A two-step strategy for the complementation of M. tuberculosis mutants

The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level

Genetic Essentiality, Biochemical and Structural Properties of Fructose 1,6-bisphosphatases II

The genetic essentiality of the glpX gene in Mycobacterium tuberculosis (Mtb) was investigated by generating an unmarked deletion mutant. This mutant was characterized by its phenotype, including growth on selective media, and its in vivo survival profile in a mouse model. The glpX gene was required for eugonic growth on gluconeogenic substrates such as glycerol, acetate, and oleic acid. Mtb lacking the glpX gene not only failed to maintain the initial inoculum density(100 counts), but also failed to replicate and survive in mouse lungs (3 log difference in bacterial count compared to the wild type strain of Mtb) during the acute phase of infection. The absence of glpX in the chronic phase of infection resulted in significant mycobacterial clearance. The glpX gene encoded a functional fructose 1,6–bisphosphatase which was successfully purified using affinity capture and size exclusion chromatography. Biochemical characterization, including determination of optimal conditions for enzymatic activity was performed. Mtb FBPase belongs to the super family of lithium sensitive phosphatases which require bivalent metal ions for enzymatic activity. The crystal structures of FBPase and its complex with catalytic product fructose 6-phosphate, were solved by a standard molecular replacement method. Mtb FBPase II exists as a functional tetramer with no allosteric regulatory mechanism as compared to the classical FBPase I present in mammals and several other bacteria. The active site of Mtb FBPase is highly conserved and 100% similar to other known FBPase II enzymes, indicative of a common catalytic mechanism. The FBPase activity of the glpX-encoded protein in Francisella tularensis was confirmed. Furthermore, the protein was purified to homogeneity and crystallized following similar methods used for Mtb FBPase. Two major bottlenecks (i.e. purification and crystallization of the protein target) in the process of structure based drug discovery (SBDD) for an already validated target were overcome. The interdisciplinary studies performed herein validate FBPase II as a drug target in pathogenic bacteria and provide critical information for SBDD

glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival.

Several enzymes involved in central carbon metabolism and gluconeogenesis play a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2-3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery

<i>glpX</i> is essential for growth in acute phase and survival during the chronic phase of <i>Mtb</i> infection in mice.

<p>Invivo growth and survival plot for <i>ΔglpX</i> compared to WT <i>Mtb</i> and the <i>glpX</i> complement. Data represents the mean±s.d. of six mice per time point.</p

In vitro growth profile of <i>ΔglpX</i>, WT <i>Mtb</i> and <i>glpX</i> complement in a) 7H9 medium + OADC enrichment, b) 7H9 medium with no additional carbon source.

<p>Growth profiles are representative of a triplicate data set.</p

In vitro growth profile of <i>ΔglpX</i>, WT <i>Mtb</i> and <i>glpX</i> complement on gluconeogenic carbon source(s).

<p>Growth profile in 7H9 medium a) 0.1% oleic acid, and b) 0.1% valeric acid Growth profiles are representative of a triplicate data set.</p

Strains and plasmids used for the generation of Δ<i>glpx</i> and <i>glpx</i> complement strains.

<p>Strains and plasmids used for the generation of Δ<i>glpx</i> and <i>glpx</i> complement strains.</p

Crystallization and preliminary X-ray characterization of the glpX-encoded class II fructose-1,6-bisphosphatase from Mycobacterium tuberculosis

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Å and belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 131.3, c = 143.2 Å. The structure has been solved by molecular replacement and is currently undergoing refinement

In vitro growth profile of <i>ΔglpX</i>, WT <i>Mtb</i> and <i>glpX</i> complement on defined (or individual) carbon source(s).

<p>Growth profile in 7H9 medium a) 0.2% glycerol, b) 0.2% Acetate, and c) 0.2% dextrose. Growth profiles are representative of a triplicate data set.</p