Saturday, November 23, 2024
11/23/2024
PROJECT SUMMARY The resurgence of severe invasive Group A Streptococcus (GAS) infections in the past decade is a major public health concern in the United States, and gaps in understanding the resilience of GAS to the immune response has impeded the development of a much-needed vaccine. The long-term goal is to understand GAS survival and persistence in macrophages to provide a cell- targeted basis for improved treatment for infection. Toward the long-term goal, the objective in this application is to examine macrophage second-line responses to GAS following the failure of lysosomes to eliminate bacteria. The central hypothesis is that GAS-induced lysosomal permeabilization enables bacterial and lysosomal proteins to enter the cytosol, which continues to alter macrophage cellular processes, including limiting the oxidative burst response and inducing histone release and non-lytic exocytosis of GAS. Guided by preliminary data produced by the applicant’s laboratory, this hypothesis will be explored in three specific aims. The work in Aim 1 will determine whether GAS NADase in the cytosol impairs the oxidative burst response. Measurements of reactive oxygen species, cellular energy pools, and NADPH oxidase complex assembly will determine whether this bacterial enzyme negatively impacts the macrophage oxidative burst response. Aim 2 will explore whether GAS-induced damage of the nuclear envelope causes DNA-free histone release and the extent to which these histones have antimicrobial activity against GAS in the macrophage cytosol. Preliminary data demonstrate that macrophages expel GAS in exosomes and the work in Aim 3 will characterize these GAS- containing exosomes. Time-lapse microscopic observations will be complemented by electron microscopy and proteomics analyses of isolated exosomes. The outcome of this work will inform approaches aimed at restoring or enhancing host cell function, which both avoids the problems of broad-spectrum antibiotic treatment and enhances the natural ability of macrophages to eradicate GAS. The proposed research is innovative because understanding these second-line responses, which are intermediate processes between lysosomal failure and disease pathology, will provide new druggable target proteins and pathways. The contribution will be a significant first step toward developing pharmacological strategies that prevent, rather than treat severe invasive disease and refractory infections.
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