Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol

The human-specific bacterial pathogen Group A Streptococcus (GAS) is a significant cause of morbidity and mortality due to its ability to cause severe invasive infection. Although macrophages are important for controlling GAS infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we examined how phagosomal perforation releases lysosomal and bacterial proteins into the cytosol and alters cytosolic protein content in the macrophage. Using IL-1β as a measure of intracellular pathogen detection, we confirmed that cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient (ΔSLO) bacteria contained new proteins that are absent in uninfected cytosol controls. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and ΔSLO-infected macrophages. M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with the IL-1β response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and ΔSLO-infected macrophages contained histone proteins H1-H4, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. Our work reveals both a mechanism for the activation of the inflammatory response on a cellular level, and the surprising consequences of phagosomal perforation in GAS infections. These responses may collectively contribute to the pathologies observed during severe invasive GAS infection, and can help inform therapies aimed at improving macrophage function and patient outcomes.