title: Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.
abstract: The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.
authors.affiliation: Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Piccaro
authors.affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Pietraforte
authors.affiliation: Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Giannoni
authors.affiliation: Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mustazzolu
authors.affiliation: Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy lanfranco.fattorini@iss.it.
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Fattorini
mesh_terms: D000995:Antitubercular Agents / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002374:Catalase / Q000737:chemistry; D003497:Cyclic N-Oxides; D012321:DNA-Directed RNA Polymerases; D004578:Electron Spin Resonance Spectroscopy; D017665:Hydroxyl Radical / Q000737:chemistry / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D004369:Pentetic Acid / Q000737:chemistry; D011485:Protein Binding; D012293:Rifampin / Q000378:metabolism / Q000494:pharmacology*; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000737:chemistry; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism*; D013890:Thiourea / Q000737:chemistry
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003497:Cyclic N-Oxides; D013113:Spin Labels; C505805:rpoB protein, Mycobacterium tuberculosis; D013481:Superoxides; D017665:Hydroxyl Radical; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D004369:Pentetic Acid; D002374:Catalase; D013482:Superoxide Dismutase; D012321:DNA-Directed RNA Polymerases; D013890:Thiourea; D012293:Rifampin