Cord Factor Research Articles

A mycobacteriophage‐derived trehalose‐6,6'‐dimycolate‐binding peptide containing both antimycobacterial and anti‐inflammatory abilities

Bacteriophages, the viruses of eubacteria, have developed unique mechanisms to interact with their host bacteria. They have been viewed as potential antibacterial therapeutics. Mycobacteriophage-derived compounds may interact with Mycobacterium tuberculosis (MTB) and/or its components, such as the cord factor, trehalose-6,6'-dimycolate (TDM), which is the most abundant glycolipid produced on the surface of MTB. TDM emulsion injected intravenously into mice induces lung immunopathology that mimics many aspects of MTB infection. Thus, TDM is an important target for anti-MTB agent development. On the basis of genomics information of mycobacteriophages, 200 peptides were synthesized. Their effects on MTB, their interactions with TDM, and anti-inflammatory activities were tested. One of them (PK34) showed MTB-killing activity with a minimal inhibitory concentration of 50 μg/ml and TDM-binding ability. In a mouse model, PK34 showed comparable ability to clear MTB as rifampin did in vivo. It also exerted strong activity to inhibit MTB or TDM-induced inflammation in vivo. PK34 significantly inhibited inflammatory cytokines secretions by inactivating MAPK and PKB signals while it maintained certain proinflammatory cytokine production. It is possible to prospect for TDM-binding and/or anti-MTB peptides by mining the mycobacteriophages genome. In addition to its direct MTB-killing ability, PK34 might be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection or a template for developing antituberculosis (TB) agents because of its immunoregulative effects. As a TDM-binding peptide, PK34 may be a promising tool to study TDM's interactions with corresponding receptors and signal pathways.

C-type Lectin MCL Is an FcRγ-Coupled Receptor that Mediates the Adjuvanticity of Mycobacterial Cord Factor

Cord factor, also called trehalose-6,6'-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle isknown to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4e(gfp) reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity ofTDM.

Ebselen: a covalent inhibitor of the Antigen 85 complex from Mycobacterium tuberculosis

The World Health Organization (WHO) reported 8.7 million new cases of tuberculosis (TB) and 1.4 million deaths in 2011. Co‐infection with Human Immunodeficiency Virus (HIV), as well as the appearance of multidrug and extensively drug‐resistance tuberculosis (MDR‐TB or XDR‐TB), stress the need of discovering new drugs to treat TB. The antigen 85 complex (Ag85) of Mycobacterium tuberculosis (M. tb) includes three mycolyltransferases: Ag85A, Ag85B and Ag85C, involved in the cell wall biosynthesis. The enzymes catalyze the transfer of mycolic acids to trehalose monomycolate (TMM) producing the cord factor trehalose dimycolate (TDM) through a transesterification reaction. The Ag85 complex appears as a valuable drug target to treat TB as inactivation of one of the enzymes affects mycobacterial cell growth in the host; cell wall organization is disrupted and an increase in sensitivity to drugs targeting the cell wall biosynthesis is observed. This study aims to improve methods for inhibitor screening using Ag85C. We report the development of a new high‐throughput assay using resorufin butyrate as an acyl donor. This fluorescence‐based assay provided a Z′ value of 0.82, validating the assay for inhibitor screening. The screening of the NIH clinical collection identified ebselen as a potent inhibitor of M. tb Ag85C. Initial dose‐dependence studies suggested an inhibitory activity in the nanomolar range. However, characterization using mass spectrometry and X‐ray crystallography, gave further insight into the mechanism of action; ebselen binds covalently to the lone cysteine located near the active site of Ag85C, locking the enzyme in an inactive conformation.Funding from the National Institutes of Health (R15AI089653–01) supported this work.

The Mincle-Activating Adjuvant TDB Induces MyD88-Dependent Th1 and Th17 Responses through IL-1R Signaling

Successful vaccination against intracellular pathogens requires the generation of cellular immune responses. Trehalose-6,6-dibehenate (TDB), the synthetic analog of the mycobacterial cord factor trehalose-6,6-dimycolate (TDM), is a potent adjuvant inducing strong Th1 and Th17 immune responses. We previously identified the C-type lectin Mincle as receptor for these glycolipids that triggers the FcRγ-Syk-Card9 pathway for APC activation and adjuvanticity. Interestingly, in vivo data revealed that the adjuvant effect was not solely Mincle-dependent but also required MyD88. Therefore, we dissected which MyD88-dependent pathways are essential for successful immunization with a tuberculosis subunit vaccine. We show here that antigen-specific Th1/Th17 immune responses required IL-1 receptor-mediated signals independent of IL-18 and IL-33-signaling. ASC-deficient mice had impaired IL-17 but intact IFNγ responses, indicating partial independence of TDB adjuvanticity from inflammasome activation. Our data suggest that the glycolipid adjuvant TDB triggers Mincle-dependent IL-1 production to induce MyD88-dependent Th1/Th17 responses in vivo.

