The Effect of Cholesterol on Mycobacterium bovis-BCG Resistance to Glutathione

When Mycobacterium tuberculosis infects its host, the human immune response produces the thiol based detoxification molecule, glutathione (GSH). The glutathione attempts to kill the invading cell by forcing it into a reduced environment. In our previously work, using Mycobacterium bovis-BCG as a model organism, we have shown that active culture attempts to control this glutathione induced reductive stress killing by over producing the oxidizing agents NAD+ and NADH,H+. The active culture cannot overcome the reductive environment and succumbs to its death. Mycobacterium also use persistence as a protective mechanism. In response to an oxygen depleted environment, BCG is able to enter non replicative persistence (NRP), a state where the cell has an active metabolism, but does not actively divide. When observing NRP BCG inoculated with GSH, it is found that the reducing agent is not bactericidal. The NAD+ and NADH, H+ levels also remain low and constant, compared to that of active BCG, indicating that there is another pathway that NRP BCG is using to resist glutathione induced reductive stress killing.

Our previous research suggests that the catabolism of cholesterol can act as an electron sink for reducing agent. It is also know that the catabolism of cholesterol causes regeneration of NAD+ when the virulence factor PDIM is synthesized. This idea leads to my project, The Impact of Cholesterol on Mycobacterium bovis-BCG Resistance to Glutathione. In my project, I hypothesize that if cholesterol causes BCG to undergo a metabolic shift similar to that of NRP BCG, and BCG is grown in a cholesterol rich environment and NAD+ and NADH,H+ levels are observed, BCG grown in a cholesterol rich environment will be able to resist glutathione induced reductive stress killing. In order to understand how cholesterol affects the active culture, I will be creating a media called 7H12T media. Once a proper media is established and the organism is able to tolerate a cholesterol rich and nutrient deprived environment, I will be conducting a NAD/NADH Glo Assay. With this data, I will be able to understand if the presence of cholesterol is pushing the cell towards an oxidative environment that will protect the BCG from reductive stress killing.

Currently, the effect of glutathione on BCG is not well understood. Without a thorough understanding of the intracellular response when exposed to glutathione, we cannot understand how to treat the tuberculosis infection. Because the scientific community is lacking this information, there has not been a new pharmaceutical created for Tuberculosis in over 10 years. I hope that with a proper understanding of the metabolic pathways being used in NRP BCG to resist the human immune response, there will be a breakthrough in targeted drug therapies for Tuberculosis.

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