Cytological and Transcript Analyses Reveal Fat and Lazy Persister-Like Bacilli in Tuberculous Sputum

Abstract

BACKGROUND: Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. METHODS AND FINDINGS: Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2) = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. CONCLUSION: As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

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Cited by papers we hold 16

• Phenotypic adaptation of Mycobacterium tuberculosis to host-associated stressors that induce persister formation · 2022
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• Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria · 2019
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• Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum · 2018
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• Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli · 2017
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• New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria · 2017
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• Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels, Growth Rate and Virulence in Mycobacterium tuberculosis. · 2016
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• Sequence comparison of six human microRNAs genes between tuberculosis patients and healthy individuals · 2015
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• The Granuloma in Tuberculosis: Dynamics of a Host–Pathogen Collusion · 2013
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• Memory T Cells in Latent Mycobacterium tuberculosis Infection Are Directed against Three Antigenic Islands and Largely Contained in a CXCR3+CCR6+ Th1 Subset · 2013
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• Proteomic Profiling of Mycobacterium tuberculosis Identifies Nutrient-starvation-responsive Toxin–antitoxin Systems · 2013
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• Metabolic Regulation of Mycobacterial Growth and Antibiotic Sensitivity · 2011
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• Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages · 2011
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• Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis · 2011
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• Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response · 2009
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• A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen · 2009
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• Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection · 2009
  Corrected

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