Innate protection of Mycobacterium smegmatis against the antimicrobial activity of nitric oxide is provided by mycothiol

Abstract

Nitric oxide (NO) is an efficient antimicrobial agent. A role for mycothiol in protecting mycobacteria from nitrosative damage was revealed by showing that a Mycobacterium smegmatis mutant is sensitive to NO. A direct correlation between NO and mycothiol levels confirmed that mycothiol is important for protecting mycobacteria from NO attack.

FIG. 1.

Antimicrobial activity of gNO on M. smegmatis mc²155 in 0.9% saline solution. Mycobacteria were exposed to 200 ppm gNO (mc²155, open squares; mshA::Tn5, open circles; mshA::Tn5 [pAL0486], open triangles) or medical air (mc²155, filled squares; mshA::Tn5, filled circles; mshA::Tn5 [pAL0486], filled triangles). (A) Killing pattern of M. smegmatis. (B) Increased sensitivity to gNO-mediated killing for MSH-deficient M. smegmatis mshA::Tn5 compared to that for its wild-type parent. (C) Complementing M. smegmatis mshA::Tn5 with cloned M. tuberculosis mshA expressed on a plasmid restored the parental level of resistance to gNO-mediated killing. In all cases, to avoid the effect of gNO on growth media compounds, bacteria were suspended in 0.9% saline. Each data point represents the mean ± standard deviation for at least three independent measurements.

FIG. 2.

MSH pool in M. smegmatis is decreased by exposure to NO. (A) MSH levels in M. smegmatis mc²155 in 0.9% saline solution decreased with continuous exposure to gNO. Organisms were exposed to 400 ppm gNO (triangles) or medical air (squares) for 7 h. The amount of MSH decreased steadily to almost zero in less than 3.5 h on gNO exposure. (B) MSH levels in M. smegmatis mc²155 decreased with exposure to the NO donor sodium nitrite, while bacterial viability remained relatively unaffected. M. smegmatis mc²155 was grown to log phase in Middlebrook 7H9 media supplemented with 0.05% Tween and 1% glucose (pH 5.5), diluted to 0.5 optical density units at 600 nm in the same medium, and treated with various concentrations of sodium nitrite for 2 hours. Numbers of CFU (bars) and MSH levels (triangles) are shown. (C) MSH levels in M. smegmatis mc²155 decreased with exposure to the NO donor GSNO, while viability remained unaffected. M. smegmatis mc²155 was grown to log phase in Middlebrook 7H9 media supplemented with 0.05% Tween and 1% glucose, diluted to 0.5 optical density units at 600 nm with the same medium, and treated with various amounts of GSNO for 2 h. CFU/ml (bars), GSH levels (circles), and MSH levels (triangles) are shown. Each data point represents the mean ± standard error for four independent measurements.