title: Cholesterol oxidase is indispensable in the pathogenesis of Mycobacterium tuberculosis.
abstract: Despite considerable research effort, the molecular mechanisms of Mycobacterium tuberculosis (Mtb) virulence remain unclear. Cholesterol oxidase (ChoD), an extracellular enzyme capable of converting cholesterol to its 3-keto-4-ene derivative, cholestenone, has been proposed to play a role in the virulence of Mtb. Here, we verified the hypothesis that ChoD is capable of modifying the bactericidal and pro-inflammatory activity of human macrophages. We also sought to determine the contribution of complement receptor 3 (CR3)- and Toll-like receptor 2 (TLR2)-mediated signaling pathways in the development of macrophage responses to Mtb. We found that intracellular replication of an Mtb mutant lacking a functional choD gene (ΔchoD) was less efficient in macrophages than that of the wild-type strain. Blocking CR3 and TLR2 with monoclonal antibodies enhanced survival of ΔchoD inside macrophages. We also showed that, in contrast to wild-type Mtb, the ΔchoD strain induced nitric oxide production in macrophages, an action that depended on the TLR2, but not the CR3, signaling pathway. Both wild-type and mutant strains inhibited the production of reactive oxygen species (ROS), but the ΔchoD strain did so to a significantly lesser extent. Blocking TLR2-mediated signaling abolished the inhibitory effect of wild-type Mtb on ROS production by macrophages. Wild-type Mtb, but not the ΔchoD strain, decreased phorbol myristate acetate-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are involved in both TLR2- and CR3-mediated signaling pathways. Our finding also revealed that the production of interleukin 10 by macrophages was significantly lower in ΔchoD-infected macrophages than in wild-type Mtb-infected macrophages. However, tumor necrosis factor-α production by macrophages was the same after infection with mutant or wild-type strains. In summary, we demonstrate here that ChoD is required for Mtb interference with the TLR2-mediated signaling pathway and subsequent intracellular growth and survival of the pathogen in human macrophages.
authors.affiliation: Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Klink
authors.forename: Marta
authors.initials: M
authors.lastname: Brzezinska
authors.forename: Izabela
authors.initials: I
authors.lastname: Szulc
authors.forename: Anna
authors.initials: A
authors.lastname: Brzostek
authors.forename: Michal
authors.initials: M
authors.lastname: Kielbik
authors.forename: Zofia
authors.initials: Z
authors.lastname: Sulowska
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Dziadek
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D002789:Cholesterol Oxidase / Q000235:genetics / Q000276:immunology*; D017353:Gene Deletion; D054884:Host-Pathogen Interactions; D006801:Humans; D016177:Macrophage-1 Antigen / Q000276:immunology; D008264:Macrophages / Q000276:immunology / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000472:pathogenicity* / Q000502:physiology*; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000276:immunology; D014376:Tuberculosis / Q000201:enzymology / Q000276:immunology / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D016177:Macrophage-1 Antigen; D051195:Toll-Like Receptor 2; D002789:Cholesterol Oxidase
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