Microbiology and Infectious Disease

Oxidative stress drives potent bactericidal activity of pyrazinamide against Mycobacterium tuberculosis

Nicholas A Dillon
Elise A Lamont
Muzafar A Rather
Anthony D Baughn author has email address

Department of Biological Sciences, University of Texas at Dallas, Richardson, United States
Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, United States

https://doi.org/10.7554/eLife.105614.1

Open access
Copyright information

Figures and data

PZA treatment synergizes with H₂O₂ to kill M. tuberculosis.
Bactericidal activity of ROS generators was examined after exposure to 200 µg/mL PZA for (A) H₂O₂, (B) clofazimine and (C) menadione. Data shown represent the mean and standard deviation from biological triplicates. Statistical significance was calculated with a multiple comparisons two-way ANOVA, *P≤0.05, **P≤0.01.

Antimycobacterial action of PZA is potentiated by oxidative stress.
Synergy between PZA and oxidizing agents H₂O₂ and diamide was assessed against M. tuberculosis H37Ra (A), M. tuberculosis H37Rv (B, E, F), and M. bovis BCG with and without a functional copy of pncA (C, D). Cultures were exposed to PZA for 72 hours prior to treatment with oxidizing agents. FIC plots were generated following outgrowth from challenge with H₂O₂ (A-D) or diamide (E, F) in 7H9 with ADS pH 5.8 (A-E) or 6.8 (F). Lowest measured FICI values are show within each plot. Data represent the mean of three biological replicates.

PZA treatment enhances H₂O₂ associated intracellular ROS production and oxidative damage in M. tuberculosis.
Intracellular ROS levels were measured by CellRox Green assay using mid-exponential phase M. tuberculosis H37Rv cultures which had been treated with either no PZA, 100 µg/mL PZA, or 200 µg/mL PZA for 3d at pH 6.8 prior to the addition of (A) no H₂O₂, (B) 0.5 mM H₂O₂, (C) 5 mM H₂O₂, or (D) 5 mM H₂O₂. Protein carbonylation was examined as a biological marker of oxidative damage. Carbonyl groups were derivatized with 2,4-dinitrophenylhydrazine and the molar ratio of carbonyl groups to protein was determined for (E) 200 µg/mL of BSA with 1 mM Fe²⁺, 25 mM H₂O₂ in 10 mM MES buffer (pH 5) with either no POA or 2 mM POA, or (F) mid-exponential phase M. tuberculosis H37Ra cultures which had been treated with either no drug or PZA at 200 µg/mL PZA for 3d at pH 5.8, cells were then subsequently exposed to no H₂O₂ or 5 mM H₂O₂ for 24 hrs. All experiments were conducted in triplicate. Statistical significance was calculated with a multiple comparisons two-way ANOVA, *≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

PZA activity is dependent on the production of host derived ROS.
PZA bactericidal activity in M. tuberculosis H37Rv infected (A) murine RAW 264.1 or (B) human THP-1 macrophages. Macrophages were either untreated or activated through preincubation with IFN-γ, and/or N-acetyl-L-cysteine (NAC) prior to the infection. After 2 hr of infection cells were washed, and PZA was added as indicated. Cultures were plated for CFU enumeration at the indicated timepoints. Statistical significance was calculated with a multiple comparisons two-way ANOVA, *≤0.05, **P≤0.01, ***P≤0.001.

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