Recognition of the mycobacterial cord factor by Mincle: relevance for granuloma formation and resistance to tuberculosis

The world's most successful intracellular bacterial pathogen, Mycobacterium tuberculosis (MTB), survives inside macrophages by blocking phagosome maturation and establishes chronic infection characterized by the formation of granulomas. Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is the most abundant cell wall lipid of virulent mycobacteria, is sufficient to cause granuloma formation, and has long been known to be a major virulence factor of MTB. Recently, TDM has been shown to activate the Syk-Card9 signaling pathway in macrophages through binding to the C-type lectin receptor Mincle. The Mincle-Card9 pathway is required for activation of macrophages by TDM in vitro and for granuloma formation in vivo following injection of TDM. Whether this pathway is also exploited by MTB to reprogram the macrophage into a comfortable niche has not been explored yet. Several recent studies have investigated the phenotype of Mincle-deficient mice in mycobacterial infection, yielding divergent results in terms of a role for Mincle in host resistance. Here, we review these studies, discuss possible reasons for discrepant results and highlight open questions in the role of Mincle and other C-type lectin receptors in the infection biology of MTB.

Development of a benzophenone and alkyne functionalised trehalose probe to study trehalose dimycolate binding proteins

Trehalose dimycolates (TDMs) are the most abundant glycolipids found in the cell wall of Mycobacterium tuberculosis (M. tb). TDMs play an important role in the pathogenesis of M. tb yet the only known receptor for TDM is the macrophage inducible C-type lectin (mincle). To understand more about the interaction of TDMs with immune cells, affinity based proteome profiling (AfBPP) can be used to determine receptors that bind TDMs. To this end, we present the synthesis of the first AfBPP-TDM probe and report on its ability to activate macrophages. By doing so, we establish that the AfBPP-TDM probe appears to be a suitable substrate for future proteomic profiling experiments.

Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species

Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.

Concise Synthesis of a Probe Molecule Enabling Analysis and Imaging of Vizantin

Trehalose 6,6'-dicorynomycolate (TDCM) was first characterized in 1963 as a cell surface glycolipid of Corynebacterium spp. by Ioneda and co-workers. TDCM shows potent anti-tumor activity due to its immunoadjuvant properties. Furthermore, the toxicity of TDCM in mice is much weaker than the related trehalose diester of mycolic acid; trehalose 6,6'-dimycolate (TDM, formerly known as cord factor). We have investigated the chemical modification of this class of compound to generate novel agents that display increased immunoadjuvant activity with minimal associated toxicity. During the course of this work we recently developed 6,6'-bis-O-(3-nonyldodecanoyl)-α,α'-trehalose (designated as vizantin). Our results show that vizantin exhibited a potent prophylactic effect on experimental lung metastasis of B16-F0 melanoma cells without a loss of body weight and death in mice. Furthermore, vizantin effectively stimulated human macrophages in an in vitro model, making it a promising candidate for a safe adjuvant in clinical applications. In order to elucidate the pharmacokinetics of vizantin, a probe molecule with similar activity was developed on the basis of a structure-activity relationship (SAR) study with vizantin. The distribution of the probe molecule after intravenous administration into a mouse was assessed by macro confocal microscopy, where it was found to accumulate in the lungs and liver.

