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. 2011 Mar 25;286(12):10276-87.
doi: 10.1074/jbc.M110.200501. Epub 2010 Dec 30.

Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species

Takahiro Yano  1 , Sacha Kassovska-Bratinova, J Shin Teh, Jeffrey Winkler, Kevin Sullivan, Andre Isaacs, Norman M Schechter, Harvey Rubin
Affiliations

Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species

Takahiro Yano et al. J Biol Chem. .

Abstract

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O(2) yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.

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Figures

FIGURE 1.
FIGURE 1.
Structures of clofazimine and a new, more soluble analog, KS6.
FIGURE 2.
FIGURE 2.
Effect of CFZ on NADH oxidation catalyzed by isolated M. smegmatis and E. coli membranes. a, NADH oxidation catalyzed by M. smegmatis membranes (Mem) was monitored at 340 nm in a cuvette. Reactions were performed in total volume of 250 μl containing 10.0 μg membrane protein, 250 μm NADH with 20 mm KCN and CFZ added as indicated. Breaks in the trace indicate additions to the reaction; the amounts of CFZ reported are cumulative values. Dashed line indicates “control” activity measured in the absence of KCN and CFZ. The sharp absorbance increases at ≥4.7 μg/ml CFZ are due in part to CFZ absorbance and possibly to light scattering upon saturation of membranes with CFZ. b, NADH oxidation by E. coli membranes was measured similarly. Reaction conditions were the same, except membrane protein added was 4.0 μg membrane protein and KCN concentration was 5.0 mm.
FIGURE 3.
FIGURE 3.
Ability of CFZ (a) or KS6 (b) to restore NADH oxidase activity in isolated M. smegmatis membrane preparations treated with KCN. a, NADH oxidase activity as a function of the amount of CFZ added to reactions was determined from time courses performed as in Fig. 2. Oxidation rates were determined from the slopes of the lines associated with the various additions. Fraction activity restored is the ratio of the NADH oxidation rates produced by CFZ addition to membrane preparations after KCN addition as in Fig. 2, divided by the NADH oxidation rate before the addition of KCN. All rates were measured as ΔA340 nm/min. The rate at 0.0 CFZ is the fractional activity remaining after KCN addition. Sets 1 and 2 refer to membrane preparations in two time-separated studies (approximately 1 year apart). The amount of membrane added to reactions was standardized to have an activity in the range of 0.1 ΔA/min at 340 nm; the measured control NADH oxidase rates averaged 0.130 ± 0.5 (n = 13) for log membranes and 0.114 ± 0.04 (n = 15) for stationary membranes. Standardized to protein, stationary phase membranes had less activity than log phase membrane; thus, to achieve similar activities, log phase reactions contained an average of 3.8 ± 1.6 μg membrane protein, and stationary phase reactions contained an average of 13.0 ± 5.5 μg membrane protein. b, experimental conditions were similar to those in a, except that KS6 was added to reactions instead of CFZ. Data are the average and S.D. of three measurements. In the case of set 2 membranes, membrane preparations from the three separate log phase growths were each assayed once, and the results were averaged, and membrane preparations from the three stationary phase growths were each assayed once, and the results were averaged. In the case of set 1, only stationary stage membranes were assayed, two different membrane preparations were assayed separately, and the results were averaged; the data reported are the average values, and the error bars are the deviation from the average. The mass of KS6 is greater than that of CFZ (562 versus 473 Da), which accounts for the difference in x axis values in the two panels.
FIGURE 4.
FIGURE 4.
TPZ inhibition of M. smegmatis NADH oxidase activity in the absence and presence of KCN/CFZ. Reactions, 250 μl total volume, contained 0.1 m HEPES pH 7.0, 225 μm NADH, and M. smegmatis membranes (10 μg membrane protein) from a preparation highly reactive with CFZ (see Fig. 2). NADH oxidase rates (loss/min of absorbance at 340 nm) were measured from time-courses as shown in Fig. 1. TPZ was added to reactions without (open circles) or with KCN/CFZ (20 mm/0.5 μg, filled circles). Fractional activity is the rate of absorbance loss in presence of TPZ relative to the control without TPZ. Line fit through the data were obtained using the Hill equation with an exponent of 3.0.
FIGURE 5.
FIGURE 5.
Reduction and re-oxidation of CFZ during a reaction with KCN-treated M. smegmatis membranes. Spectra were obtained from a single reaction in a total volume of 500 μl monitored over time; background absorbance due to membranes was subtracted. Membranes (Memb, 80 μg membrane protein) from a highly CFZ-responsive preparation were preincubated with 10 mm KCN and 4.8 μg of CFZ; the spectrum did not change with time (thick line). NADH (225 μm) was then added, and the spectrum was recorded immediately (+ NADH). Additional spectra were then recorded at ∼1.0-min intervals. Absorbance in the region of 350–400 nm is from NADH and that from 400–550 nm is from CFZ. Spectra depicted with a solid line show change in CFZ (reduction phase) correlating with decrease in NADH absorbance, and those depicted with a dotted line show CFZ absorbance change (reoxidation phase) after virtually complete oxidation of NADH.
FIGURE 6.
FIGURE 6.
