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A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen.

Deb C, Lee CM, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, Pawar S, Rogers L, Kolattukudy PE - PLoS ONE (2009)

Bottom Line: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb.A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established.Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT

Background: Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen.

Methodology/principal findings: We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO(2) (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells.

Conclusions/significance: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

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Accumulation of lipid bodies and loss of acid-fastness in Mtb cells under multiple-stress.(A), Acid-fast staining cells (green) decreased and lipid body staining cells (red) increased with time under multiple-stress. Cells were stained with Auramine-O (acid-fast stain) and Nile Red (neutral lipid stain) and examined by confocal laser scanning microscopy (Leica TCS SP5) at the same laser intensity for all the samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed by LAS AF software for image projection. Overlaid images of the dual-stained Mtb are shown. Bar = 4 µM. (B), Magnified view of three different Mtb cells, representing three different subsets of Mtb cells in terms of acid-fast and neutral lipid staining property, observed in the Mtb population under multiple-stress: only acid-fast positive without any Nile Red stain (green), both acid-fast and lipid stain positive (orange yellow) and acid-fast negative cells with only Nile Red staining lipid bodies (right). The only acid-fast stain (green) positive cells gradually decreased and the other two types steadily increased during multiple-stress treatment. These cells selected from a day 9 sample were stained with both dyes and examined by confocal scanning as stated above in (A). Bar = 5 µM.
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pone-0006077-g003: Accumulation of lipid bodies and loss of acid-fastness in Mtb cells under multiple-stress.(A), Acid-fast staining cells (green) decreased and lipid body staining cells (red) increased with time under multiple-stress. Cells were stained with Auramine-O (acid-fast stain) and Nile Red (neutral lipid stain) and examined by confocal laser scanning microscopy (Leica TCS SP5) at the same laser intensity for all the samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed by LAS AF software for image projection. Overlaid images of the dual-stained Mtb are shown. Bar = 4 µM. (B), Magnified view of three different Mtb cells, representing three different subsets of Mtb cells in terms of acid-fast and neutral lipid staining property, observed in the Mtb population under multiple-stress: only acid-fast positive without any Nile Red stain (green), both acid-fast and lipid stain positive (orange yellow) and acid-fast negative cells with only Nile Red staining lipid bodies (right). The only acid-fast stain (green) positive cells gradually decreased and the other two types steadily increased during multiple-stress treatment. These cells selected from a day 9 sample were stained with both dyes and examined by confocal scanning as stated above in (A). Bar = 5 µM.

Mentions: Dual staining of Mtb with the combination of Auramine-O and Nile Red has been used to reveal acid-fast staining property and neutral lipid accumulation in the same cell [36]. We applied this dual staining procedure to examine acid-fastness of Mtb cells and lipid body accumulation within the Mtb cells under multiple-stress. When we subjected a young, synchronous culture of Mtb to the multiple-stress condition for increasing periods of time, we observed a steady decrease in Auramine-O stained green-fluorescing acid-fast cells with a corresponding increase in Nile Red stained red-fluorescing lipid-body containing cells (Fig. 3). Initially, in the freshly grown starter culture about 90% of the population was acid-fast positive by retaining the green-fluorescing Auramine-O stain, and only a few lipid-body accumulating (Nile Red) cells could be detected. Most of these Nile Red positive cells also retained the acid-fast specific Auramine-O stain in the heterogeneous population at day 0. After 18 days under multiple-stress, acid-fast positive cells decreased to about 30% of the population while Nile Red-stained cells with internal red spherical bodies increased from 10% to about 70% (Fig. 4). Overlaid green and red color images of dual-stained Mtb showed some cells staining with both Auramine-O and Nile Red and fluoresced at both green and red wavelengths to give an orange appearance while other cells stained exclusively for Auramine-O or Nile Red (Fig. 3). This difference in dual staining property indicated generation of at least three different sub-populations in the Mtb cultures under multiple-stress condition: a subset that stained only with Auramine-O: (probably actively multiplying), a second subset which stained with both Auramine-O and Nile Red (probably transitioning to non-replicating state) and a third subset that stained only with Nile Red (probably non-replicating and dormant) (Fig. 3B).


A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen.

