Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
- Washington University in St. Louis, United States
- Washington University Medical Center, United States
Abstract
For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelop integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1β and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.
Data availability
RNA-seq data can be accessed in the Gene Expression Omnibus database (Accession GSE206485; ID: 200206485).
Article and author information
Author details
Jennifer A Philips
Funding
NIAID/NIH (R01 AI087682)
- Jennifer A Philips
NIAID/NIH (R01 AI30454)
- Jennifer A Philips
National Cancer Institute -Innovative Molecular Analysis Technologies (R21CA236652)
- Srikanth Singamaneni
National Science Foundation (CBET-1900277)
- Srikanth Singamaneni
NIH/NHLBI (T32 HL007317-37)
- Andrew T Roth
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Bavesh D Kana, University of the Witwatersrand, South Africa
Ethics
Animal experimentation: All work with mice were approved by the Washington University School of Medicine Institutional Animal Care and Use Committee (IACUC protocol # 21-0245). Euthanasia was performed prior to bone marrow harvest in accordance with the 2020 AVMA Guidelines for the Euthanasia of Animals prior to tissue harvest.
Version history
- Preprint posted: June 16, 2022 (view preprint)
- Received: December 7, 2022
- Accepted: January 11, 2023
- Accepted Manuscript published: February 28, 2023 (version 1)
- Version of Record published: March 9, 2023 (version 2)
Copyright
© 2023, Mittal 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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Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
eLife 12:e85416.
https://doi.org/10.7554/eLife.85416
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