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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 storage lipids by Mtb under multiple-stress in vitro.Mtb was subjected to a combination of four stresses - high CO2, low O2, acidic pH and nutrient starvation. Total lipids were extracted at 0, 3, 9 and 18 days and resolved on silica-TLC using hexane-diethyl ether-formic acid (90∶10∶1, v/v/v). Lipids were visualized by charring after spraying with dichromate-sulfuric acid and quantified by densitometry using Alpha Innotech Gel documentation system and AlphaImager 2200 software (Alpha Innotech, USA). TG, triglycerides; WE, wax esters.
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pone-0006077-g001: Accumulation of storage lipids by Mtb under multiple-stress in vitro.Mtb was subjected to a combination of four stresses - high CO2, low O2, acidic pH and nutrient starvation. Total lipids were extracted at 0, 3, 9 and 18 days and resolved on silica-TLC using hexane-diethyl ether-formic acid (90∶10∶1, v/v/v). Lipids were visualized by charring after spraying with dichromate-sulfuric acid and quantified by densitometry using Alpha Innotech Gel documentation system and AlphaImager 2200 software (Alpha Innotech, USA). TG, triglycerides; WE, wax esters.

Mentions: Since the hypoxic or NO-stress conditions caused accumulation of storage lipids but did not result in detectable Rif-resistance against moderate concentrations of Rif (5 µg/ml) (unpublished results, [24], [35],), an important indicator of true dormancy, we tested a combination of stress conditions which the pathogen is thought to encounter in the host [10], [21], [31], [33], [34]. After growing Mtb in complete Dubos medium (OD600 nm = 0.2) the cells were transferred to a low-nutrient medium (10% Dubos medium without glycerol) at acidic pH (pH 5.0) in an atmosphere containing high (10%) CO2 and low (5%) O2 for 18 days and the lipid accumulation in those Mtb cells was analyzed at 3, 9 and 18 days. We found that TG accumulation reached maximal level by day 9 (Fig. 1). WE levels in Mtb cells also increased significantly and reached maximal levels by 3 days (Fig. 1). The aerobic control samples at pH 5.0 or 7.0 did not show an increase in WE or TG (data not shown). Capillary-GC analysis revealed that palmitate (C16:0) and stearate (C18:0) were the major fatty acid constituents of the TG and WE fractions (Fig 2). Metabolic incorporation of 14C-oleic acid into lipids by the Mtb cells after 0, 9 and 18 days under multiple-stress revealed that 14C was incorporated predominantly into TG, and polar lipids (Table 1). Incorporation of 14C-oleic acid into TG increased from 12% of the label in total lipids on day 0 to 43% on day 9 decreasing slightly to 37% on day 18. Incorporation of the radiolabeled oleic acid into polar lipids decreased from 51% on day 0 to 17% on day 9 reflecting down regulation of the biosynthesis of membrane lipids and channeling of fatty acids into storage lipids as the pathogen goes into the dormant state. Incorporation of 14C-oleic acid into WE increased significantly on day 9 and remained at the same level at day 18 (Table 1).


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 storage lipids by Mtb under multiple-stress in vitro.Mtb was subjected to a combination of four stresses - high CO2, low O2, acidic pH and nutrient starvation. Total lipids were extracted at 0, 3, 9 and 18 days and resolved on silica-TLC using hexane-diethyl ether-formic acid (90∶10∶1, v/v/v). Lipids were visualized by charring after spraying with dichromate-sulfuric acid and quantified by densitometry using Alpha Innotech Gel documentation system and AlphaImager 2200 software (Alpha Innotech, USA). TG, triglycerides; WE, wax esters.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006077-g001: Accumulation of storage lipids by Mtb under multiple-stress in vitro.Mtb was subjected to a combination of four stresses - high CO2, low O2, acidic pH and nutrient starvation. Total lipids were extracted at 0, 3, 9 and 18 days and resolved on silica-TLC using hexane-diethyl ether-formic acid (90∶10∶1, v/v/v). Lipids were visualized by charring after spraying with dichromate-sulfuric acid and quantified by densitometry using Alpha Innotech Gel documentation system and AlphaImager 2200 software (Alpha Innotech, USA). TG, triglycerides; WE, wax esters.
Mentions: Since the hypoxic or NO-stress conditions caused accumulation of storage lipids but did not result in detectable Rif-resistance against moderate concentrations of Rif (5 µg/ml) (unpublished results, [24], [35],), an important indicator of true dormancy, we tested a combination of stress conditions which the pathogen is thought to encounter in the host [10], [21], [31], [33], [34]. After growing Mtb in complete Dubos medium (OD600 nm = 0.2) the cells were transferred to a low-nutrient medium (10% Dubos medium without glycerol) at acidic pH (pH 5.0) in an atmosphere containing high (10%) CO2 and low (5%) O2 for 18 days and the lipid accumulation in those Mtb cells was analyzed at 3, 9 and 18 days. We found that TG accumulation reached maximal level by day 9 (Fig. 1). WE levels in Mtb cells also increased significantly and reached maximal levels by 3 days (Fig. 1). The aerobic control samples at pH 5.0 or 7.0 did not show an increase in WE or TG (data not shown). Capillary-GC analysis revealed that palmitate (C16:0) and stearate (C18:0) were the major fatty acid constituents of the TG and WE fractions (Fig 2). Metabolic incorporation of 14C-oleic acid into lipids by the Mtb cells after 0, 9 and 18 days under multiple-stress revealed that 14C was incorporated predominantly into TG, and polar lipids (Table 1). Incorporation of 14C-oleic acid into TG increased from 12% of the label in total lipids on day 0 to 43% on day 9 decreasing slightly to 37% on day 18. Incorporation of the radiolabeled oleic acid into polar lipids decreased from 51% on day 0 to 17% on day 9 reflecting down regulation of the biosynthesis of membrane lipids and channeling of fatty acids into storage lipids as the pathogen goes into the dormant state. Incorporation of 14C-oleic acid into WE increased significantly on day 9 and remained at the same level at day 18 (Table 1).

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