Blog Post 1-The influence of Cholesterol on NAD+ production in Mycobacterium bovis-BCG

According to the US Centers for Disease Control (CDC) approximately one-third of the world’s population is infected with the bacterium, Mycobacterium tuberculosis, which causes tuberculosis (TB). Once a person has Tuberculosis the bacterium still remains in the body. This is because when the active mycobacteria enter our body, our immune response activates and tries to combat the foreign invaders with cells called macrophages, but the macrophages are not able to kill all of the bacteria. For the bacteria, the macrophage cannot kill the macrophages surrounds it and trap it in an airtight sac, forming a granuloma. The macrophages release harmful chemical agents, Glutathione, in the granuloma. In the granuloma, the M. tuberculosis is completely harmless, but it entered into the non-replicative persistence stage (NRP). In this stage, the bacteria basically dormant and drastically decreases its metabolism drastically. The bacteria can be dormant for years and years and if our immune system is weakened or compromised nutrients can enter the granuloma and the bacteria can “reawaken” and behaves as if it was never trapped. The title of my project is “The influence of Cholesterol on NAD+ production in Mycobacterium bovis-BCG”. Mycobacterium bovis-BCG shares 99% of its genome with M.tuberculosis, but it is safe to work in an undergraduate lab. We believe that the when the bacteria enters NRP it starts to create NAD+ in order to offset the reductive stress caused by glutathione.                                 The purpose of my project is to better understand the mechanism, non-replicative persistence, that allows for Mycobacterium bovis-BCG to survive in a nutrient deprived environment. If we figure out how non-replicative persistence works we can end Tuberculosis.

I am working with Dr. Kelly with growth curves, testing what media would expose Mycobacterium bovis-BCG to glutathione without killing it. Then we would conduct an NAD+ Assay to test the concentration of NAD+ using Promega. After the assay, we would run a Polymerase Chain Reaction in order to find out what genes are up-regulated and down-regulated.