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
Share this article
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
  2. Download BibTeX
  3. Download .RIS

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.

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.

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.

Metrics

  • 3,048
    views
  • 413
    downloads
  • 13
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

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)

  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

Categories and tags

Research organism

Further reading

    1. Microbiology and Infectious Disease

    Telomerase RNA component knockout exacerbates Staphylococcus aureus pneumonia by extensive inflammation and dysfunction of T cells

    Yasmina Reisser, Franziska Hornung ... Stefanie Deinhardt-Emmer
    Research Article

    The telomerase RNA component (Terc) constitutes a non-coding RNA critical for telomerase function, commonly associated with aging and pivotal in immunomodulation during inflammation. Our study unveils heightened susceptibility to pneumonia caused by Staphylococcus aureus (S. aureus) in Terc knockout (Tercko/ko) mice compared to both young and old infected counterparts. The exacerbated infection in Tercko/ko mice correlates with heightened inflammation, manifested by elevated interleukin-1β (IL-1β) levels and activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome within the lung. Employing mRNA sequencing methods alongside in vitro analysis of alveolar macrophages (AMs) and T cells, our study elucidates a compelling correlation between Tercko/ko, inflammation, and impaired T cell functionality. Terc deletion results in compromised T cell function, characterized by dysregulation of the T cell receptor and absence of CD247, potentially compromising the host’s capacity to mount an effective immune response against S. aureus. This investigation provides insights into the intricate mechanisms governing increased vulnerability to severe pneumonia in the context of Terc deficiency, which might also contribute to aging-related pathologies, while also highlighting the influence of Terc on T cell function.

    1. Microbiology and Infectious Disease

    Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

    Li Zhang, Fen Hu ... Hang Yang
    Research Article

    Phage-derived peptidoglycan hydrolases (i.e. lysins) are considered promising alternatives to conventional antibiotics due to their direct peptidoglycan degradation activity and low risk of resistance development. The discovery of these enzymes is often hampered by the limited availability of phage genomes. Herein, we report a new strategy to mine active peptidoglycan hydrolases from bacterial proteomes by lysin-derived antimicrobial peptide-primed screening. As a proof-of-concept, five peptidoglycan hydrolases from the Acinetobacter baumannii proteome (PHAb7-PHAb11) were identified using PlyF307 lysin-derived peptide as a template. Among them, PHAb10 and PHAb11 showed potent bactericidal activity against multiple pathogens even after treatment at 100°C for 1 hr, while the other three were thermosensitive. We solved the crystal structures of PHAb8, PHAb10, and PHAb11 and unveiled that hyper-thermostable PHAb10 underwent a unique folding-refolding thermodynamic scheme mediated by a dimer-monomer transition, while thermosensitive PHAb8 formed a monomer. Two mouse models of bacterial infection further demonstrated the safety and efficacy of PHAb10. In conclusion, our antimicrobial peptide-primed strategy provides new clues for the discovery of promising antimicrobial drugs.

    1. Ecology
    2. Microbiology and Infectious Disease

    Variation in thermal physiology can drive the temperature-dependence of microbial community richness

    Tom Clegg, Samraat Pawar
    Research Article Updated

    Predicting how species diversity changes along environmental gradients is an enduring problem in ecology. In microbes, current theories tend to invoke energy availability and enzyme kinetics as the main drivers of temperature-richness relationships. Here, we derive a general empirically-grounded theory that can explain this phenomenon by linking microbial species richness in competitive communities to variation in the temperature-dependence of their interaction and growth rates. Specifically, the shape of the microbial community temperature-richness relationship depends on how rapidly the strength of effective competition between species pairs changes with temperature relative to the variance of their growth rates. Furthermore, it predicts that a thermal specialist-generalist tradeoff in growth rates alters coexistence by shifting this balance, causing richness to peak at relatively higher temperatures. Finally, we show that the observed patterns of variation in thermal performance curves of metabolic traits across extant bacterial taxa is indeed sufficient to generate the variety of community-level temperature-richness responses observed in the real world. Our results provide a new and general mechanism that can help explain temperature-diversity gradients in microbial communities, and provide a quantitative framework for interlinking variation in the thermal physiology of microbial species to their community-level diversity.