Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum

ABSTRACT Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for lo...

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Autores principales:	Paras Jain, Brian C. Weinrick, Eric J. Kalivoda, Hui Yang, Vanisha Munsamy, Catherine Vilcheze, Torin R. Weisbrod, Michelle H. Larsen, Max R. O’Donnell, Alexander Pym, William R. Jacobs
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/32a454e686594896ba898650dbcbbb01
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spelling	oai:doaj.org-article:32a454e686594896ba898650dbcbbb012021-11-15T15:50:15ZDual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum10.1128/mBio.01023-162150-7511https://doaj.org/article/32a454e686594896ba898650dbcbbb012016-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01023-16https://doaj.org/toc/2150-7511ABSTRACT Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ2DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ2GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ2DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ2DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ2DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells. IMPORTANCE Tuberculosis (TB) is again the leading cause of death from a single infectious disease, having surpassed HIV. The recalcitrance of the TB pandemic is largely due to the ability of the pathogen Mycobacterium tuberculosis to enter a persistent state in which it is less susceptible to antibiotics and immune effectors, necessitating lengthy treatment. It has been difficult to study persister cells, as we have lacked tools to isolate these rare cells. In this article, we describe the development of dual-reporter mycobacteriophages that encode a green fluorescent marker of viability and in which the promoters of genes we have identified as induced in the persister state are fused to a gene encoding a red fluorescent protein. We show that these tools can identify heterogeneity in a cell population that correlates with propensity to survive antibiotic treatment and that the proportions of these subpopulations change in M. tuberculosis cells within human sputum during the course of treatment.Paras JainBrian C. WeinrickEric J. KalivodaHui YangVanisha MunsamyCatherine VilchezeTorin R. WeisbrodMichelle H. LarsenMax R. O’DonnellAlexander PymWilliam R. JacobsAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 5 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Paras Jain Brian C. Weinrick Eric J. Kalivoda Hui Yang Vanisha Munsamy Catherine Vilcheze Torin R. Weisbrod Michelle H. Larsen Max R. O’Donnell Alexander Pym William R. Jacobs Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
description	ABSTRACT Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ2DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ2GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ2DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ2DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ2DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells. IMPORTANCE Tuberculosis (TB) is again the leading cause of death from a single infectious disease, having surpassed HIV. The recalcitrance of the TB pandemic is largely due to the ability of the pathogen Mycobacterium tuberculosis to enter a persistent state in which it is less susceptible to antibiotics and immune effectors, necessitating lengthy treatment. It has been difficult to study persister cells, as we have lacked tools to isolate these rare cells. In this article, we describe the development of dual-reporter mycobacteriophages that encode a green fluorescent marker of viability and in which the promoters of genes we have identified as induced in the persister state are fused to a gene encoding a red fluorescent protein. We show that these tools can identify heterogeneity in a cell population that correlates with propensity to survive antibiotic treatment and that the proportions of these subpopulations change in M. tuberculosis cells within human sputum during the course of treatment.
format	article
author	Paras Jain Brian C. Weinrick Eric J. Kalivoda Hui Yang Vanisha Munsamy Catherine Vilcheze Torin R. Weisbrod Michelle H. Larsen Max R. O’Donnell Alexander Pym William R. Jacobs
author_facet	Paras Jain Brian C. Weinrick Eric J. Kalivoda Hui Yang Vanisha Munsamy Catherine Vilcheze Torin R. Weisbrod Michelle H. Larsen Max R. O’Donnell Alexander Pym William R. Jacobs
author_sort	Paras Jain
title	Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
title_short	Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
title_full	Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
title_fullStr	Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
title_full_unstemmed	Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum
title_sort	dual-reporter mycobacteriophages (φ<sup>2</sup>drms) reveal preexisting <named-content content-type="genus-species">mycobacterium tuberculosis</named-content> persistent cells in human sputum
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/32a454e686594896ba898650dbcbbb01
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Dual-Reporter Mycobacteriophages (Φ<sup>2</sup>DRMs) Reveal Preexisting <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> Persistent Cells in Human Sputum

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