Deciphering the role of autophagy receptor phosphorylation and host kinases in the targeting of M. tuberculosis to autophagy

摘要 结核病是传染病死亡的主要原因，也是多药耐药的主要原因。 结核分枝杆菌感染的治疗具有挑战性。为了开发替代疗法，这项提议 寻求寻找新的免疫途径，负责控制巨噬细胞中结核病的存活。与生俱来的 可以抗细菌的免疫反应是自噬。在选择性自噬中，巨噬细胞感觉 进入细胞质并触发激活的激酶，从而使自噬磷酸化的病原体 适配器，从而针对溶酶体中的微生物进行破坏。坦克结合蛋白1(TBK1)和 PTEN诱导的PINK1是两种与自噬靶向有关的巨噬细胞激酶 细菌，但它们的磷酸化底物的特性和PINK1在结核病自噬中的潜在作用是 不清楚。为了确定激酶底物，我们将完成对 蛋白激酶缺陷小鼠骨髓巨噬细胞结核感染过程中的磷酸蛋白质组(目的) 1)。磷蛋白质组学和免疫荧光显微镜显示四种自噬受体共存 其中三个是磷酸化的。以确定这些以前未被认识到的角色 自噬适配器在自噬靶向结核病中，我们将获得靶向敲除小鼠或使用 CRISPR/Cas9创建具有自噬适配器突变的巨噬细胞并量化自噬靶向， 炎症反应和适配器缺陷细胞中的细菌生长(目标2)。通过理解 自噬靶向结核病的机制，我们可能能够设计出宿主导向的结核病治疗方法。 在正规课程和导师的指导下，布齐克博士的目标是了解激酶的机制-- 依赖于针对结核病的自噬，并发展技能和填补知识空白，以便成为 独立科学家。Budzik博士的多样化指导团队包括加州大学的主要导师Jeffery Cox博士 伯克利大学免疫学和病理学教授，国际公认的结核病宿主专家- 病原体相互作用，共同导师，加州大学旧金山分校医学教授，内科科学家 在结核病临床试验和与诊断学相关的转化研究方面的专业知识，以及委员会成员Dr。 内万·克罗根，加州大学旧金山分校细胞和分子药理学教授，这项提议的合作者之一 在将高通量网络生物学应用于微生物发病机制方面的专业知识方面，Dr。 Jayanta Debnath，加州大学旧金山分校教授兼病理学主席，他的研究涵盖了 自噬靶向，加州大学旧金山分校医学教授，真核基因组编辑专家David Erle博士， 和加州大学伯克利分校生物化学、生物物理学和结构生物学教授Daniel Portnoy博士 李斯特氏菌先天免疫力国际专家。Budzik博士的研究建议重点是激酶介导的 针对结核病进行自噬，再加上他的有组织的培训计划，将使他能够获得数据和 R01应用程序的必备技能，并作为独立调查员发展独特的科学利基。

ABSTRACT 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.