Deb C, Lee CM, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, Pawar S, Rogers L, Kolattukudy PE - PLoS ONE (2009)

Accumulation of lipid bodies and loss of acid-fastness in Mtb cells under multiple-stress.(A), Acid-fast staining cells (green) decreased and lipid body staining cells (red) increased with time under multiple-stress. Cells were stained with Auramine-O (acid-fast stain) and Nile Red (neutral lipid stain) and examined by confocal laser scanning microscopy (Leica TCS SP5) at the same laser intensity for all the samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed by LAS AF software for image projection. Overlaid images of the dual-stained Mtb are shown. Bar = 4 µM. (B), Magnified view of three different Mtb cells, representing three different subsets of Mtb cells in terms of acid-fast and neutral lipid staining property, observed in the Mtb population under multiple-stress: only acid-fast positive without any Nile Red stain (green), both acid-fast and lipid stain positive (orange yellow) and acid-fast negative cells with only Nile Red staining lipid bodies (right). The only acid-fast stain (green) positive cells gradually decreased and the other two types steadily increased during multiple-stress treatment. These cells selected from a day 9 sample were stained with both dyes and examined by confocal scanning as stated above in (A). Bar = 5 µM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698117&req=5

pone-0006077-g003: Accumulation of lipid bodies and loss of acid-fastness in Mtb cells under multiple-stress.(A), Acid-fast staining cells (green) decreased and lipid body staining cells (red) increased with time under multiple-stress. Cells were stained with Auramine-O (acid-fast stain) and Nile Red (neutral lipid stain) and examined by confocal laser scanning microscopy (Leica TCS SP5) at the same laser intensity for all the samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed by LAS AF software for image projection. Overlaid images of the dual-stained Mtb are shown. Bar = 4 µM. (B), Magnified view of three different Mtb cells, representing three different subsets of Mtb cells in terms of acid-fast and neutral lipid staining property, observed in the Mtb population under multiple-stress: only acid-fast positive without any Nile Red stain (green), both acid-fast and lipid stain positive (orange yellow) and acid-fast negative cells with only Nile Red staining lipid bodies (right). The only acid-fast stain (green) positive cells gradually decreased and the other two types steadily increased during multiple-stress treatment. These cells selected from a day 9 sample were stained with both dyes and examined by confocal scanning as stated above in (A). Bar = 5 µM.
Mentions: Dual staining of Mtb with the combination of Auramine-O and Nile Red has been used to reveal acid-fast staining property and neutral lipid accumulation in the same cell [36]. We applied this dual staining procedure to examine acid-fastness of Mtb cells and lipid body accumulation within the Mtb cells under multiple-stress. When we subjected a young, synchronous culture of Mtb to the multiple-stress condition for increasing periods of time, we observed a steady decrease in Auramine-O stained green-fluorescing acid-fast cells with a corresponding increase in Nile Red stained red-fluorescing lipid-body containing cells (Fig. 3). Initially, in the freshly grown starter culture about 90% of the population was acid-fast positive by retaining the green-fluorescing Auramine-O stain, and only a few lipid-body accumulating (Nile Red) cells could be detected. Most of these Nile Red positive cells also retained the acid-fast specific Auramine-O stain in the heterogeneous population at day 0. After 18 days under multiple-stress, acid-fast positive cells decreased to about 30% of the population while Nile Red-stained cells with internal red spherical bodies increased from 10% to about 70% (Fig. 4). Overlaid green and red color images of dual-stained Mtb showed some cells staining with both Auramine-O and Nile Red and fluoresced at both green and red wavelengths to give an orange appearance while other cells stained exclusively for Auramine-O or Nile Red (Fig. 3). This difference in dual staining property indicated generation of at least three different sub-populations in the Mtb cultures under multiple-stress condition: a subset that stained only with Auramine-O: (probably actively multiplying), a second subset which stained with both Auramine-O and Nile Red (probably transitioning to non-replicating state) and a third subset that stained only with Nile Red (probably non-replicating and dormant) (Fig. 3B).

Bottom Line: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb.A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established.Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA.

ABSTRACT

Background: Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen.

Methodology/principal findings: We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO(2) (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells.

Conclusions/significance: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

Show MeSH
Related in: MedlinePlus