Using proteomics to identify host cell death factors during M. tuberculosis infection

Infections from M. tuberculosis (Mtb) are a persistent global health threat. To develop therapies that augment protective host immune responses against Mtb and shorten TB treatment, this proposal seeks to test our central hypothesis that Mtb infection triggers autophagy factor phosphorylation that regulates host cell death mechanisms. Understanding these regulatory mechanisms will enable the development of new host cell death targets for Mtb treatment. In an unbiased phosphoproteomic analysis, we discovered Tax1bp1 (Tax1-binding protein 1) and Optineurin were significantly more phosphorylated in Mtb- versus mock-infected bone marrow- derived macrophages (BMDMs). Cytosolic autophagy receptors target pathogens for elimination by selective autophagy, which inhibits necrosis. As phosphorylation leads to signal amplification and enables protein function, we sought to test the role of Tax1bp1 and Optineurin during Mtb infection. As expected, we found Optineurin restricts Mtb growth. However, surprisingly we found that Tax1bp1 enhances Mtb growth in most cell types and during animal infection. Additionally, we found that the two autophagy receptors have opposite impacts on host cell death. Tax1bp1 enhances early host cell necrosis and delays apoptosis whereas Optineurin limits necrosis. To further determine how Tax1bp1 promotes necrosis during Mtb infection, here we will investigate whether Tax1bp1 activates inflammasomes to cause pyroptosis, a programmed form of necrosis. We will also test whether inhibition of the kinase that phosphorylates Tax1bp1 blocks Mtb growth and necrosis. To elucidate whether Tax1bp1’s function in innate immune cells promotes Mtb growth in vivo, we will take advantage of a new floxed Tax1bp1 mouse. To further test the function of Optineurin’s previously unrecognized phosphosites in controlling host cell death, we will express Optineurin phosphomutant alleles in macrophages for host cell death assays during Mtb infection. To identify additional phosphorylated host cell death factors in other macrophage types relevant to Mtb infection, we performed phosphoproteomic profiling of Mtb-infected primary human and murine alveolar macrophages (AMs). We identified five host cell death factors phosphorylated in both BMDMs and AMs that are not already known to respond to Mtb infection involved in the mTOR (mammalian target of rapamycin) pathway, which regulates autophagy and apoptosis. To test the possible roles of these five host factors in cell death during Mtb infection, we will engineer knockout macrophages for cell death assays. A better understanding of the mechanisms by which Tax1bp1, Optineurin, and previously unrecognized host cell death factors impact Mtb pathogenesis may lead to the development of host-directed therapies for TB infection that shorten the treatment duration. Project Number: 1R01AI194696-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jonathan Budzik | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $819,998 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 IIDB-P (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19469601