Streptolysin S Promotes Programmed Cell Death and Enhances Inflammatory Signaling in Epithelial Keratinocytes during Group A Streptococcus Infection

Abstract

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...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

“…The results in Figures 2 and 3 indicate that both SLS-dependent inflammatory signaling responses do not require direct contact between the bacteria and host cells. As previous experiments have already demonstrated SLS-dependent p38 and NFκB activation in direct infection models 21 , similar levels of p38 or NFκB activation among the three GAS strains in the permeable membrane insert-based system would have indicated that the response required direct contact between the bacteria and host cells. Figure 4 demonstrates that this infection system may be applied to assess toxin-dependent changes in host cytotoxicity via ethidium homodimer and LDH release assays.…”

“…Though very consistent host responses have been observed when comparing the results of the permeable membrane insert-based infection studies with traditional direct infection studies, some notable differences in the kinetics of these host responses have been observed 21 . For example, changes in host signaling and membrane-based cytotoxicity take 30-50% longer to occur in the permeable membrane insert-based infection model than corresponding direct infection models.…”

“…This system can also be applied to visualize the effects of secreted bacterial factors on host protein localization by immunofluorescence microscopy (Figure 3). The data show SLS-dependent activation of the key inflammatory mediator Nuclear Factor kappa B (NFκB), which translocates from the cytoplasm to the nucleus upon activation 21 . The results in Figures 2 and 3 indicate that both SLS-dependent inflammatory signaling responses do not require direct contact between the bacteria and host cells.…”

“…Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest 21 . Note: The GASM1T1 5448 strains utilized for these experiments included WT GAS', a sagAΔcat SLS-deficient mutant (abbreviated in text as ΔsagA), and a sagA complemented strain (abbreviated in text as ΔsagA+sagA) 21 . 2.…”

“…This system has allowed for effective evaluation of host responses that are solely due to sustained secreted bacterial components while eliminating any responses that may occur through direct contact during Group A Streptococcal infection. Though other secreted bacterial factors besides SLS can also pass through the porous membrane, the use of an isogenic mutant panel including wild-type (WT), an SLS-knockout (ΔsagA) and a sagA complemented strain (ΔsagA+sagA) allows for precise evaluation of host responses that are strictly SLS-dependent 21 .…”