Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) binds triacylated glycolipid agonists of Toll-like receptor 2

Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (MTB) in mice. MTB lipoprotein LprG has TLR2 agonist activity, which is thought to be dependent on its N-terminal triacylation. Unexpectedly, here we find that nonacylated LprG retains TLR2 activity. Moreover, we show LprG association with triacylated glycolipid TLR2 agonists lipoarabinomannan, lipomannan and phosphatidylinositol mannosides (which share core structures). Binding of triacylated species was specific to LprG (not LprA) and increased LprG TLR2 agonist activity; conversely, association of glycolipids with LprG enhanced their recognition by TLR2. The crystal structure of LprG in complex with phosphatidylinositol mannoside revealed a hydrophobic pocket that accommodates the three alkyl chains of the ligand. In conclusion, we demonstrate a glycolipid binding function of LprG that enhances recognition of triacylated MTB glycolipids by TLR2 and may affect glycolipid assembly or transport for bacterial cell wall biogenesis.


Nature Structural & Molecular Biology
Published online: 8 August 2010 | doi:10.1038/nsmb.1869

Multiple M. tuberculosis Phenotypes in Mouse and Guinea Pig Lung Tissue Revealed by a Dual-Staining Approach

unique hallmark of TB is the granulomatous lesions formed in the lung. Granulomas can be heterogeneous in nature and can develop a necrotic, hypoxic core, which is surrounded by an acellular, fibrotic rim. Studying bacilli in this in vivo microenvironment is problematic as Mycobacterium tuberculosis can change its phenotype and also become acid-fast negative. Under in vitro models of differing environments, M. tuberculosis alters its metabolism, transcriptional profile, and rate of replication. In this study, the researchers investigated whether these phenotypic adaptations of M. tuberculosis are unique for certain environmental conditions and if they could therefore be used as differential markers. Bacilli were studied using fluorescent acid-fast auramine-rhodamine targeting the mycolic acid containing cell wall, and immunofluorescence targeting bacterial proteins using an anti-M. tuberculosiswhole cell lysate polyclonal antibody. These techniques were combined and simultaneously applied to M. tuberculosis in vitro culture samples and to lung sections of M. tuberculosis infected mice and guinea pigs. Two phenotypically different subpopulations of M. tuberculosis were found in stationary culture whilst three subpopulations were found in hypoxic culture and in lung sections. Bacilli were either exclusively acid-fast positive, exclusively immunofluorescent positive or acid-fast and immunofluorescent positive. These results suggest that M. tuberculosis exists as multiple populations in most conditions, even within seemingly a single microenvironment. This is relevant information for approaches that study bacillary characteristics in pooled samples (using lipidomics and proteomics) as well as in M. tuberculosis drug development.

Ryan, G.J., Hoff, D.R., Driver, E.R., Voskuil, M.I., et al.

PLoS One. 2010 Jun 14; Volume 5, Number 6: e11108.

Incorporation of a Dietary Omega 3 Fatty Acid Impairs Murine Macrophage Responses to Mycobacterium tuberculosis

Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance toMycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. The researchers hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNgamma-mediated signaling pathway. Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNgamma, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFalpha, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNgamma receptor with lipid rafts, without affecting surface expression of IFNgamma receptor. The researchers conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNgamma, by modulation of IFNgamma receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to TB.

Bonilla, D.L., Ly, L.H., Fan, Y.Y., Chapkin, R.S., et al.

PLoS One. 2010 May 28; Volume 5, Number 5: e10878.

PubMed

A Collaborative Database and Computational Models for Tuberculosis Drug Discovery;

The search for molecules with activity against Mycobacterium tuberculosis (Mtb) is employing many approaches in parallel including high throughput screening and computational methods. The researchers have developed a database (CDD TB) to capture public and private Mtb data while enabling data mining and collaborations with other researchers. They have used the public data along with several cheminformatics approaches to produce models that describe active and inactive compounds. The researchers have compared these datasets to those for known FDA approved drugs and between Mtb active and inactive compounds. The distribution of polar surface area and pK(a) of active compounds was found to be a statistically significant determinant of activity against Mtb. Hydrophobicity was not always statistically significant. Bayesian classification models for 220, 463 molecules were generated and tested with external molecules, and enabled the discrimination of active or inactive substructures from other datasets in the CDD TB. Computational pharmacophores based on known Mtb drugs were able to map to and retrieve a small subset of some of the Mtb datasets, including a high percentage of Mtb actives. The combination of the database, dataset analysis, Bayesian and pharmacophore models provides new insights into molecular properties and features that are determinants of activity in whole cells. This study provides novel insights into the key 1D molecular descriptors, 2D chemical substructures, and 3D pharmacophores which can be used to mine the chemistry space, prioritizing those molecules with a higher probability of activity against Mtb.

