PROJECT SUMMARY Tuberculosis is a global disease affecting millions of people in primarily developing regions of the world. Central nervous system tuberculosis (CNS-TB) is the most severe extra pulmonary form of the disease, which, despite aggressive therapeutic intervention, can have a fatality rate of up to 80%. Significant barriers to understanding disease pathogenesis exist, which translates to poor diagnosis and treatment outcomes. Thus, improved insight into neuro-glial-immune cell interactions in CNS TB, and the factors that regulate brain pathology require a global molecular and cellular perspective. Our long-term goal is to understand the mechanisms associated with the development of CNS-TB, and different CNS-TB outcomes, in order to develop novel, effective, and broadly accessible therapeutics. Single cell/nucleus RNA sequencing (sc/snRNA-seq) gene expression analysis allows for a large-scale view of the cells and molecular pathways involved in neuropathogenesis, with the potential to identify novel differences between TB diseased states. In this study, we will investigate transcriptional changes induced by M. tuberculosis infection of the brain under clinical and experimental conditions to determine how CNS-TB starts and progresses to different outcomes. This is made possible by preliminary snRNA-seq data generated during our Global Brain R21 that we recently completed. We hypothesize that M. tuberculosis invades the CNS due to the failed generation of an appropriate neuroimmune response in CNS resident and peripherally recruited immune cells. This is strongly supported by our preliminary data from human and rodent snRNA-seq studies. By exploring the transcriptional differences in cells from normal and diseased states we will be able to identify the cell type-specific molecular and cellular processes that underpin the pathogenesis of CNS-TB, including neuroinflammation and immune system engagement. Understanding cell type-specific CNS and immune signaling in CNS-TB will enable us to develop novel therapies and improve clinical outcomes. To test our central hypothesis, we will determine the molecular pathways across CNS and immune cells that define clinical phenotypes in patients with CNS-TB and create a full disease spectrum signaling framework of CNS-TB using multiple mouse models. In addition to our scientific aims, we will expand the single cell gene expression analysis platform, established at the University of Cape Town (UCT) during our Global Brain R21 support, to the broader African scientific community. We aim to provide training in project planning, library preparation, and data analysis. When complete, we will have a novel understanding of the cell type-specific inflammatory signaling events that drive CNS-TB and will be poised to engage novel molecular targets to improve CNS-TB outcomes. Furthermore, UCT will be established as a hub of single cell biology, to launch scientific careers of the next generation of African scient...