title: The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis.
abstract: BACKGROUND
  A 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.
  
  
  RESULTS
  A 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.
  
  
  CONCLUSIONS
  The 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.
authors: Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106 93-232, Lodz, Poland.
authors: Marta
authors: M
authors: Brzezinska
authors: Izabela
authors: I
authors: Szulc
authors: Anna
authors: A
authors: Brzostek
authors: Magdalena
authors: M
authors: Klink
authors: Michal
authors: M
authors: Kielbik
authors: Zofia
authors: Z
authors: Sulowska
authors: Jakub
authors: J
authors: Pawelczyk
authors: Jaroslaw
authors: J
authors: Dziadek
mesh_terms: D002460:Cell Line; D015169:Colony Count, Microbial; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D009569:Nitric Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000172:deficiency / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D037521:Virulence Factors; D016753:Interleukin-10; D009569:Nitric Oxide; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase