TY - JOUR
T1 - Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum
AU - Jain, Paras
AU - Weinrick, Brian C.
AU - Kalivoda, Eric J.
AU - Yang, Hui
AU - Munsamy, Vanisha
AU - Vilcheze, Catherine
AU - Weisbrod, Torin R.
AU - Larsen, Michelle H.
AU - O’Donnell, Max R.
AU - Pym, Alexander
AU - Jacobs, William R.
N1 - Funding Information:
This work, including the efforts of Paras Jain, was funded by Potts Memorial Foundation. This work, including the efforts of William R. Jacobs, was funded by HHS | National Institutes of Health (NIH) (AI26170). This work, including the efforts of William R. Jacobs, was funded by HHS | National Institutes of Health (NIH) (U19AI111276). This work, including the efforts of Paras Jain, was funded by Stony Wold-Herbert Fund (Stony Wold-Herbert Fund, Inc.). This work, including the efforts of Michelle H. Larsen, was funded by HHS | National Institutes of Health (NIH) (1R01AI114900-01). This work, including the efforts of Torin R. Weisbrod, was funded by HHS | National Institutes of Health (NIH) (R01 AI09898). This work, including the efforts of William R. Jacobs, was funded by CFAR grant (P30AI051519). This work, including the efforts of Max O’Donnell, was funded by Clinical Scientist Development Award from the Doris Duke Charitable Foundation. The funders have had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2016 Jain et al.
PY - 2016/9/1
Y1 - 2016/9/1
N2 - 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 inΦ2GFP10, 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 dualreporter 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.
AB - 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 inΦ2GFP10, 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 dualreporter 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.
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U2 - 10.1128/mBio.01023-16
DO - 10.1128/mBio.01023-16
M3 - Article
C2 - 27795387
AN - SCOPUS:84994440906
VL - 7
JO - mBio
JF - mBio
SN - 2161-2129
IS - 5
M1 - e01023-16
ER -