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 FindingsWe developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (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/SignificanceThe 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.

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O 2 and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K m of 7.57 mM and V max of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37°C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrientdeprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.Tuberculosis caused by the organism Mycobacterium tuberculosis is one of the major public health threats in the world. The emergence of multidrug-resistant strains poses serious threats to the control of this disease due to the complex nature of second line drug treatment (1). Upon infection, the bacterium goes through an initial replicative phase inside the alveolar macrophages, after which it enters a nonreplicative, drug-resistant state of dormancy. This state of dormancy is probably induced by the environmental stress exerted upon the pathogen by the host's immune response. The bacterium is able to survive in this dormant state for decades until the host's immune system is weakened when it reactivates and causes the infectious disease (2). The current antimycobacterial drugs are able to kill only the actively replicating mycobacteria and do not clear the latent bacteria from the host (3). Thus, latency is a major problem in tuberculosis control. One-third of the world population is infected with the latent microorganism, and nearly 2 million deaths occur annually (4, 5). Individuals carrying a latent infection are estimated to harbor a 2-23% lifetime risk of reactivation (6).There is strong evidence that fatty acids may be the energy source for the mycobacterium during persistence (7-10). However, the source of these fatty acids is not known. We proposed that M. tuberculosis stores energy in the form of triacylglyce...

Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14 C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta-and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by twodimensional thin-layer chromatographic analysis of lipids derived from [1-14 C]propionic acid and 35 SO 4 Ϫ2 . With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C 26 was a major fatty acid in this TG, indicating that the TGS1 gene product uses C 26 fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C 26 : 0 -CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.