Blog Post #2

The first step in understanding the impact of cholesterol on Mycobacterium bovis-BCG is creating a suitable environment for the organism to grow in. Basing our recipe off of a liquid medium used to culture Mycobacterium species (7H9 media), we made 7H12T media that contains added cholesterol. This process involved a lot of trial and error because we needed to find a way to dissolve the cholesterol in the liquid without having to heat ethanol, the solvent, to dangerous temperatures. We also had to find a detergent that would prevent the naturally sticky bacteria from clumping together, while still being affordable. I performed three different growth curves each with a different recipe before I saw any growth.

When preforming the growth curve, 1 mL of BCG from frozen stock was allowed to grow for 24 hours in 7H9 media, then transferred into 24 mL of 7H12T media. Each day for four days, the optical density was tested to determine if the BCG was growing in the cholesterol rich media. The data was compared against BCG grown solely in 7H9 media. Initially the data showed growth, but we quickly realized the cultures were contaminated due to their high growth rate.

To test for contamination, we streaked out the culture onto TSA plates, but we did not see any growth on the plates. Although this would indicate that there was no contamination, we wanted to confirm by using a crystal violet stain. Mycobacterium is an acid fast organism with a mycolic acid outer layer. The crystal violet stain is not able to penetrate this outer layer, so if the culture only contained BCG, we would not see anything on the slide. Unfortunately, we found three different types of contamination including yeast spores which we were able to identify by their shape and stain. We were able to trace the contamination back to our original frozen stocks, which forced us to have to purchase a new pure culture of BCG. Due to the regulations of buying a biosafety level two organism, it took almost a month for it to arrive. We are now working on culturing the new BCG and creating frozen stocks of pure culture which will take three weeks.

Although I was not able to gain any insight on my hypothesis, I did learn a lot about contamination and working in a microbiology lab. I was able to learn about warning signs for contamination, different types of tests for contamination like the crystal violet stain and how to control the situation. Once we finish culturing the new BCG, I plan to continue working towards my original goal, testing BCG grown in 7H12T media using a NAD/NADH-GloTM Assay to determine if cholesterol causes a metabolic shift and will protect the organism in a similar way that NRP BCG protects itself. I also want to further our understanding of the metabolic processes that are happening when someone becomes infected with Mycobacteirum tuberculosis.

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

Currently, one-third of the world’s population is infected with Mycobacterium tuberculosis. Of those infections, 10% are characterized by their dormant latent phase and 50% of them are multidrug resistant. As tuberculosis is one of the top ten causes of death throughout the world (World Health Organization), it is pertinent to understand the intracellular response that the human immune response has on M. tuberculosis. When the immune response is activated glutathione (GSH), a thiol based detoxification molecule, is produced to protect the host tissue (Patel et al., 2016). When GSH is secreted it induces uncontrollable reductive stress in the mycobacterial cell, leading to its death.
M. tuberculosis has the ability to enter a latent stage which is characterized by a metabolic shift that allows it to remain dormant inside the host. This is also known as “non replicative persistence,” (NRP). Additionally, being able to hide and remain safe inside the host makes it very difficult to treat. Our research has shown that when BCG is in non replicative persistence, it is resistant to GSH induced reductive stress killing (Patel et al., 2016). Other labs have demonstrated that cholesterol, the sole carbon source for latent Tuberculosis, can be built up inside the mycobacterial cell creating excess NAD+ and NADP that can draw in excess electrons from GSH induced reductive stress (Vandervan et al., 2015). This leads us to hypothesize that the cholesterol induced metabolism will protect M. bovis-BCG from GSH killing similar to how NRP mycobacteria resists GSH. This connection led us to the idea for my project, The Impact of Cholesterol on Mycobacterium bovis-BCG Resistance to Glutathione.
In order to further understand the impact of a cholesterol rich environment, we will be using a NAD/NADH-GloTM Assay. Mycobacterium bovis-BCG will be used as a model organism as it is 99% genetically similar to M. tuberculosis. We expect to see BCG accumulating more NAD+/NADP when it is exposed to the cholesterol. This accumulation would suggest that there was a metabolic shift towards an oxidative environment in the bacterial cytoplasms that is preventing reductive stress killing. This project will further our understanding of the metabolic processes taking place during the infection, and would be beneficial to the development of new vaccinations.