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

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 exa...

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Autores principales: Mittal, Ekansh, Roth, Andrew T, Seth, Anushree, Singamaneni, Srikanth, Beatty, Wandy, Philips, Jennifer A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9998084/
https://www.ncbi.nlm.nih.gov/pubmed/36852737
http://dx.doi.org/10.7554/eLife.85416
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author Mittal, Ekansh
Roth, Andrew T
Seth, Anushree
Singamaneni, Srikanth
Beatty, Wandy
Philips, Jennifer A
author_facet Mittal, Ekansh
Roth, Andrew T
Seth, Anushree
Singamaneni, Srikanth
Beatty, Wandy
Philips, Jennifer A
author_sort Mittal, Ekansh
collection PubMed
description 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 envelope 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.
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spelling pubmed-99980842023-03-10 Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions Mittal, Ekansh Roth, Andrew T Seth, Anushree Singamaneni, Srikanth Beatty, Wandy Philips, Jennifer A eLife Microbiology and Infectious Disease 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 envelope 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. eLife Sciences Publications, Ltd 2023-02-28 /pmc/articles/PMC9998084/ /pubmed/36852737 http://dx.doi.org/10.7554/eLife.85416 Text en © 2023, Mittal, Roth et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Microbiology and Infectious Disease
Mittal, Ekansh
Roth, Andrew T
Seth, Anushree
Singamaneni, Srikanth
Beatty, Wandy
Philips, Jennifer A
Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title_full Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title_fullStr Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title_full_unstemmed Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title_short Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
title_sort single cell preparations of mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions
topic Microbiology and Infectious Disease
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9998084/
https://www.ncbi.nlm.nih.gov/pubmed/36852737
http://dx.doi.org/10.7554/eLife.85416
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