Tuberculosis is a major cause of mortality from infectious disease, and multi-drug resistant (MDR) Mycobacterium tuberculosis infections are challenging to treat. To develop alternative therapies, this proposal seeks to find new immune pathways responsible for controlling TB survival in macrophages. One innate immune response that can be anti-bacterial is autophagy. In selective autophagy, macrophages sense pathogens that access the cytosol and trigger the activation of kinases, which phosphorylate autophagy adaptors and thereby target the microbe for destruction in the lysosome. TANK-binding kinase 1 (TBK1) and PTEN-induced putative kinase 1 (PINK1) are two macrophage kinases implicated in autophagy targeting of bacteria, but the identity of their phosphorylated substrates and potential role of PINK1 in autophagy of TB are unclear. To determine the kinase substrates, we will complete our analysis of changes in the phosphoproteome during TB infection in bone marrow-derived macrophages from kinase-deficient mice (Aim 1). Phosphoproteomics and immunofluorescence microscopy revealed four autophagy receptors colocalize with TB and three of them are phosphorylated. To determine the role of these formerly unrecognized autophagy adaptors in autophagosomal targeting of TB, we will obtain targeted knockout mice or use CRISPR/Cas9 to create macrophages with autophagy adaptor mutations and quantify autophagy targeting, inflammatory responses, and bacterial growth in the adaptor deficient cells (Aim 2). By understanding the mechanism of autophagy targeting of TB, we may be able to design host-directed therapies for TB. Guided by formal coursework and mentorship, Dr. Budzik?s goals are to understand the mechanism of kinase- dependent targeting of TB to autophagy, and develop skills and fill knowledge gaps in order to become an independent scientist. Dr. Budzik?s diverse mentoring team includes primary mentor Dr. Jeffery Cox, UC Berkeley Professor of Immunology and Pathogenesis, an internationally recognized expert on TB host- pathogen interactions, co-mentor Dr. Payam Nahid, UCSF Professor of Medicine, a physician scientist with expertise on TB clinical trials and translational research relating to diagnostics, and committee members Dr. Nevan Krogan, UCSF Professor of Cellular and Molecular Pharmacology, a collaborator on this proposal with expertise in applying high throughput network biology to mechanistic insights on microbial pathogenesis, Dr. Jayanta Debnath, UCSF Professor and Chair of Pathology, whose research encompasses mechanisms of autophagy targeting, Dr. David Erle, UCSF Professor of Medicine, an expert on eukaryotic genome editing, and Dr. Daniel Portnoy, UC Berkeley Professor of Biochemistry, Biophysics, and Structural Biology, an international expert on innate immunity to Listeria. Dr. Budzik?s research proposal focusing on kinase-mediated targeting of TB to autophagy together with his organized training plan will allow him to obtain data and prerequisite skills for a R01 application and develop a unique scientific niche as an independent investigator.
Tuberculosis is a leading cause of death from infectious disease and new treatments are needed to combat multi-drug resistant isolates. In this proposal, we aim to discover the mechanism of kinase-dependent targeting of M. tuberculosis to autophagy, a process that restricts M. tuberculosis growth in host cells. A better understanding of the host pathogen interaction will allow for the design of novel host-directed therapies for tuberculosis.
