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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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Microarray analysis demonstrated changes in expression of genes involved in glyoxylate cycle and energy metabolism.(A), The expression ratio of genes involved in glyoxylate shunt cycle was shown in the red-green-display according to the log2-tranformed color code. Experimental time-points were shown at the top of the column. Genes were selected based on their annotation in TubercuList database, and grouped into those that were either regulated at least one of the time-points under multiple- stress condition. (B), Energy generation and NAD regeneration under multiple-stress. Genes involved in energy generation were selected based on their annotation. Red denotes induction and green denotes repression.
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pone-0006077-g007: Microarray analysis demonstrated changes in expression of genes involved in glyoxylate cycle and energy metabolism.(A), The expression ratio of genes involved in glyoxylate shunt cycle was shown in the red-green-display according to the log2-tranformed color code. Experimental time-points were shown at the top of the column. Genes were selected based on their annotation in TubercuList database, and grouped into those that were either regulated at least one of the time-points under multiple- stress condition. (B), Energy generation and NAD regeneration under multiple-stress. Genes involved in energy generation were selected based on their annotation. Red denotes induction and green denotes repression.

Mentions: We analyzed changes in the gene expression profiles using oligonucleotide microarray. Analysis of variance (ANOVA) and significance analysis of microarrays (SAM) [37] were conducted to identify significant gene expression changes at selected time points during the 18 days of multiple-stress application to Mtb cells. The genes that were differentially expressed more than 2-fold include a total of 331 targets, representing ∼7% of ORFs on the chip. Under the multiple-stress condition, genes that encode enzymes of glyoxylate cycle such as isocitrate lyase (icl or aceA/Rv0467) and citrate synthase (gltA1/Rv1131c) showed significant increase in expression for all the time points examined (Fig. 7A). Mtb showed shutdown of both ATP and NAD energy regeneration systems (Fig. 7B). All the genes encoding NADH dehydrogenase I subunits (nuoABEFHIJKLMN) and ubiquinol–cytochrome C complex (qcrA/B/C) were repressed. In addition, the gene expression of ATP synthase subunits was repressed, indicating the major shutdown of ATP generation in the cells. Moreover, slowdown of the overall activity in transcription/translation apparatus was manifested under the multiple-stresses. For instance, rhlE (ATP-dependent RNA helicase homolog) was repressed, demonstrating reduced activity in transcription machineries. However, genes required for anaerobic respiration (frdA, narG/H/X, nirA) were continuously expressed until the later time points examined (18 days) and aerobic respiration was significantly repressed throughout the period of multiple-stress treatment. We also found significant induction of the genes classified as the stress-response genes (e.g. hspX/acr; Rv2031c) that may play a role in maintaining long term survival within the host [38].


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)

Microarray analysis demonstrated changes in expression of genes involved in glyoxylate cycle and energy metabolism.(A), The expression ratio of genes involved in glyoxylate shunt cycle was shown in the red-green-display according to the log2-tranformed color code. Experimental time-points were shown at the top of the column. Genes were selected based on their annotation in TubercuList database, and grouped into those that were either regulated at least one of the time-points under multiple- stress condition. (B), Energy generation and NAD regeneration under multiple-stress. Genes involved in energy generation were selected based on their annotation. Red denotes induction and green denotes repression.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006077-g007: Microarray analysis demonstrated changes in expression of genes involved in glyoxylate cycle and energy metabolism.(A), The expression ratio of genes involved in glyoxylate shunt cycle was shown in the red-green-display according to the log2-tranformed color code. Experimental time-points were shown at the top of the column. Genes were selected based on their annotation in TubercuList database, and grouped into those that were either regulated at least one of the time-points under multiple- stress condition. (B), Energy generation and NAD regeneration under multiple-stress. Genes involved in energy generation were selected based on their annotation. Red denotes induction and green denotes repression.
Mentions: We analyzed changes in the gene expression profiles using oligonucleotide microarray. Analysis of variance (ANOVA) and significance analysis of microarrays (SAM) [37] were conducted to identify significant gene expression changes at selected time points during the 18 days of multiple-stress application to Mtb cells. The genes that were differentially expressed more than 2-fold include a total of 331 targets, representing ∼7% of ORFs on the chip. Under the multiple-stress condition, genes that encode enzymes of glyoxylate cycle such as isocitrate lyase (icl or aceA/Rv0467) and citrate synthase (gltA1/Rv1131c) showed significant increase in expression for all the time points examined (Fig. 7A). Mtb showed shutdown of both ATP and NAD energy regeneration systems (Fig. 7B). All the genes encoding NADH dehydrogenase I subunits (nuoABEFHIJKLMN) and ubiquinol–cytochrome C complex (qcrA/B/C) were repressed. In addition, the gene expression of ATP synthase subunits was repressed, indicating the major shutdown of ATP generation in the cells. Moreover, slowdown of the overall activity in transcription/translation apparatus was manifested under the multiple-stresses. For instance, rhlE (ATP-dependent RNA helicase homolog) was repressed, demonstrating reduced activity in transcription machineries. However, genes required for anaerobic respiration (frdA, narG/H/X, nirA) were continuously expressed until the later time points examined (18 days) and aerobic respiration was significantly repressed throughout the period of multiple-stress treatment. We also found significant induction of the genes classified as the stress-response genes (e.g. hspX/acr; Rv2031c) that may play a role in maintaining long term survival within the host [38].

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