Ekins, S., Bradford, J., Dole, K., Spektor, A., et al.

Molecular BioSystems. 2010 May 10; Volume 6, Number 5: 840-51. Epub 2010 Feb 9.

Altered Protein Expression Patterns ofMycobacterium tuberculosis Induced by ATB107;

ATB107 is a potent inhibitor of indole-3-glycerol phosphate synthase (IGPS). It can effectively inhibit the growth of clinical isolates of drug-resistant Mycobacterium tuberculosis strains as well as M. tuberculosis H37Rv. To investigate the mechanism of ATB107 action in M. tuberculosis, two-dimensional gel electrophoresis coupled with MALDI-TOF-MS analysis (2-DE-MS) was performed to illustrate alterations in the protein expression profile in response to ATB107. Results show that ATB107 affected tryptophan biosynthesis by decreasing the expression of protein encoded by Rv3246c, the transcriptional regulatory protein of MtrA belonging to the MtrA-MtrB two-component regulatory system, in both drug-sensitive and drug-resistant virulent strains. ATB107 might present a stress condition similar to isoniazid (INH) or ethionamide for M. tuberculosis since the altered expression in response to ATB107 of some genes, such as Rv3140, Rv2243, and Rv2428, is consistent with INH or ethionamide treatment. After incubation with ATB107, the expression of 2 proteins encoded by Rv0685 and Rv2624c was down-regulated while that of protein encoded by Rv3140 was up-regulated in all M. tuberculosis strains used in this study. This may be the common response to tryptophan absence; however, relations to ATB107 are unknown and further evaluation is warranted.

Shen, H., Yang, E., Wang, F., Jin, R., et al.

Journal of Microbiology. 2010 Jun; Volume 48, Number 3: 337-46. Epub 2010 Jun 23.

PubMed

Tuberculosis Breakthrough Announced (Canada)

Dr. Maziar Divangahi of McGill University and the Research Institute of the McGill University Health Center stated that there may be an opportunity to improve TB vaccination and treatment using existing drugs. He explained the process by which TB-causing bacteria enter the body through the oral cavity, and white blood cells or macrophages engulf and encapsulate, but are unable to kill the bacteria. The bacteria undergo a type of hibernation and propagate inside the engulfment until the macrophages can hold them no more. The macrophages break apart, allowing the spread of the infection in the body. Normally macrophages trap bacteria inside their cell membranes and the bacteria die slowly, but this does not work for TB bacteria. TB bacteria have a system called necrosis, which destroys cell membranes aiding the bacteria’s escape. Dr. Divangahi focused on exploring the use of elcosanoids. He noted that elcosanoids can work with or against macrophages in relation to TB bacteria. Analysis of human genes found that changes occurring in the presence of elcosanoids will either produce immunity or susceptibility to TB. Elcosanoid-producing drugs are available and are used in treating other inflammatory diseases. Dr. Divangahi predicted that the next steps will be to determine how these drugs can be used to treat TB.

News Fire, www.news-fire.com, July 30, 2010, by Robert Valenzuela

Evasion of innate immunity by Mycobacterium tuberculosis: is death an exit strategy?

Samuel M. Behar, Maziar Divangahi & Heinz G. Remold

Abstract

Virulent Mycobacterium tuberculosis inhibits apoptosis and triggers necrosis of host macrophages to evade innate immunity and delay the initiation of adaptive immunity. By contrast, attenuated M. tuberculosis induces macrophage apoptosis, an innate defence mechanism that reduces bacterial viability. In this Opinion article, we describe how virulent M. tuberculosisblocks production of the eicosanoid lipid mediator prostaglandin E₂ (PGE₂). PGE₂ production by infected macrophages prevents mitochondrial damage and initiates plasma membrane repair, two processes that are crucial for preventing necrosis and inducing apoptosis. Thus, M. tuberculosis-mediated modulation of eicosanoid production determines the death modality of the infected macrophage, which in turn has a substantial impact on the outcome of infection.

Nature Reviews Microbiology , | doi:10.1038/nrmicro2387