Streptococcus pyogenes, or group A Streptococcus (GAS), is a pathogen that causes a multitude of human diseases from pharyngitis to severe infections such as toxic shock syndrome and necrotizing fasciitis. One of the primary virulence factors produced by GAS is the peptide toxin streptolysin S (SLS). In addition to its well-recognized role as a cytolysin, recent evidence has indicated that SLS may influence host cell signaling pathways at sublytic concentrations during infection. We employed an antibody arraybased approach to comprehensively identify global host cell changes in human epithelial keratinocytes in response to the SLS toxin. We identified key SLS-dependent host responses, including the initiation of specific programmed cell death and inflammatory cascades with concomitant downregulation of Akt-mediated cytoprotection. Significant signaling responses identified by our array analysis were confirmed using biochemical and protein identification methods. To further demonstrate that the observed SLS-dependent host signaling changes were mediated primarily by the secreted toxin, we designed a Transwell infection system in which direct bacterial attachment to host cells was prevented, while secreted factors were allowed access to host cells. The results using this approach were consistent with our direct infection studies and reveal that SLS is a bacterial toxin that does not require bacterial attachment to host cells for activity. In light of these findings, we propose that the production of SLS by GAS during skin infection promotes invasive outcomes by triggering programmed cell death and inflammatory cascades in host cells to breach the keratinocyte barrier for dissemination into deeper tissues. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a common colonizer of the skin and mucosal surfaces of humans (1-3). GAS is typically innocuous in these locations or else leads to fairly minor and generally self-limiting infections of the skin or respiratory tract, such as impetigo or pharyngitis (1-3). In cases where an initial infection is left untreated, GAS may cause one of several severe postinfection (p.i.) sequelae, including rheumatic fever or glomerulonephritis (1-3). Furthermore, in rare cases, this exclusively human pathogen breaches the epithelial barrier and invades deeper tissues and blood, resulting in outcomes such as necrotizing fasciitis and Streptococcus toxic shock (1-3). The World Health Organization (WHO) estimates that GAS is responsible for about 18 million cases of severe postinfection sequelae and 700,000 cases of invasive disease each year (2, 4). Combined, GAS infections lead to approximately 500,000 deaths annually (2, 4).The success of GAS in causing both mild and severe infections is due largely to the myriad of secreted and surface-bound virulence factors expressed by this pathogen. One of the most potent virulence factors produced by GAS is streptolysin S (SLS), a small, ribosomally produced peptide whose mature product is predicted to be 2.7 kDa in size (5-8...