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 O 2 · ؊ 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 O 2 · ؊ , 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 O 2 · ؊ -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.T uberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, remains one of the most important public health problems, particularly in low-and middle-income countries. Current treatment for TB includes administration of first-line drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for 2 months, followed by RIF and INH for 4 months. While molecular targets of these drugs are known (1), less information has been reported on the cellular responses triggered after target interaction. In Gram-negative and -positive bacteria, it is known that bactericidal antibiotics induced a mechanism of cellular death stimulating the production of reactive oxygen species (ROS) (2-5). The most common ROS include superoxide anion (O 2 · Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH) (6). Using the dye hydroxyphenyl fluorescein (HPF) and flow cytometric enumeration, it was found that aminoglycosides, quinolones, and beta-lactams induced ·OH in Escherichia coli and Staphylococcus aureus through pathways involving alterations in central and iron metabolism driving the Fenton reaction (2). This reaction leads to formation of ·OH through the reduction of H 2 O 2 by ferrous iron and causes damage to DNA, proteins, and lipids that ultimately results in the death of the bacterial cell. ...

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

“…Gram-negative and Gram-positive bacteria that were exposed to lethal concentrations of antibiotics have been found to have a high degree of oxidative stress due to elevated levels of ROS (68,(93)(94)(95)(96)(97). M. tuberculosis, which was exposed to 8 g/ml of RIF but only up to 3 days (pertaining only to the early part of killing phase) in vitro, showed generation of hydroxyl radical, the levels of which increased with the increase in RIF concentration (98). However, our study involving prolonged and continued exposure to RIF showed that the levels of hydroxyl radical generation were steadily increasing during the killing phase, reaching significantly high levels in the RIF persistence phase.…”

“…The powdered cell lysate was resuspended in 200 l of 100 mM sodium acetate (pH 5.2) and centrifuged at 12,000 ϫ g for 5 min at room temperature. The supernatant (180 l) was then mixed with 20 l of 1 M DMPO to achieve a 100 mM final concentration (98). Samples were immediately loaded into an aqueous flat cell (ES-LC12), and a reading was taken in a JEOL JES-X3 ESR spectrometer exactly 2 min after the addition of DMPO using the following parameters: X-band frequency, 9428.401 (MHz); power, 4.00 (mW), field center, 337.275 (mT); sweep time, 2.0 (min).…”

The evaluation of the anti‐cancer drug elesclomol that forms a redox‐active copper chelate as a potential anti‐tubercular drug

“…In Mtb , RIF inhibits the beta subunit of RNA polymerase (RpoB) encoded by the rpoB gene . The cellular responses to RIF involve generation of ROS, mainly the hydroxyl radical . As elesclomol through its copper chelate also generates oxidative stress, this may explain the observed synergism.…”

Redox‐guided small molecule antimycobacterials

“…After binding to its target, RIF produces superoxide radical anion, which further mediates metal‐dependent generation of hydroxyl radical, confirmed by electron paramagnetic resonance (EPR). This is in‐part, responsible for its antimycobacterial activity . Thus, this illustrates the potential for design and development of redox‐based drugs against TB.…”