Dynamic post-translational modification profiling of M. tuberculosis-infected primary macrophages
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.
Reviewing Editor
- Bavesh D Kana, University of the Witwatersrand, South Africa
Version history
- Received: August 29, 2019
- Accepted: January 16, 2020
- Accepted Manuscript published: January 17, 2020 (version 1)
- Version of Record published: February 19, 2020 (version 2)
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.
Metrics
-
- 5,170
- Page views
-
- 770
- Downloads
-
- 27
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Computational and Systems Biology
- Microbiology and Infectious Disease
Candida albicans, an opportunistic human pathogen, poses a significant threat to human health and is associated with significant socio-economic burden. Current antifungal treatments fail, at least in part, because C. albicans can initiate a strong drug tolerance response that allows some cells to grow at drug concentrations above their minimal inhibitory concentration. To better characterize this cytoprotective tolerance program at the molecular single-cell level, we used a nanoliter droplet-based transcriptomics platform to profile thousands of individual fungal cells and establish their subpopulation characteristics in the absence and presence of antifungal drugs. Profiles of untreated cells exhibit heterogeneous expression that correlates with cell cycle stage with distinct metabolic and stress responses. At 2 days post-fluconazole exposure (a time when tolerance is measurable), surviving cells bifurcate into two major subpopulations: one characterized by the upregulation of genes encoding ribosomal proteins, rRNA processing machinery, and mitochondrial cellular respiration capacity, termed the Ribo-dominant (Rd) state; and the other enriched for genes encoding stress responses and related processes, termed the Stress-dominant (Sd) state. This bifurcation persists at 3 and 6 days post-treatment. We provide evidence that the ribosome assembly stress response (RASTR) is activated in these subpopulations and may facilitate cell survival.
-
- Immunology and Inflammation
- Microbiology and Infectious Disease
Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. When a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we engineered S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. To clear an infection, cells may have specific tasks that they must complete before they die; different modes of cell death could initiate these ‘bucket lists’ in either convergent or divergent ways.