Project Summary/Abstract Mycobacterium tuberculosis’s (Mtb) ability to survive and replicate in mononuclear phagocytes (MPs) is central to its pathogenicity and capability to infect a quarter of the world’s population. The laboratory of Dr. Joel Ernst (co-mentor) and others have demonstrated differential Mtb growth and replication between populations of MPs. Yet, the complexity and diversity of MP subsets is only recently being defined at transcriptional resolution and not well described during Mtb infection. It is therefore unclear which subsets are most permissive to Mtb growth and if Mtb directs monocyte differentiation. Using flow cytometry and single cell RNA-sequencing (scRNA-seq) from infected mice, preliminary data demonstrates differential infection rates and responses to Mtb within two recruited macrophage subpopulations. This has directed the central hypothesis that Mtb virulence factors drive differentiation states of recruited MPs in the lung during infection, providing a fertile niche for Mtb growth and replication. The long-term goal of this work is to delineate the relationship between Mtb and MPs in the context of a recent revolution in understanding lung immune cell heterogeneity, enabling more effective host-directed therapies. Utilizing fluorescently expressing Mtb, flow cytometry, and single cell-RNA sequencing in mice infected with a laboratory strain of Mtb (H37Rv), and a strain lacking a functional ESX-1 secretion system, specific Aim 1 will test the hypothesis that an Mtb virulence system drives differentiation of monocytes to specific macrophage subsets. With use of flow cytometry, immunohistochemistry, and ex vivo infection models, specific Aim 2 will test the hypothesis that recruited macrophage subsets have differential ability to restrict Mtb replication. The training plan is focused to develop expertise in immunologic responses to mycobacterium, mononuclear phagocyte and lung milieu biology, genetic manipulation of mycobacterium, scRNA-seq and bioinformatics, leadership, and responsible research practices. Training will include didactic courses, workshops, seminars, local and international conferences. The primary mentor and co-mentors are Dr. Oren Rosenberg and Dr. Joel Ernst, both successful physician-scientists with combined expertise in immunology, Mtb biology, and host- pathogen interactions. An overarching team of mentors also include a scientific advisor in bioinformatics, collaborator in next generation sequencing, and accomplished physician-scientist career mentors. Dr. Zha has laboratory and office space at UCSF Mission Bay, with shared space in a BSL2 and BSL3 laboratories, with access to equipment and mouse models necessary for this research. This Career Development Award application has an innovative research proposal, rigorous training plan, mentorship of leaders in mycobacterial immunology and biology, team of physician-scientist advisors with a track record of transitioning scientists to independen...