Characterization of CFZ reduction and reoxidation in reactions with KCN-treated M. smegmatis membranes. a, time course showing reduction (absorbance decrease) and spontaneous oxidation of CFZ. Reaction conditions were similar to those in Fig. 5, except that absorbance was monitored continuously at a single wavelength of 470 nm. Breaks in the trace are due to raising/closing the door of the spectrophotometer to make additions. The order of addition to the 250-μl reaction were 1) membranes (40 μg membrane protein) and 2.4 μg CFZ, 2) 20 mm KCN, 3) 225 μm NADH, 4) second addition of 225 μm NADH after return of absorbance, and 5) manual agitation of the reaction by vigorous shaking of the cuvette. b, the reactions was performed similar to a, except that the NADH and CFZ concentrations were increased. The order of addition to the 250 μl reaction were 1) membranes (Memb, 85 μg membrane protein) and 6.0 μg CFZ, 2) 20 mm KCN, 3) 500 μm NADH, and 4) agitation of the cuvette. c, same as b, except for the addition of SDS during the slow phase of absorbance loss. Additions to reaction were 1) membranes, KCN, and CFZ as in b, 2) 225 μm NADH, 3) SDS (0.4% final) with agitation, and 4) second addition of NADH.
FIGURE 7.
FIGURE 7.
ROS production catalyzed by M. smegmatis log-phase membranes in the absence and presence of CFZ. a, demonstration of CFZ-mediated ROS production. NADH oxidation and ROS production were measured in separate reactions following absorbance at either 340 nm (right axis, dashed line) or 563 nm (left axis, solid lines), respectively. Reactions were preformed in 1.0 ml containing 20 μg membrane protein, either NADH (0.2 mm) or succinate (1.0 mm) as electron donors, CFZ (0.5 μg) or TPZ (0.2 mm), and ROS detection reagents as described under “Experimental Procedures.” b, dependence of ROS production on the amount of CFZ. This study was performed in 50 μl total volume using a plate reader (Tecan Corp.). The reaction contained 1.0 μg of log-phase membranes and different amounts of CFZ as indicated in the panel. Parallel reactions were monitored at 340 nm and 563 nm. At each concentration, initial velocities for the rate of NADH consumption and resorufin production were used to calculate flux of electrons producing resorufin relative to NADH oxidation rate (y axis) as described under “Experimental Procedures.”
FIGURE 8.
FIGURE 8.
Reduction of CFZ (a) and ROS production (b) catalyzed by purified recombinant M. tuberculosis NDH-2. a, spectra showing reduction of CFZ by NDH-2 under anaerobic conditions and subsequent reoxidation. Stock CFZ in Me2SO was added to a solution of 50 mm MOPS/Na+, pH 6.5, 2.0 mm MgCl2 to produce a final concentration of 1.18 μg/ml. Based on spectra taken during a 40-min period (CFZ in buffer), ∼70% of the CFZ remained in solution exhibiting a spectrum consistent with cationic CFZ. NADH (200 μm) was then added; it did not produce a significant change during a 10-min incubation period (+ NADH). Purified NDH-2 (2.7 μg/ml, 0.01% Big CHAPS at final) was then added to start the reaction (1 min), and additional spectra were taken at the indicated times. After the absorbance loss was complete, the cuvette was exposed to air for 20 min after which a spectrum was recorded (air-oxidized). b, production of ROS by purified NDH-2. NADH oxidation (340 nm absorbance decrease) and ROS generation (absorbance increase, 563 nm) catalyzed by NDH-2 was measured under aerobic conditions similar to the reactions in Fig. 7. Reactions were performed in a total volume of 1.0 ml and contained 200 μm NADH with an ROS detection system. At the indicated times, NDH-2 was added to final concentration of 2.0 μg/ml, followed by CFZ to a final of 1.0 μg/ml or TPZ to a final of 200 μm.
FIGURE 9.
FIGURE 9.
CFZ-mediated (a) and KS6-mediated (b) restoration of NADH oxidation by membranes isolated from nonmycobacterial organisms. Measurements and data reported were made as described in Figs. 2 and 3. Bacterial membranes in the assays had a starting NADH oxidase activity of ∼0.1 ΔA/min at 340 nm/min. The mitochondrial preparations had an activity of 0.05 ΔA/min at 340 nm.
FIGURE 10.
FIGURE 10.
Inhibition of M. smegmatis growth by CFZ and INH and effect of antioxidants. a, effect of CFZ on colony counts of wild type M. smegmatis mc2 155 plated on 7H9 agar. Data at each concentration represent the average colony number on three plates ± S.D. b, effect of free radical scavengers on CFZ inhibition of M. smegmatis growth. M. smegmatis was grown on agar plates containing various antioxidants (5.0 mm 4-hydroxy-TEMPO, 5.0 mm N-acetylcysteine (NAC), and 12.5 μg/ml α-tocopherol) with or without 0.5 μg/ml CFZ or 10 μg/ml INH. Representative plates demonstrating bacterial growth under the various conditions are shown.
FIGURE 11.
FIGURE 11.
Depiction of CFZ-mediated redox cycling and ROS production. Diagram depicting menaquinone (MQ) of the respiratory chain and CFZ as competing substrates of NDH-2. The electron transport chain (ETC) in M. smegmatis is primarily composed of two oxidoreductases in addition to NDH-2: cytochrome bc1 complex, which is reduced by menaquinol, and cytochrome aa3, which obtains electrons from cytochrome bc1 and transfers them to O2 in a coupled reaction that produces water and the translocation of protons from the cytoplasm to periplasmic space. Oxidation of reduced CFZ by oxygen occurs nonenzymatically and produces ROS. The cell wall of M. smegmatis is much thicker than depicted in the diagram.
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