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

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

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 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 (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

Human Granuloma In Vitro Model, for TB Dormancy and Resuscitation

Tuberculosis (TB) is responsible for death of nearly two million people in the world annually. Upon infection, Mycobacterium tuberculosis (Mtb) causes formation of granuloma where the pathogen goes into dormant state and can live for decades before resuscitation to develop active disease when the immune system of the host is weakened and/or suppressed. In an attempt to better understand host-pathogen interactions, several groups have been developing in vitro models of human tuberculosis granuloma. However, to date, an in vitro granuloma model in which Mtb goes into dormancy and can subsequently resuscitate under conditions that mimic weakening of the immune system has not been reported. We describe the development of a biomimetic in vitro model of human tuberculosis granuloma using human primary leukocytes, in which the Mtb exhibited characteristics of dormant mycobacteria as demonstrated by (1) loss of acid-fastness, (2) accumulation of lipid bodies (3) development of rifampicin-tolerance and (4) gene expression changes. Further, when these micro granulomas were treated with immunosuppressant anti-tumor necrosis factor-alpha monoclonal antibodies (anti-TNF alpha mAbs), resuscitation of Mtb was observed as has been found in humans. In this human in vitro granuloma model triacylglycerol synthase 1deletion mutant (Delta tgs1) with impaired ability to accumulate triacylglycerides (TG), but not the complemented mutant, could not go into dormancy. Deletion mutant of lipY, with compromised ability to mobilize the stored TG, but not the complemented mutant, was unable to come out of dormancy upon treatment with anti-TNF alpha mAbs. In conclusion, we have developed an in vitro human tuberculosis granuloma model that largely exhibits functional features of dormancy and resuscitation observed in human tuberculosis

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

Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains ofThiobacillus ferrooxidans.The 1.4-kb fragment hybridized with IST2,an insertion sequence ofT. ferrooxidans.The 1.2-kb fragment was cloned and sequenced. The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with ISAE1ofAlcaligenes eutrophus.The IST445contains three open reading frames which have codon usage almost similar to 56 different coding genes ofT. ferrooxidans.In Southern blots of restricted genomic DNAs probed with IST445,each of the several strains ofT. ferrooxidansgives a distinctive fingerprint. IST445is present in the range of 10-20 copies per genome in the four strains studied