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A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen

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 dor...

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Autores principales: Deb, Chirajyoti, Lee, Chang-Muk, Dubey, Vinod S., Daniel, Jaiyanth, Abomoelak, Bassam, Sirakova, Tatiana D., Pawar, Santosh, Rogers, Linda, Kolattukudy, Pappachan E.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698117/
https://www.ncbi.nlm.nih.gov/pubmed/19562030
http://dx.doi.org/10.1371/journal.pone.0006077
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author Deb, Chirajyoti
Lee, Chang-Muk
Dubey, Vinod S.
Daniel, Jaiyanth
Abomoelak, Bassam
Sirakova, Tatiana D.
Pawar, Santosh
Rogers, Linda
Kolattukudy, Pappachan E.
author_facet Deb, Chirajyoti
Lee, Chang-Muk
Dubey, Vinod S.
Daniel, Jaiyanth
Abomoelak, Bassam
Sirakova, Tatiana D.
Pawar, Santosh
Rogers, Linda
Kolattukudy, Pappachan E.
author_sort Deb, Chirajyoti
collection PubMed
description 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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spelling pubmed-26981172009-06-29 A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen Deb, Chirajyoti Lee, Chang-Muk Dubey, Vinod S. Daniel, Jaiyanth Abomoelak, Bassam Sirakova, Tatiana D. Pawar, Santosh Rogers, Linda Kolattukudy, Pappachan E. PLoS One Research Article 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. Public Library of Science 2009-06-29 /pmc/articles/PMC2698117/ /pubmed/19562030 http://dx.doi.org/10.1371/journal.pone.0006077 Text en Deb et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Deb, Chirajyoti
Lee, Chang-Muk
Dubey, Vinod S.
Daniel, Jaiyanth
Abomoelak, Bassam
Sirakova, Tatiana D.
Pawar, Santosh
Rogers, Linda
Kolattukudy, Pappachan E.
A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title_full A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title_fullStr A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title_full_unstemmed A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title_short A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen
title_sort novel in vitro multiple-stress dormancy model for mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698117/
https://www.ncbi.nlm.nih.gov/pubmed/19562030
http://dx.doi.org/10.1371/journal.pone.0006077
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