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

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.

SUBMITTER: Deb C

PROVIDER: S-EPMC2698117 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Deb Chirajyoti C Lee Chang-Muk CM Dubey Vinod S VS Daniel Jaiyanth J Abomoelak Bassam B Sirakova Tatiana D TD Pawar Santosh S Rogers Linda L Kolattukudy Pappachan E PE

PloS one 20090629 6

<h4>Background</h4>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 require ...[more]

PMID: 19562030

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Project description:Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of( 14)C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of (14)C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.

Project description:UnlabelledStrong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium's metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs' activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules.ImportanceMycobacterium tuberculosis establishes long-lasting highly prevalent infection inside the human body, called latent tuberculosis. The known involvement of fatty acids is changing our understanding of that silent infection; however, question of how tubercle bacilli globally adapt to a lipid-enriched environment is still an unanswered. With the single change of providing fatty acids as carbon sources, the bacilli switch on their program related to dormant stage: slowed growth, accumulation of lipid bodies, and development of drug tolerance. In this stage, unexpected and previously unknown participants were found to play putatively important roles during the process. For the first time, this work compares the global transcriptomics of bacteria by using strand-specific RNA sequencing under two different growth conditions. This study suggests novel targets for the control of tuberculosis and provides a new straightforward in vitro model that could help to test the activity of drugs against dormant bacilli from a novel perspective.

Project description:Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by 'non-culturability'-a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, 'non-culturable' M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions-the raw data are available via ProteomeXchange with identifier PXD028849. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

Project description:Dormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in K(+)-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K(+) reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis.RNA-seq-based analysis of the dormant non-culturable population obtained under rifampicin exposure revealed a 30-50-fold decrease of the total mRNA level, indicating global transcriptional repression. However, the analysis of persisting transcripts displayed a cohort of mRNA molecules coding for biosynthetic enzymes, proteins involved in adaptation and repair processes, detoxification, and control of transcription initiation. This 'dormant transcriptome' demonstrated considerable stability during M. tuberculosis persistence and mRNA de novo synthesis arrest. On the contrary, several small non-coding RNAs showed increased abundance on dormancy. Interestingly, M. tuberculosis entry into dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point located outside the ribosome catalytic center.Dormant non-culturable M. tuberculosis bacilli are characterized by a global transcriptional repression. At the same time, the dormant bacilli retain low-abundant mRNAs, which are considerably stable during in vitro persistence, reflecting their readiness for translation upon early resuscitation steps. Increased abundance of non-coding RNAs on dormancy may indicate their role in the entry into and maintenance of M. tuberculosis dormant non-culturable state.

Dataset Information

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

Publications

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

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