An oxidative environment promotes growth of Mycobacterium abscessus

Abstract

Mycobacterium abscessus infections, particularly those causing chronic lung diseases, are becoming more prevalent worldwide. M. abscessus infections are difficult to treat because of antibiotic resistance. Thus, new treatment options are urgently needed. M. abscessus is an intracellular pathogen that primarily infects macrophages and fibroblasts. Because this bacterium has only recently been identified as a separate species, very little is known about M. abscessus-host interactions and how M. abscessus growth is regulated. Oxidative stress has long been shown to inhibit the growth of bacterial organisms. However, some intracellular bacteria, such as Mycobacterium tuberculosis, grow well in oxidizing environments. In this study, we show that M. abscessus infection causes the host cell environment to become more oxidizing. Furthermore, we show that a more oxidizing environment leads to enhanced growth of M. abscessus inside macrophages. In the presence of antioxidants, MnTE-2-PyP (chemical name: manganese(II) meso-tetrakis-(N-methylpyridinium-2-yl) porphyrin) or N-acetyl-l-cysteine, M. abscessus growth is inhibited. These results lead us to postulate that antioxidants may aid in the treatment of M. abscessus infections.

Figure 1. SOD mRNA levels are altered with M. abscessus infection

Differentiated THP-1 cells were uninfected (control), infected with M. abscessus for the indicated times. Cells were harvested at the times indicated and mRNA was isolated. A. Real Time RT-PCR analysis for MnSOD mRNA levels. MnSOD mRNA was significantly elevated with infection. B. Real Time RT-PCR analysis for Cu/ZnSOD mRNA levels. Cu/ZnSOD mRNA levels were unchanged early during infection; however, at 48 hours post-infection Cu/ZnSOD levels declined (not significant). All data represent the mean ± SEM, n=4. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 2. SOD protein levels are altered with M. abscessus infection

Differentiated THP-1 cells were uninfected (control), infected with M. abscessus or infected in the presence of MnTE-2-PyP for the indicated times. Cells were harvested at the times indicated and protein was isolated. A. A representative western blot for MnSOD and Cu/ZnSOD proteins. B&C. Densitometric analysis of MnSOD and Cu/ZnSOD blots. B. MnSOD protein levels are significantly enhanced at 48 and 72 hours post-infection as compared to uninfected cells for their respective time points. C. Cu/ZnSOD protein levels were significantly reduced 72 hours post-infection. D. MnSOD protein measured by ELISA. E. Cu/ZnSOD protein levels measured by ELISA. The ELISA data was normalized to total protein levels in each sample. All western blot data were normalized to the 24 hour uninfected time point for the respective SOD protein. For a protein loading control, blots were incubated with a β-actin antibody. Western blot data represent the mean ± SEM, n=4 and ELISA data represent the mean ± SEM, n=3. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 3. SOD activity is altered with M. abscessus infection

Differentiated THP-1 cells were uninfected (control), infected with M. abscessus (M. abs) or infected in the presence of MnTE-2-PyP (M. abs + MnTE-2-PyP) for 24, 48 or 72 hours. A. A representative nitroblue tetrazolium gel. B. Densitometric analysis of MnSOD activity. MnSOD activity was significantly enhanced in infected cells as compared to uninfected cells at 24 hours post-infection. At 48 and 72 hours post-infection MnSOD activity was enhanced in infected cells as compared to control (not significant). C. Densitometric analysis of Cu/ZnSOD activity. Cu/ZnSOD activity was significantly decreased at 72 hours post-infection in M. abscessus infected cells as compared to uninfected cells. All data was made relative to the 24 hour uninfected time point for the respective SOD. For controls, media, FBS and M. abscessus were analyzed for SOD activity. All data represent the mean ± SEM, n=4. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 4. Catalase activity is decreased with M. abscessus infection

Differentiated THP-1 cells were uninfected (control), infected with M. abscessus (M. abs) or infected in the presence of MnTE-2-PyP (M. abs + MnTE-2-PyP) for 0, 24, 48 or 72 hours. A. A representative catalase activity gel. B. Densitometric analysis of catalase activity. Catalase activity was significantly reduced in infected cells as compared to non-infected cells at 72 hours post-infection. Catalase activity was decreased with infection at 24 and 48 hours post-infection (not significant). MnTE-2-PyP did not affect catalase activity. All data represent the mean ± SEM, n=4. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 5. Glutathione peroxidase (GPx) activity is decreased with M. abscessus infection

Differentiated THP-1 cells were uninfected, infected with M. abscessus or infected in the presence of MnTE-2-PyP for 0, 24, 48 or 72 hours. GPx activity was significantly decreased in cells infected with M. abscessus alone when compared to uninfected cells or cells infected in the presence of MnTE-2-PyP at 48 and 72 hours post-infection. Each experiment was normalized to the control 0 time point. All data represent the mean ± SEM, n=5. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 6. Glutathione (GSH) levels are decreased with M. abscessus infection

Differentiated THP-1 cells were uninfected or infected with M. abscessus for 0, 5, 30, 60, 120 or 240 minutes post-infection. GSH was measured using a colorimetric assay and was decreased by infection with M. abscessus. All data represent the mean ± SEM, n=5. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 7. An oxidative environment promotes the intracellular viability of M. abscessus

A. Hydrogen peroxide (H₂O₂) significantly enhanced M. abscessus growth at day 8 post-infection. B. PMA enhanced growth of M. abscessus at days 4 and 8 post-infection; however this increase was not statistically significant. All data represent the mean ± SEM, n=3. The asterisk (*) denotes significant change from the respective control (p< 0.05).

Figure 8. N-acetyl-L-cysteine (NAC) inhibits intracellular growth of M. abscessus

Differentiated THP-1 cells were pre-incubated for one hour in RPMI medium or medium containing NAC (5, 10, 15 mM) and infected with M. abscessus for one hour (MOI=10). Cells were harvested one hour post-infection (d0), 4 and 8 days post-infection. Cells incubated with NAC (10 and 15 mM) had significantly reduced bacterial numbers on days 4 and 8 post-infection. All data represent the mean ± SEM, n=5. The asterisk (*) denotes significant change from the infected alone condition (control, p< 0.05).