1. Microbiology and Infectious Disease

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions

  1. Ekansh Mittal
  2. Andrew T Roth
  3. Anushree Seth
  4. Srikanth Singamaneni
  5. Wandy Beatty
  6. Jennifer A Philips  Is a corresponding author
  1. Washington University in St. Louis, United States
  2. Washington University Medical Center, United States
https://doi.org/10.7554/eLife.85416
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Cite this article
  1. Ekansh Mittal
  2. Andrew T Roth
  3. Anushree Seth
  4. Srikanth Singamaneni
  5. Wandy Beatty
  6. Jennifer A Philips
(2023)
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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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).

The following data sets were generated

Article and author information

Author details

  1. Ekansh Mittal

    Department of Medicine, Washington University in St. Louis, St Louis, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9034-033X

  2. Andrew T Roth

    Department of Medicine, Washington University in St. Louis, St Louis, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4239-7926

  3. Anushree Seth

    Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St Louis, United States
    Competing interests
    Anushree Seth, is currently employed with Auragent Bioscience LLC. The plasmonic-fluor technology used in the manuscript has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC..

  4. Srikanth Singamaneni

    Department of Mechanical Engineering and Materials Science, Washington University Medical Center, St Louis, United States
    Competing interests
    Srikanth Singamaneni, is an inventor on a provisional patent related to plasmonic-fluor technology, and the technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC. SS is a co-founder/shareholder of Auragent Bioscience LLC. SS along with Washington University may have financial gain through Auragent Bioscience LLC through this licensing agreement. These potential conflicts of interest have been disclosed and are being managed by Washington University in St. Louis..

  5. Wandy Beatty

    Department of Molecular Microbiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    No competing interests declared.

  6. Jennifer A Philips

    Department of Medicine, Washington University in St. Louis, St Louis, United States
    For correspondence
    philips.j.a@wustl.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9476-0240

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

  1. 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

  1. Preprint posted: June 16, 2022 (view preprint)
  2. Received: December 7, 2022
  3. Accepted: January 11, 2023
  4. Accepted Manuscript published: February 28, 2023 (version 1)
  5. 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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  1. Ekansh Mittal
  2. Andrew T Roth
  3. Anushree Seth
  4. Srikanth Singamaneni
  5. Wandy Beatty
  6. Jennifer A Philips
(2023)
Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
eLife 12:e85416.
https://doi.org/10.7554/eLife.85416

Share this article

https://doi.org/10.7554/eLife.85416

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