The Effects of Glutathione and Its Derivatives on the Survival of Mycobacterium bovis-BCG Vegetative and Persistent Organisms

Mycobacterium tuberculosis is responsible for nearly 2 million deaths yearly. Upon inhalation, mycobacteria are engulfed by alveolar macrophages. These cells produce reactive oxygen and nitrogen intermediates (ROI’s and RNI’s), which normally kill pathogens, but are ineffective against invading mycobacteria. What ensues, is the formation of a tubercle to sequester the infected macrophages, and the initiation of a latent tuberculosis infection, in which the mycobacteria enter a state of non-replicative persistence (NRP). Glutathione (GSH), a host tripeptide thiol-based detoxification molecule, protects host cells from ROI/RNI toxicity. Recently, it has been demonstrated that GSH is toxic to in vitro and early infection mycobacteria, but no studies determined whether GSH is toxic to latent mycobacteria. To further elucidate the impact of GSH on mycobacteria, we exposed mid-logarithmic (mid-log) and NRP-induced M. bovis-BCG to glutathione in its reduced (GSH), oxidized (GSSG), and nitric oxide associated (GSNO) forms, for five days. We have demonstrated that the growth of mid-log M. bovis-BCG (BCG) is inhibited by 4 mM GSH, and killed by 8 mM GSH. The growth of NRP mycobacteria exposed to 4 mM GSH was inhibited similar to that of unexposed NRP mycobacteria. In contrast, the growth of NRP BCG is stimulated four fold following exposure to 8 mM GSH. We conclude that exposure of NRP BCG to 8 mM GSH stimulates exit from the NRP state into an actively metabolizing state. 4 and 8 mM GSSG and GSNO inhibited the growth of mid-log BCG; however, NRP BCG exposed to GSSG and GSNO inhibited growth similar to that of unexposed NRP BCG. These results were confirmed by performing viability studies, and analysis of free-intracellular cytoplasmic ATP concentrations. Initially hypothesized, the toxicity to GSH was due to redox potential imbalances within mycobacterial cytoplasm. Mycobacteria contain MSH detoxification system (5), to cope with GSH. However, the mechanism of interaction between the activation of the system and growth inhibition in mycobacteria is poorly understood. HPLC analyses on mid-log and NRP BCG cytoplasms exposed to 4 and 8 mM GSH demonstrated that the activation of the detoxification system and the oxidative stress of the mid-log and NRP cytoplasms were similar. This suggests that the activation of the detoxification system and the oxidative state of the mycobacterial cytoplasm may not play a direct role in mycobacterial growth inhibition and/or exit from the NRP state.