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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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Related in: MedlinePlus

Decrease in buoyant density of Mtb cells subjected to multiple-stress.Mtb cells subjected to the multiple-stresses were placed on the preformed gradient and centrifuged at 400 g for 20 min. The center tube is a 3 day cell sample mixed with density marker (M) beads. Percoll gradients were self-formed by centrifugation from a starting solution with a density (ρ) of 1.0925 gm/ml. The densities of selected bead layers (ρ, in gm/ml) are given on the right, and the positions of one ml fractions collected for analyses are at the left. Numbers below the tubes indicate the number of days under multiple-stress.
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pone-0006077-g005: Decrease in buoyant density of Mtb cells subjected to multiple-stress.Mtb cells subjected to the multiple-stresses were placed on the preformed gradient and centrifuged at 400 g for 20 min. The center tube is a 3 day cell sample mixed with density marker (M) beads. Percoll gradients were self-formed by centrifugation from a starting solution with a density (ρ) of 1.0925 gm/ml. The densities of selected bead layers (ρ, in gm/ml) are given on the right, and the positions of one ml fractions collected for analyses are at the left. Numbers below the tubes indicate the number of days under multiple-stress.

Mentions: We investigated whether the lipid accumulation in Mtb cells might be reflected in changes of buoyant density. Mtb cultures subjected to multiple-stress for different periods of time were fractionated on a Percoll density gradient and subsets of Mtb population were separated at different density levels at different time periods during multiple-stress treatment (Fig. 5). With increasing incubation time under multiple-stress, the bands of floating Mtb cells representing the major fraction of the Mtb population shifted towards lower buoyant density regions of the gradient in the upper phase of the tube (Fig. 5). This reflected the increase in lipid-loaded and non acid-fast-staining cells in the population with time under multiple-stress. Percoll density gradient fractionation of the Mtb culture, after 18 days under multiple-stress, showed a population of cells as a diffuse band in the lighter density region and the great majority of the lipid-droplet containing cells were distributed in the lighter fractions (Fig. 5; fractions 6, 7 and 8). Auramine-O/Nile Red staining of the different fractions showed that with increasing periods under the multiple-stress condition, increasing number of cells became lipid-loaded and lost acid-fast staining property. Nile Red staining of Percoll density gradient fractions from 18 day-stressed cultures showed that the lighter fractions were more enriched in lipid-loaded cells that lost acid-fastness (Fig. 6). These changes are consistent with the conclusion that application of multiple-stress caused progressive changes in lipid accumulation resulting in increasing percentages of presumably dormant cells in the lighter fractions.


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)

Decrease in buoyant density of Mtb cells subjected to multiple-stress.Mtb cells subjected to the multiple-stresses were placed on the preformed gradient and centrifuged at 400 g for 20 min. The center tube is a 3 day cell sample mixed with density marker (M) beads. Percoll gradients were self-formed by centrifugation from a starting solution with a density (ρ) of 1.0925 gm/ml. The densities of selected bead layers (ρ, in gm/ml) are given on the right, and the positions of one ml fractions collected for analyses are at the left. Numbers below the tubes indicate the number of days under multiple-stress.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006077-g005: Decrease in buoyant density of Mtb cells subjected to multiple-stress.Mtb cells subjected to the multiple-stresses were placed on the preformed gradient and centrifuged at 400 g for 20 min. The center tube is a 3 day cell sample mixed with density marker (M) beads. Percoll gradients were self-formed by centrifugation from a starting solution with a density (ρ) of 1.0925 gm/ml. The densities of selected bead layers (ρ, in gm/ml) are given on the right, and the positions of one ml fractions collected for analyses are at the left. Numbers below the tubes indicate the number of days under multiple-stress.
Mentions: We investigated whether the lipid accumulation in Mtb cells might be reflected in changes of buoyant density. Mtb cultures subjected to multiple-stress for different periods of time were fractionated on a Percoll density gradient and subsets of Mtb population were separated at different density levels at different time periods during multiple-stress treatment (Fig. 5). With increasing incubation time under multiple-stress, the bands of floating Mtb cells representing the major fraction of the Mtb population shifted towards lower buoyant density regions of the gradient in the upper phase of the tube (Fig. 5). This reflected the increase in lipid-loaded and non acid-fast-staining cells in the population with time under multiple-stress. Percoll density gradient fractionation of the Mtb culture, after 18 days under multiple-stress, showed a population of cells as a diffuse band in the lighter density region and the great majority of the lipid-droplet containing cells were distributed in the lighter fractions (Fig. 5; fractions 6, 7 and 8). Auramine-O/Nile Red staining of the different fractions showed that with increasing periods under the multiple-stress condition, increasing number of cells became lipid-loaded and lost acid-fast staining property. Nile Red staining of Percoll density gradient fractions from 18 day-stressed cultures showed that the lighter fractions were more enriched in lipid-loaded cells that lost acid-fastness (Fig. 6). These changes are consistent with the conclusion that application of multiple-stress caused progressive changes in lipid accumulation resulting in increasing percentages of presumably dormant cells in the lighter fractions.

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