Dynamic post-translational modification profiling of M. tuberculosis-infected primary macrophages
- University of California, San Francisco, United States
- University of California, Berkeley, United States
- University of California, San Diego, United States
Abstract
Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015361.
Article and author information
Author details
Funding
National Institute of Allergy and Infectious Diseases (P01 AI063302)
- Jeffery S Cox
Cystic Fibrosis Foundation (Harry Shwachman Award)
- Jonathan M Budzik
National Institute of Allergy and Infectious Diseases (P01 AI063302)
- Nevan J Krogan
National Institute of General Medical Sciences (P50 GM082250)
- Nevan J Krogan
National Institute of Allergy and Infectious Diseases (U19 AI106754)
- Nevan J Krogan
National Institute of Allergy and Infectious Diseases (U19 AI106754)
- Jeffery S Cox
National Institute of Allergy and Infectious Diseases (DP1 AI124619)
- Jeffery S Cox
National Institute of Allergy and Infectious Diseases (R01 AI120694)
- Jeffery S Cox
National Institute of Allergy and Infectious Diseases (R01 AI120694)
- Nevan J Krogan
National Institute of Allergy and Infectious Diseases (1K08AI146267)
- Jonathan M Budzik
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: An animal use protocol (AUP-2015-11-8096) for mouse use was approved by the Office of Laboratory and Animal Care at the University of California, Berkeley, in adherence with guidelines from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Copyright
© 2020, Budzik et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Dynamic post-translational modification profiling of M. tuberculosis-infected primary macrophages
eLife 9:e51461.
https://doi.org/10.7554/eLife.51461
Further reading
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
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- Immunology and Inflammation
- Microbiology and Infectious Disease
The members of the Mycobacterium tuberculosis complex (MTBC) causing human tuberculosis comprise 10 phylogenetic lineages that differ in their geographical distribution. The human consequences of this phylogenetic diversity remain poorly understood. Here, we assessed the phenotypic properties at the host-pathogen interface of 14 clinical strains representing five major MTBC lineages. Using a human in vitro granuloma model combined with bacterial load assessment, microscopy, flow cytometry, and multiplexed-bead arrays, we observed considerable intra-lineage diversity. Yet, modern lineages were overall associated with increased growth rate and more pronounced granulomatous responses. MTBC lineages exhibited distinct propensities to accumulate triglyceride lipid droplets—a phenotype associated with dormancy—that was particularly pronounced in lineage 2 and reduced in lineage 3 strains. The most favorable granuloma responses were associated with strong CD4 and CD8 T cell activation as well as inflammatory responses mediated by CXCL9, granzyme B, and TNF. Both of which showed consistent negative correlation with bacterial proliferation across genetically distant MTBC strains of different lineages. Taken together, our data indicate that different virulence strategies and protective immune traits associate with MTBC genetic diversity at lineage and strain level.