Cord Factor and Peptidoglycan Recapitulate the Th17-Promoting Adjuvant Activity of Mycobacteria through Mincle/CARD9 Signaling and the Inflammasome

Cord Factor and Peptidoglycan Recapitulate the Th17-Promoting Adjuvant Activity of Mycobacteria through Mincle/CARD9 Signaling and the Inflammasome

Psoriasis cured in biological dietary therapy with a lymphopoietic Ti-lipid spinal cord factor simulating anti-angiogenesis, also analyzed with microRNA profiling

Background. Psoriasis is a chronic disease with genetic predisposition lacking curative treatments. It is triggered by stress and depression. Objective. With cancer patients in Bio-Immunotherapy ingestion of a lymphopoietic central nervous system (CNS) titanium-lipid in synergy with certain L-amino acids and trace element ions were surprisingly also found to cure psoriasis. This study was aimed to empirically delineate these necessary synergistic metabolic functional food components. Materials and Methods. The function of various dietary natural functional-food ingredients were analyzed in several patients over decades. The effect of specific natural food supplements have been characterized centered on alimentary nontoxic nutritional factors aimed to compensate this genetic, metabolic and immunological deficiency disease. Different forms of psoriasis, from mild, to cases with nail deformities and polyarthritis were also analyzed by epigenetic microRNA profiling for changes by the Agilent microarray system. Ready-made powders containing the functional food components were prepared to improve patient compliance. The prize is only ≈ 3€/day. Continuous ingestion of Tin chloride (Sn) in mgs, Isoleusine (Ile) in grams was included in powder No.1, while No.2 contained the lymphopoietic CNS-lipids equivalent to ≈ 50g of brain CNS-titanium extracted from carnivorous animals (piglets). These powders were complied to compensate the etiological metabolic deficiency. Results. Continuous ingestion of these powders gave positive clinical results in all forms of psoriasis in some months. The minimal dose required to prevent recurrent disease is now sought. In miRNA profiling three miRNAs (miR-218, miR-410, and miR-495) were down-regulated in psoriatic patients, but not in controls. No dysregulated miRNA was related to the treatment. Individual curative dose-levels varied. The only side-effect was intense itching if the lymphopoietic oral supplementation was too low or excessively high. Endogenous production of this Ti-vitamin in the patient could also biologically be activated. Thin needle biopsies were taken from active skin lesions. The redness of the skin deformities was due to excessive erythrocyte infiltration. The treatment decreased rapidly the erythrocyte content in these skin lesions. Curtailed capillary permeability resulted rapidly in a visual change and the skin lesions became pale. The treatment triggered a rapid anti-angiogenetic reaction. This paleness was followed by healing of all skin lesions. Stress could cause recurrent disease, as well as excessive alcohol ingestion. The white cell (B-Lymph-T cell) distribution was different in patients suffering from sterile inflammatory reactions as compared to patients who suffered from infectious Bowel disease. The immunereactivity was clearly different in patients who suffered from auto-inflammatory as compared with patients who suffered from an infectious Bowel disease. Sterile inflammatory reactions show a different B-Lymph-T profile as caused by standard infectious reactions. The immune-reaction has a different pattern if the inflammatory reaction is not caused by bacteria or viruses, while the reactivity is clearly different if the inflammatory response is caused by infectious agents. Biological dietary treatment of psoriasis, and sterile inflammatory diatheses 491 Conclusions. These inexpensive bio-modulating factors cured ≈ 90 % of patients in follow-up over several years. The lymphopoietic CNS titanium lipid is an essential functional food item required to normalize the immune-response in psoriatic patients as well as in other sterile inflammatory diatheses. Increased serum L-amino-acid levels may affect epigenetic ribosomal transcription and the phylogenetic expression. The necessary triggering components activating a cure seem to be different and regulate the pathophysiologic expression. Further cooperative trials are required to characterize the triggering factors e.g. for Crohn s disease and ulcerative colitis.

New Perspectives on Ancient Granulomas

EDITORIAL article Front. Immunol., 15 November 2013Sec. Inflammation Volume 4 - 2013 | https://doi.org/10.3389/fimmu.2013.00345

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

Trehalose 6,6'-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-μm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis.

Diversion of phagosome trafficking by pathogenic R hodococcus equi depends on mycolic acid chain length

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

English

In this study, a sandwich enzyme linked immunosorbent assay (ELISA) was developed to measure the concentration of cord factor (CF) antigen of M. tuberculosis in the pleural fluids of 42 patients with tuberculous pleural effusion (TPE). The assay was also simultaneously performed in 43 pleural fluid specimens with malignant pleural effusion (MPE). The assay detected CF antigen in 83.3% in patients with TPE effusion in whom M. tuberculosis was not demonstrated in pleural fluid specimens by bacteriological methods. The data also indicated that cord factor antigen was not present in significant titres in any one of the 43 pleural fluids of patients with MPE. It is concluded that estimation of cord factor of M. tuberculosis in pleural fluid specimens by the sandwich ELISA as developed in this study will support the diagnosis of tuberculous aetiology in pleural fluids, particularly in those patients in whom M. tuberculosis was not demonstrated by bacteriological methods.   Key words: Pulmonary tuberculosis, tuberculous pleural effusion (TPE), Mycobacterium tuberculosis, cord factor (CF) antigen, trehalose 6,6' dimycolate, enzyme-linked-immunosorbent assay (ELISA), malignant pleural effusion (MPE) specificity.

