Mycobacterium tuberculosisKeto-Mycolic Acid and Macrophage Nuclear Receptor TR4 Modulate Foamy Biogenesis in Granulomas: A Case of a Heterologous and Noncanonical Ligand-Receptor Pair

Abstract

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA–TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor

“…Macrophage Infection and Determination of Colony-forming Units (cfu)-As described previously, peritoneal macrophages and monocyte-derived macrophages (MDMs) were used as primary cell lines for intracellular infection (36,37), and THP-1 and RAW 264.7 macrophages were used as secondary cell lines. These cell lines were infected with either wild-type or recombinant strains of M. smegmatis expressing LprI, HbN, and LprIHbN at a multiplicity of infection of 1:5 for 2 h. For the intracellular survival assay in the presence of lysozyme, 100 g/ml HEWL was exogenously supplemented at the time of infection.…”

A human xenobiotic nuclear receptor contributes to nonresponsiveness of Mycobacterium tuberculosis to the antituberculosis drug rifampicin

“…Aerosol infection and determination of M. tuberculosis burden-Experimental mice were exposed to aerosol inhalation of M. tuberculosis H37Rv (∼100 CFU per lung, as per the standardized dose, observed after day 1 of infection) using the inhalation exposure system (Glas-Col; Terre Haute, IN) in the BSL3 facility, at IMTECH as described previously (28). Following infection, mice were divided into different treatment group (five mice per group) and were allowed to establish the infection for Bacterial viability assaysMacrophages were washed with incomplete RPMI medium after 4 h of infection with M. tuberculosis, and incubated in complete RPMI media for 48 h. After 48 h the cells were then solubilized in 100 µL of 0.06% SDS in PBS, and the bacterial suspension was serially diluted and 100 μl of each diluted sample was plated on 7H11 agar plates.…”

Human Xenobiotic Nuclear Receptor PXR AugmentsMycobacterium tuberculosisSurvival

“…PBMCs were isolated from fresh blood drawn from donors by Ficoll-Hypaque gradient centrifugation (Sigma-Aldrich). PBMCs were cultured and obtained, as described previously (21). Cells were incubated for 3 h at 37˚C and then washed with PBS to remove nonadherent cells.…”

“…hMDMs were obtained by culturing PBMCs with M-CSF (50 ng/ml) for 7 d. Plasmids pSG5-hPXR, pSG5-mouse PXR (mPXR), and CYP3A4-XREM-Luc were provided by S. Kliewer (University of Texas South Western Medical Center) and R. Kim (University of Western Ontario, London, Canada), respectively. GAL4 plasmid system was used, as described earlier (21).…”

“…Among the host factors, NRs in particular have been shown to modulate host effector functions that are imperative for clearance or survival of intracellular bacteria and parasites, including Listeria monocytogenes, Salmonella typhimurium, Leishmania donovani, and M. tuberculosis (17)(18)(19)(20)(21).…”