Interactions of Streptococcus equi and Mast Cells: In the Search of Virulence Factors

Sammanfattning: Mast cells are key players of the innate immune system due to their location at the interface of host and pathogen encounters, such as on mucosal surfaces or the skin. Secreting a variety of compounds, they communicate with other immune cells in a highly specific manner. Subsequently, reinforcements against foreign invaders are recruited while also defending the host, using bacteriolytic effector molecules. One type of pathogens which are competent challengers of the host’s immune system are Streptococci, causing a burden for humans and animals. Streptococcus equi subspecies equi is one example, a highly contagious horse pathogen with a silent carrier subset, causing “strangles”, a disease resulting in equine morbidity and mortality all over the world. The present study aimed to explore the virulence factors in S. equi responsible for immune system activation, represented by mast cells. Knockout mutants of the genes aroB, hasA, pyrC, recA, sagA and a combination of those, including a deletion strain of all superantigens (seeHILM), were co-cultivated with murine bone-marrow-derived mast cells (BMMCs). Mast cells alone and S. equi strain 4047 (wild-type) were used as controls. It was shown that 4 h after encounter of the bacteria, BMMCs responded with IL-6, TNF-α and MCP-1 secretion, indicating an inflammatory response to all strains except against the sagA mutant (ΔsagA) or the multi-deletion strain, the latter lacking several virulence factors including sagA. These results were confirmed at the mRNA-level where IL-6, TNF-α and Nr4a3 gene expression was significantly upregulated in BMMCs after 4 h incubation with wild-type S. equi. In contrast, when BMMCs were co-cultivated with sagA-deficient S. equi, no detectable upregulation was seen. These results were further confirmed in peritoneal-derived mast cells. After 24 h no secretion of cytokines was detected in response to ΔsagA mutants, in contrast to the strong cytokine output in response to wild-type S. equi. To elucidate the role of SagA, the precursor of streptolysin S (SLS), lysis was determined, and it was observed that ΔsagA does not lyse mast cells in contrast to wild-type with intact SLS. Transwell-based experiments indicated a partially contact-dependent response of mast cells to bacteria. Taken together, this study shows for the first time that SLS is the major mast cell activator produced by S. equi. I suggest the possible mechanism of cell death by lysis and reprogrammed signaling pathways of the host by sublytic concentrations of SLS, resulting in damage associated pattern-mediated signaling as well as auto- and paracrine amplification by inflammatory cytokines and other messenger molecules.