Targeting of long chain triacylglycerol hydrolase gene for tuberculosis treatment (CON)

Disclosed herein are novel methods for screening for compounds useful in treating or preventing tuberculosis. In exemplary embodiments, screening methods are based on the implementation or manipulation of triacylglycerol hydrolase like polypeptides or polynucleotides encoding the same. The methods are useful in identifying agents active against TB infection

Targeting of long chain triacylglycerol hydrolase gene for tuberculosis treatment

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. Upon infection, the bacterium goes through an initial replication phase after which it enters a nonreplicative, drug-resistant state of dormancy. The bacterium is able to survive in this dormant state for decades until the hosts immune system is weakened when it reactivates and causes the infectious disease

In vitro model of latent mycobacterial infection (DIV II)

A new in vitro culture model for latency of tuberculosis pathogen suitable for screening chemical libraries was developed by subjecting the pathogen to multiple stresses that the pathogen is thought to encounter in the host. When subjected to these stress factors, the pathogen develops key features characteristic of latency, drug resistance and lipid storage. Using the culture, a method to screen chemicals was developed. This method is suitable for high through put screening

In vitro model of latent mycobacterial infection

A new in vitro culture model for latency of tuberculosis pathogen suitable for screening chemical libraries was developed by subjecting the pathogen to multiple stresses that the pathogen is thought to encounter in the host. When subjected to these stress factors, the pathogen develops key features characteristic of latency, drug resistance and lipid storage. Using the culture, a method to screen chemicals was developed. This method is suitable for high through put screening

In Vitro Model of Latent Mycobacterial Infection

A new in vitro culture model for latency of tuberculosis pathogen suitable for screening chemical libraries was developed by subjecting the pathogen to multiple stresses that the pathogen is thought to encounter in the host. When subjected to these stress factors, the pathogen develops key features characteristic of latency, drug resistance and lipid storage. Using the culture, a method to screen chemicals was developed. This method is suitable for high through put screening

In vitro model of latent mycobacterial infection (DIV I)

A new in vitro culture model for latency of tuberculosis pathogen suitable for screening chemical libraries was developed by subjecting the pathogen to multiple stresses that the pathogen is thought to encounter in the host. When subjected to these stress factors, the pathogen develops key features characteristic of latency, drug resistance and lipid storage. Using the culture, a method to screen chemicals was developed. This method is suitable for high through put screening

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium 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 degrees 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 nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen

Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent Acyl-CoA carboxylase in Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa alpha 3-subunit (AccA3, Rv3285), the 59-kDa beta 5-subunit (AccD5, Rv3280), and the 56-kDa beta 4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the beta 5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered epsilon-subunits of Streptomyces coelicolor, suggesting that it might be an epsilon-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted alpha 3-beta 5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the epsilon-subunit stimulated the carboxylation by 3.2- and 6.3-fold, respectively. The alpha 3-beta 4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This epsilon-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of epsilon-protein was highest during the log phase and decreased during the stationary phase

Expression of Fungal Cutinase and Swollenin in Tobacco Chloroplasts Reveals Novel Enzyme Functions and/or Substrates

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis

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 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 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 an