C3-Dependent Intracellular Killing in Innate Immunity and Bacterial Pathogenesis

SUMMARYBacterial infections remain a leading cause of morbidity and mortality worldwide and a critical public health issuedue to increasing antibiotic resistance and limited vaccines. Many of the most consequential bacterial infectionsoriginate at mucosal surfaces, such as the gut, respiratory tract or skin, then disseminate to other tissues via thebloodstream. Two preeminent human pathogens causing both mucosal and invasive diseases are Gram-negative Salmonella enterica (e.g., serovar Typhimurium, STm) and Gram-positive group A Streptococcus(GAS). Pivotal to innate host defense against bloodstream infection is the function of complement systemproteins and their activation cascades, especially opsonization by C3, coupled with the bactericidal activity ofphagocytic cells including macrophages (M?) and neutrophils. Pathogenic strains of STm and GAS can subvertphagolysosome function to survive intracellularly in M? ex vivo and in vivo, whereupon the autophagy systememerges as a critical battleground for pathogen survival/killing. This project brings together two highlyexperienced and productive physician-scientist investigators with complementary expertise: Gram-negativebacterial pathogenesis, mucosal immunity and gnotobiotic mouse models (MPI, M. Raffatellu) coupled to Gram-positive bacterial pathogenesis, innate immunity, and bacterial-phagocyte interactions (MPI, V. Nizet). Togetherwe have recently discovered a novel, essential intracellular function of C3: targeting of bacteria to the autophagysystem for killing in M? ? a discovery that may challenge conclusions of countless studies of M?-bacterialinteractions performed in the absence of active serum. Further, we have discovered that the STm serine proteasePgtE, the GAS serine protease SpyCEP, and the GAS cysteine protease SpeB allow the respective pathogensto inactivate C3 and to replicate intracellularly in M?. Our central hypothesis is that intracellular C3-dependentautophagy is critical to host innate defense, and that the ability of invasive pathogens such as STm and GAS tocounteract this process substantially increases their disease-causing potential. Recent data also indicate themicrobiome plays an essential role in skin and mucosal complement production, which may represent anothercrucial factor by which commensal microbes affect invasive bacterial infection risk. Here we propose to continueto investigate the role of intracellular C3 during infection, to understand the ramifications of this new principle ofinnate immunity on host-pathogen interactions and the outcome of two of the most important human infectiousdiseases. Our approaches are likely to reveal new virulence genes and host immune pathways that will connectmechanisms, resolve longstanding knowledge gaps, and lead to new avenues of investigation of broad relevanceto bacterial pathogenesis including potential novel therapeutic targets and leads.