Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol 2256825

Abstract

Abstract Introduction Although macrophages are important for controlling Group A Streptococcus (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 identified lysosomal and bacterial proteins released into the cytosol as a consequence of phagosomal perforation. Methods We prepared cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient (α”SLO) bacteria and uninfected controls and verified lysosomal or bacterial proteins were present using IL-1β as a measure of intracellular pathogen detection. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and α”SLO-infected macrophages. Results Bacterial M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with an 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 α”SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. We confirmed the presence of histones and the absence of contaminating nuclei in the cytosolic fraction by Western blot. DNA was not detected in the cytosol of GAS-infected cells, but histones were secreted into the extracellular medium. We are currently exploring the mechanism and purpose of this histone release. Conclusion Our data both confirms host cell detection of bacterial proteins after phagosomal perforation, as well as reveals the surprising profile of proteins altered during GAS infection. Funding Source NIH R15AI176429, AHA 17GRNT33410851, Occidental College Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)