Role of Ubiquitin Ligases and Deubiquitinases in Host Defense against Mycobacterium tuberculosis

Project summary One mechanism that is central to innate immunity against intracellular pathogens such as Mycobacterium tuberculosis (Mtb) is engagement and targeting of bacteria for degradation by the cellular ubiquitination machinery, a process that also directly impacts immune responses. Ubiquitin ligases expressed by host innate immune cells attach the protein ubiquitin to intracellular pathogens or the organelles containing them, and such ubiquitination events then trigger recruitment of the host autophagy-lysosomal degradation machinery to engulf and destroy the invading pathogen. Ubiquitin ligases such as SMURF1 and PARKIN tag Mtb-containing structures for autophagic degradation in a process termed xenophagy. Conversely, host deubiquitinating enzymes (DUBs) may remove these ubiquitin tags, potentially aiding the pathogen's survival. A major gap in our understanding of the cellular mechanisms of xenophagy is that we do not have a complete understanding of the mechanisms of ubiquitin attachment and removal from Mtb, and we hypothesize that the extent of Mtb ubiquitination, mediated by the relative balance between the activities of ubiquitin ligases and deubiquitinases, directly impacts the outcome of Mtb infection. Bridging the knowledge gap around the roles of individual ubiquitin ligases and DUBs may yield novel strategies for host-directed therapies, crucial in the current climate of growing antibiotic resistance. Here, we will apply genetic, biochemical, immunologic, transcriptomic and animal approaches to determine the functional role of specific ubiquitination and deubiquitination mechanisms in the context of Mtb infection. Thus, in the proposed research we will: (1) Elucidate the role of ubiquitin ligases SMURF1 and SMURF2 in targeting Mtb for autophagy and how they regulate interferon-β production during infection. (2) Delineate the roles of K63 and K48 deubiquitination during macrophage infection, focusing on the K63-specific DUB USP15 and the yet unidentified DUBs responsible for K48 deubiquitination, critical steps that negatively regulate autophagy. (3) Determine the function of linear M1-ubiquitination by the LUBAC system and its negative regulation by the DUB CYLD in cellular immunity to Mtb. The proposed work is expected to identify the molecular mechanisms that target Mtb for degradation and host proteins whose activities regulate protective immune responses.