Cord factors from atypical mycobacteria (Mycobacterium alvei, Mycobacterium brumae) stimulate the secretion of some pro-inflammatory cytokines of relevance in tuberculosis

The ability to induce several cytokines relevant to tuberculosis (TNF-α, IL-1β, IL-6, IL-12p40 and IL-23) by cord factor (trehalose dimycolate) from Mycobacterium alvei CR-21(T) and Mycobacterium brumae CR-270(T) was studied in the cell lines RAW 264.7 and THP-1, and compared to the ability of cord factor from Mycobacterium tuberculosis H37Rv, where this glycolipid appears to be implicated in the pathogenesis of tuberculosis. Details of the fine structure of these molecules were obtained by NMR and MS. The mycoloyl residues were identified as α and (ω-1)-methoxy in M. alvei CR-21(T) and α in M. brumae CR-270(T); in both cases they were di-unsaturated instead of cyclopropanated as found in M. tuberculosis. In RAW 264.7 cells, cord factors from M. alvei CR-21(T), M. brumae CR-270(T) and M. tuberculosis differed in their ability to stimulate IL-6, the higher levels corresponding to the cord factor from M. tuberculosis. In THP-1 cells, a similar overall profile of cytokines was found for M. alvei CR-21(T) and M. brumae CR-270(T), with high proportions of IL-1β and TNF-α, and different from M. tuberculosis, where IL-6 and IL-12p40 prevailed. The data obtained indicate that cord factors from the atypical mycobacteria M. alvei CR-21(T) and M. brumae CR-270(T) stimulated the secretion of several pro-inflammatory cytokines, although there were some differences with those of M. tuberculosis H37Rv. This finding seems to be due to their particular mycoloyl substituents and could be of interest when considering the potential adjuvanticity of these molecules.

Pulmonary TCR γδ T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6′-dimycolate (TDM) isolated from Mycobacterium tuberculosis

We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6′-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.

The mycobacterial cord factor adjuvant analogue trehalose-6,6′-dibehenate (TDB) activates the Nlrp3 inflammasome

The success of a vaccine consists in the induction of an innate immune response and subsequent activation of the adaptive immune system. Because antigens are usually not immunogenic, the addition of adjuvants that activate innate immunity is required. The mycobacterial cord factor trehalose-6,6′-dimycolate (TDM) and its synthetic adjuvant analogue trehalose-6,6′-dibehenate (TDB) rely on the C-type lectin Mincle and the signaling molecules Syk and Card9 to trigger innate immunity. In this study, we show that stimulation of bone marrow-derived dendritic cells (BMDCs) with TDB induces Nlrp3 inflammasome-dependent IL-1β secretion. While Card9 is required for NF-κB activation by TDB, it is dispensable for TDB-induced activation of the Nlrp3 inflammasome. Additionally, efflux of intracellular potassium, lysosomal rupture, and oxygen radical (ROS) production are crucial for caspase-1 processing and IL-1β secretion by TDB. In an in vivo inflammation model, we demonstrate that the recruitment of neutrophils by TDB is significantly reduced in the Nlrp3-deficient mice compared to the wild-type mice, while the production of chemokines in vitro is not influenced by the absence of Nlrp3. These results identify the Nlrp3 inflammasome as an essential mediator for the induction of an innate immune response triggered by TDB.

