Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum

Paras Jain, Brian C. Weinrick, Eric J. Kalivoda, Hui Yang, Vanisha Munsamy, Catherine Vilcheze, Torin Weisbrod, Michelle H. Larsen, Max R. O’Donnell, Alexander Pym, William R. Jacobs

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 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.

Original languageEnglish (US)
Article numbere01023-16
JournalmBio
Volume7
Issue number5
DOIs
StatePublished - Sep 1 2016

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Mycobacteriophages
Sputum
Mycobacterium tuberculosis
Anti-Bacterial Agents
Tuberculosis
Population
Genes
Isoniazid
Pandemics
Green Fluorescent Proteins
Treatment Failure
Fluorescence Microscopy
Communicable Diseases
Cause of Death
Flow Cytometry
Cell Culture Techniques
Alleles
HIV

ASJC Scopus subject areas

  • Microbiology
  • Virology

Cite this

Jain, P., Weinrick, B. C., Kalivoda, E. J., Yang, H., Munsamy, V., Vilcheze, C., ... Jacobs, W. R. (2016). Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum. mBio, 7(5), [e01023-16]. https://doi.org/10.1128/mBio.01023-16

Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum. / Jain, Paras; Weinrick, Brian C.; Kalivoda, Eric J.; Yang, Hui; Munsamy, Vanisha; Vilcheze, Catherine; Weisbrod, Torin; Larsen, Michelle H.; O’Donnell, Max R.; Pym, Alexander; Jacobs, William R.

In: mBio, Vol. 7, No. 5, e01023-16, 01.09.2016.

Research output: Contribution to journalArticle

Jain, P, Weinrick, BC, Kalivoda, EJ, Yang, H, Munsamy, V, Vilcheze, C, Weisbrod, T, Larsen, MH, O’Donnell, MR, Pym, A & Jacobs, WR 2016, 'Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum', mBio, vol. 7, no. 5, e01023-16. https://doi.org/10.1128/mBio.01023-16
Jain, Paras ; Weinrick, Brian C. ; Kalivoda, Eric J. ; Yang, Hui ; Munsamy, Vanisha ; Vilcheze, Catherine ; Weisbrod, Torin ; Larsen, Michelle H. ; O’Donnell, Max R. ; Pym, Alexander ; Jacobs, William R. / Dual-reporter mycobacteriophages (Φ2DRMS) reveal preexisting mycobacterium tuberculosis persistent cells in human sputum. In: mBio. 2016 ; Vol. 7, No. 5.
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