Mycobacterium tuberculosis whiB3 and Lipid Metabolism Genes Are Regulated by Host Induced Oxidative Stress

Abstract

The physiological state of the human macrophage may impact the metabolism and the persistence of Mycobacterium tuberculosis. This pathogen senses and counters the levels of O₂, CO, reactive oxygen species (ROS), and pH in macrophages. M. tuberculosis responds to oxidative stress through WhiB3. The goal was to determine the effect of NADPH oxidase (NOX) modulation and oxidative agents on the expression of whiB3 and genes involved in lipid metabolism (lip-Y, Icl-1, and tgs-1) in intracellular mycobacteria. Human macrophages were first treated with NOX modulators such as DPI (ROS inhibitor) and PMA (ROS activator), or with oxidative agents (H₂O₂ and generator system O₂^•-), and then infected with mycobacteria. We determined ROS production, cell viability, and expression of whiB3, as well as genes involved in lipid metabolism. PMA, H₂O₂, and O₂^•- increased ROS production in human macrophages, generating oxidative stress in bacteria and augmented the gene expression of whiB3, lip-Y, Icl-1, and tgs-1. Our results suggest that ROS production in macrophages induces oxidative stress in intracellular bacteria inducing whiB3 expression. This factor may activate the synthesis of reserve lipids produced to survive in the latency state, which allows its persistence for long periods within the host.

Keywords: Mycobacterium tuberculosis; WhiB3; lipid metabolism; oxidative stress.

Figure 1

whib3 expression of M. tuberculosis in intracellular bacteria in MDM and AM compared to aerobic in vitro 7H9 growth during mid-exponential growth phase (control, dashed line). Total RNA was isolated and real-time qRT-PCR was performed. Relative expression levels of whiB3 were estimated using the RNA 16S transcript as internal control for normalization of RNA amounts; expression levels from an in vitro culture were used as control. MDM: Monocyte-derived macrophages and AM: alveolar macrophages. The data show the maximums and minimums by quartiles. * p < 0.05 significant difference compared to the expression of control (bacteria cultivated in vitro), n = 6. The dashed horizontal line represents the median, and the Wilcoxon test was used to compare against the control.

Figure 2

Effect of NOX activity modulation on gene expression of intracellular mycobacteria. The macrophages were treated with PMA (750 ng/mL) or DPI (20 µM) for 1 h. Subsequently, they were infected with M. tuberculosis (MOI 1:15) in the presence of PMA or DPI and incubated for another hour. The non-phagocytized bacteria were eliminated and fresh medium with PMA or DPI was added for 2 more hours. We detected cell viability (A) and ROS levels (C) in MDM. Furthermore, expression levels of whiB3 and sodA (B,D) were determined in intracellular bacteria. The data show the maximums and minimums by quartiles. * p < 0.05 significant difference compared to the expression of uninfected macrophages (A,C) or intracellular bacteria without treatment (B,D), n = 4–7. The Wilcoxon test was used to compare to the control. In panels (A,C), the dashed horizontal line represents the median of the uninfected macrophages without treatment, and panels (B,D), represents the median of the intracellular bacteria without treatment.

Figure 3

Effect of oxidizing agents on whiB3 and sodA gene expression of intracellular mycobacteria. The MDM were treated with 5 mM H₂O₂ and O₂^•− generator system for 15 min, and then infected with M. tuberculosis H37Ra (MOI 1:15) in the presence of these oxidants for 1 h. Non-phagocytized bacteria were removed, and fresh medium plus oxidants was added for 2 h. We detected cell viability (A) and ROS levels (C) in macrophages. Furthermore, the RNA expression levels of whiB3 and sodA (B,D) were determined in intracellular bacteria. The data show the maximums and minimums by quartiles. * p < 0.05 significant difference compared to the expression of uninfected macrophages (A,C) or intracellular bacteria without treatment (B,D), n = 4–8. The Wilcoxon test was used to compare to the control. In panels (A,C), the dashed horizontal line represents the median of the uninfected macrophages without treatment, and in panels (B,C), it represents the median of the intracellular bacteria without treatment.

Figure 4

Expression of the lip-Y, tgs-1 and icl-1 in intracellular mycobacteria under oxidant conditions. The MDM were treated with NOX modulators (DPI and PMA) for 1 h or oxidizing agents (H₂O₂ and O₂^•−) for 15 min, and then infected with M. tuberculosis H37Ra (MOI 1:15) in presence of these oxidants for 1 h. Non-phagocyted bacteria were removed, and fresh medium containing NOX modulators or oxidants were added for 2 h. Then, we determined the expression of lip-Y (A,B), tgs-1 (C,D) and icl-1 (E,F). The data show the maximums and minimums by quartiles. * p < 0.05 significant difference compared to intracellular bacteria without treatment (control), n = 7–8. The dashed horizontal line represents the median of the control, and the Wilcoxon test was used to compare against the control (without treatment).

Figure 5

Effect of oxidizing agents on bacterial survival and gene expression of M. tuberculosis. Bacteria were grown in the presence of 5 mM H₂O₂ and O₂^•− generator system for 2 h. Then, we determined the survival (A) and the expression of sodA (B), whiB3 (C), lipY (D), tgs-1 (E) and icl-1 (F). The data show the maximums and minimums by quartiles. * p < 0.05 significant difference compared to the expression of the control, n = 4. The dashed horizontal line represents the median of the control, and the Wilcoxon test was used to compare against the control (untreated mycobacterial culture).

Figure 6

Effect of oxidants on gene expression of free-living mycobacteria cultured in 7H9 (1) and intracellular mycobacteria (2). The superoxide anion (O₂^●−) is converted into hydrogen peroxide (H₂O₂) (3). Both oxidants damage the cell wall and membrane, generating lipoperoxidation (4). H₂O₂ is an electrically neutral molecule that crosses membranes (5), increasing its concentration inside the cell causing an imbalance in the redox state. The MosR regulator senses oxidative stress and induces whiB3 expression (6). Oxidative conditions favor the [4Fe-4S] cluster oxidation of WhiB3 (7). [4Fe-4S]ox-WhiB3 can bind to promoters of its target genes (8), generating increased expression of lip-Y, tgs-1, and icl-1, causing a metabolic change that leads to a state of dormancy.