Glutathione and nitrosoglutathione in macrophage defense against Mycobacterium tuberculosis

Abstract

We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.

FIG. 1.

Growth of M. tuberculosis in the presence and absence of GSH (A) and GSNO (B) as determined by measuring OD. The H37Rv (Rv) cell suspension was treated with 5 mM GSH or 5 mM GSNO or left untreated. The OD was measured every day for 5 days (d.) (A). All experiments were repeated three times in triplicate.

FIG. 2.

Growth of H37Rv in untreated (A) and IFN-γ-LPS-treated (B) J774.1 cells. Experiments with H37Rv-infected macrophages, maintained in the absence (A) and presence (B) of IFN-γ plus LPS, were terminated at 1 and 72 h to determine the growth of H37Rv inside J774.1 cells. * denotes a statistically significant increase in the number of CFU between 1 and 72 h (P < 0.0006). Data are averages of results from six different experiments performed in triplicate.

FIG. 3.

(A) Growth of H37Rv in IFN-γ-LPS-BSO-treated J774.1 cells. Experiments with H37Rv-infected macrophages treated with IFN-γ plus LPS and BSO were terminated at 1 and 72 h to determine the levels of growth of H37Rv inside J774.1 cells. Statistical significance was calculated with the Statview program. * represents a statistically significant increase in the number of CFU between 1 and 72 h (P < 0.0083). Data are averages of results from from six different experiments performed in triplicate. (B) NO estimation in J774.1 cells. Nitrite levels in macrophage supernatants were determined spectrophotometrically by a Greiss reaction. Data are averages of results from five different experiments. * indicates a statistically significant increase in nitrite levels between control and IFN-γ-LPS- or IFN-γ-LPS-BSO-treated macrophages. For values for the control versus those after IFN-γ-LPS treatment, P was <0.0001. For values for the control versus those after IFN-γ-LPS-BSO treatment, P was <0.0030.

FIG. 4.

Estimation of GSH levels in J774.1 cells by flow cytometry (A) and spectrophotometry (B). (A) GSH was quantitated in J774.1 cells by staining with monochlorobimane. * signifies a statistically significant decrease in the number of fluorescent cells in IFN-γ-LPS-BSO-treated macrophages compared to that for control or IFN-γ-LPS-treated macrophages. For values for the control versus those after IFN-γ-LPS-BSO treatment, P was <0.0001. For values after IFN-γ-LPS treatment versus those after IFN-γ-LPS-BSO treatment, P was <0.0026. Data are averages of results from three different experiments. (B) GSH was also assayed by spectrophotometry. Data are averages from three different experiments. * designates a statistically significant decrease in intracellular GSH levels in IFN-γ-LPS-BSO-treated macrophages compared to those in control macrophages or IFN-γ-LPS-treated macrophages. For values for the control versus those after IFN-γ-LPS—BSO treatment, P was <0.0024. For values after IFN-γ-LPS treatment versus IFN-γ-LPS-BSO treatment, P was <0.0028.