSK-3β is also associated with bacterial internalization and pathogenesis of infection 13, 14, 15.
GSK-3β mediates the opening of mitochondrial permeability transition pores (mPTP) and induces the change of mitochondrial membrane potential (ΔΨm) 11, 12. Glycogen synthesis kinase-3β (GSK-3β), an important regulator of multiple cellular functions, controls metabolism, survival signaling and death signaling of cells 10. However, the detailed mechanisms involved in SLS-induced cell death are not well-understood. The cytotoxic effects of SLS have been reported in different types of cells, in which it mediated apoptosis, oncosis, and activation of the proinflammatory signaling pathways during the invasive GAS infection 5, 6, 7, 8, 9. Anti-SLS antibody induced by immunizing with the SLS peptide, coupled to keyhole limpet hemocyanin or with the SLS epitope-contained recombinant protein, can reduce the SLS-mediated hemolysis and mortality of GAS-infected mice 3, 4. SLS is the other potent cytolysin with a wide range of targets, but the immunogenicity of SLS is lower compared with SLO. The anti-SLO titer is used as an indicator of streptococcal infection because SLO is a potent immunogen to humans 2. SLO is a well-documented cytolysin with cell-lytic and tissue-destructive activity, and is involved in severe tissue damage induced by GAS infection. Two leukocidal toxins are produced by GAS, one is oxygen-labile streptolysin O (SLO) and the other is oxygen-stable streptolysin S (SLS). GAS produces several virulence factors, such as M protein, fibronectin-binding proteins, streptococcal pyrogenic exotoxin B (SPE B), Ig-degrading enzyme, and leukocidal toxins which hinder the host immune system-mediated opsonophagocytosis 1. Group A Streptococcus (GAS Streptococcus pyogenes) infection leads to a wide spectrum of human diseases from noninvasive impetigo to invasive necrotizing fasciitis. These results suggest that a new mechanism of SLS-induced macrophage death was through inhibiting GSK-3β degradation and further enhancing mitochondrial damage. Furthermore, the sagB mutant, but not the wild type or the sagB-complementary mutant, could induce GSK-3β degradation via a proteasome-dependent pathway. Compared to the wild type GAS with its isogenic sagB mutant (SLS mutant)-infected macrophages, we found sagB mutant infection caused less mitochondrial ROS generation and cell death than those of the wild type GAS-infected ones. Streptolysin S (SLS), a GAS toxin, played the important role on GAS-induced macrophage death. Inhibition of GSK-3β activity by lithium chloride or decreasing GSK-3β expression by lentivirus-mediated short hairpin RNA for GSK-3β could not only decrease the wild type GAS-induced mitochondrial ROS generation, mitochondria damage and cell death, but also reduced GAS intracellular replication. The wild type GAS infection could activate glycogen synthase kinase-3β (GSK-3β). Here, analyzing the timing of reactive oxygen species (ROS) production and using mitochondrial ROS scavenger, we found the wild type GAS-induced RAW264.7 cell death was associated with mitochondrial ROS. Previous studies indicate GAS infection leads to RAW264.7 cell death, but the mechanism is unclear. Group A Streptococcus (GAS) infection is associated with a variety of human diseases.