Nocardia brasiliensis Cell Wall Lipids Modulate Macrophage and Dendritic Responses That Favor Development of Experimental Actinomycetoma in BALB/c Mice

Nocardia brasiliensis is a Gram-positive facultative intracellular bacterium frequently isolated from human actinomycetoma. However, the pathogenesis of this infection remains unknown. Here, we used a model of bacterial delipidation with benzine to investigate the role of N. brasiliensis cell wall-associated lipids in experimental actinomycetoma. Delipidation of N. brasiliensis with benzine resulted in complete abolition of actinomycetoma without affecting bacterial viability. Chemical analyses revealed that trehalose dimycolate and an unidentified hydrophobic compound were the principal compounds extracted from N. brasiliensis with benzine. By electron microscopy, the extracted lipids were found to be located in the outermost membrane layer of the N. brasiliensis cell wall. They also appeared to confer acid-fastness. In vitro, the extractable lipids from the N. brasiliensis cell wall induced the production of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and CCL-2 in macrophages. The N. brasiliensis cell wall extractable lipids inhibited important macrophage microbicidal effects, such as tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, phagocytosis, bacterial killing, and major histocompatibility complex class II (MHC-II) expression in response to gamma interferon (IFN-γ). In dendritic cells (DCs), N. brasiliensis cell wall-associated extractable lipids suppressed MHC-II, CD80, and CD40 expression while inducing tumor growth factor β (TGF-β) production. Immunization with delipidated N. brasiliensis induced partial protection preventing actinomycetoma. These findings suggest that N. brasiliensis cell wall-associated lipids are important for actinomycetoma development by inducing inflammation and modulating the responses of macrophages and DCs to N. brasiliensis.

Computational screening for new inhibitors of M. tuberculosis mycolyltransferases antigen 85 group of proteins as potential drug targets

The group of antigen 85 proteins of Mycobacterium tuberculosis is responsible for converting trehalose monomycolate to trehalose dimycolate, which contributes to cell wall stability. Here, we have used a serial enrichment approach to identify new potential inhibitors by searching the libraries of compounds using both 2D atom pair descriptors and binary fingerprints followed by molecular docking. Three different docking softwares AutoDock, GOLD, and LigandFit were used for docking calculations. In addition, we applied the criteria of selecting compounds with binding efficiency close to the starting known inhibitor and showing potential to form hydrogen bonds with the active site amino acid residues. The starting inhibitor was ethyl-3-phenoxybenzyl-butylphosphonate, which had IC50 value of 2.0 μM in mycolyltransferase inhibition assay. Our search from more than 34 million compounds from public libraries yielded 49 compounds. Subsequently, selection was restricted to compounds conforming to the Lipinski rule of five and exhibiting hydrogen bonding to any of the amino acid residues in the active site pocket of all three proteins of antigen 85A, 85B, and 85C. Finally, we selected those ligands which were ranked top in the table with other known decoys in all the docking results. The compound NIH415032 from tuberculosis antimicrobial acquisition and coordinating facility was further examined using molecular dynamics simulations for 10 ns. These results showed that the binding is stable, although some of the hydrogen bond atom pairs varied through the course of simulation. The NIH415032 has antitubercular properties with IC90 at 20 μg/ml (53.023 μM). These results will be helpful to the medicinal chemists for developing new antitubercular molecules for testing.

Mincle is not essential for controlling Mycobacterium tuberculosis infection

Individually and combined, Toll-like receptors (TLR)-2, -4, -9, nucleotide oligomerization domain (NOD) 2 and NALP3 contribute to the Mycobacterium tuberculosis (Mtb)-induced innate immune response only to a limited extent, particularly in terms of inducing antibacterial protection and granuloma formation in vivo. A singular essential sensory component of this initial response has not been discovered yet. Trehalose-6,6′-dimycolate (TDM), a well known mycobacterial cell wall glycolipid, is believed to be involved in these early inflammatory processes after Mtb infection. Only recently the macrophage inducible C-type lectin (Mincle) was demonstrated as an essential receptor for TDM. However, not much is known about the sensing capacity of Mincle during infection with live mycobacteria. To determine the significance of Mincle during tuberculosis (TB), we analyzed the outcome of Mtb infection in Mincle-deficient mice. Whereas in the absence of Mincle macrophages did not respond to TDM, Mincle-deficient mice were capable of mounting an efficient granulomatous and protective immune response after low and high dose infections with Mtb. Mutant mice generated a normal T helper (TH) 1 and TH17 immune response followed by the induction of efficient macrophage effector mechanisms and control of mycobacterial growth identical to wildtype mice. From our results we conclude that absence of the innate receptor Mincle can be fully compensated for in vivo in terms of sensing Mtb and mounting a protective inflammatory immune response.