The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis

Abstract

BackgroundA growing body of evidence suggests that Mycobacterium tuberculosis (Mtb) uses the host’s cholesterol as a source of carbon and energy during infection. Strains defective in cholesterol transport or degradation exhibit attenuated growth in activated macrophages and diminished infectivity in animal models. The aim of this study was to evaluate intracellular replication of a cholesterol degradation-deficient Mtb mutant in human macrophages (MØ) in vitro and assess the functional responses of Mtb mutant-infected MØ.ResultsA mutant Mtb H37Rv strain containing an inactivated kstD gene (∆kstD), which encodes 3-ketosteroid 1(2)-dehydrogenase (KstD), was previously prepared using the homologous recombination-based gene-replacement technique. A control strain carrying the kstD gene complemented with an intact kstD was also previously constructed. In this study, human resting MØ were obtained after overnight differentiation of the human monocyte-macrophage cell line THP-1. Resting MØ were further activated with interferon-γ (IFN-γ). The ability of the kstD-defective Mtb mutant strain to replicate intracellularly in human MØ was evaluated using a colony-forming assay. Nitric oxide (NO) and reactive oxygen species (ROS) production by MØ infected with wild-type or ∆kstD strains was detected using Griess reagent and chemiluminescence methods, respectively. The production of tumor necrosis factor-α and interleukin-10 by MØ after infection with wild-type or mutant Mtb was examined using enzyme-linked immunosorbent assays.We found that replication of mutant Mtb was attenuated in resting MØ compared to the wild-type or complemented strains. Moreover, the mutant was unable to inhibit the NO and ROS production induced through Toll-like receptor 2 (TLR2) signaling in infected resting MØ. In contrast, mutant and wild-type Mtb behaved similarly in MØ activated with IFN-γ before and during infection.ConclusionsThe Mtb mutant ∆kstD strain, which is unable to use cholesterol as a source of carbon and energy, has a limited ability to multiply in resting MØ following infection, reflecting a failure of the ∆kstD strain to inhibit the TLR2-dependent bactericidal activity of resting MØ.

Cholesterol Oxidase Is Indispensable in the Pathogenesis of Mycobacterium tuberculosis

“…THP-1 cells were cultured in CM consisting of RPMI-1640 supplemented with 1 mM sodium pyruvate, 10% FBS, 0.05 mM 2-ME, 100 U/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in a humidified 5% CO 2 atmosphere. Monocytes were differentiated into macrophages as described previously [14] by incubating with PMA (20 ng/ml) for 24 hours (37°C/5% CO 2 ). The ability of these macrophages to adhere to plastic dishes (an indicator of monocyte differentiation to macrophages) was examined under a light microscope.…”

“…It is well known that Mtb is able to accumulate, and/or degrade cholesterol and use it as a source of carbon and energy and cholesterol utilization is an important determinant of Mtb survival in macrophages [9]–[14]. The initial step in cholesterol degradation is its oxidation and isomerization to cholestenone.…”

Statins Increase Rifampin Mycobactericidal Effect

“…Concerning the isolated effect of statins against mycobacteria inside macrophages, other events must be taken into consideration. For example, the metabolism of M. tuberculosis isolated from infected mouse lungs is stimulated by fatty acids and is unresponsive to carbohydrates (29), and the generation of M. tuberculosis mutants with knockouts of some enzymes involved in cholesterol metabolism, such as KshA, KshB, FadA5, ChoD, and KstD, leads to inefficient mouse and macrophage infection, demonstrating the pivotal relevance of this pathway to tuberculosis infection and persistence inside the host (30)(31)(32)(33).…”

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

“…In particular, cholesterol degradation appeared to be essential for the survival of M. tuberculosis in the normally adverse environment of the host macrophages (19,20). For this purpose, M. tuberculosis H37Rv contains a large gene cluster coding for enzymes catalyzing cholesterol degradation, including a gene for ⌬ 1 -KSTD (Rv3537) (19) that is important for its pathogenicity (21). Thus, these first three-dimensional structures of a ⌬ 1 -KSTD may facilitate the design of inhibitors that may be developed into efficacious drugs to combat pathogenic bacteria.…”