Due to the fastidious nature of Mycobacterium bovis-BCG and the minimal media that I am attempting to culture it in, I have run into multiple different problems. Once I was finally able to establish a media that was suitable for my bacterium and experiment, I began growth trials. Initially, I was dealing with a contamination problem, so I was unsure if my media was successful, or if another organism was using it for itself. After weeks of waiting, my research team was able to culture pure, uncontaminated BCG, that we are now able to use for all our experiments.
Normally when growth trials are conducted, we dilute our cultures down to an optical density of 0.1, where after a week we are able to see a steady growth curve. Following this procedure, I conducted two more growth trials with the new BCG. I noticed that all my samples were remaining at an optical density of 0.1 for more than a week, which is abnormal. Assuming that the bacteria needed more time to adjust to the minimal, nutrient depleted media, I allowed it to continue to grow for another week. After two weeks the optical density remained the same. With both of my growth trials following this pattern, I started to think my media was unsuccessful. Then I thought maybe the bacteria weren’t dying after all. Maybe they were protecting themselves from their harsh environment by entering non replicative persistence.
I am now conducting experiments to determine if the BCG has become dormant. I am currently conducting viability trials, where I allow the bacteria to grow in the cholesterol media for a week (hopefully they will have entered NRP), and plate them on 7H11 plates. After two weeks of incubation, I can count their colonies and see if the bacteria are still alive, or if I need to alter my 7H12 media.
In the beginning of this year, I aimed to understand the effects of the human immune response on persistent Mycobacterium tuberculosis. Using Mycobacterium bovis-BCG as a model organism, I planned to conduct growth trials for active and persistent culture grown in a cholesterol rich 7H12T media and treat them with glutathione to determine if active culture could undergo a metabolic shift similar to that of NRP BCG. If the active BCG was able to shift towards an NRP metabolism, it would indicate that the active BCG was able to induce a persistent state solely through the presence of cholesterol. I also planned to conduct a NAD+/NADH, H+ Glo Assay to understand how cholesterol is impacting the cell.
Over the past year our laboratory has been suffering from contamination issues, preventing the growth of our BCG and the ability to obtain accurate data from pure culture. Luckily, we were able to order new BCG and grow it up into new pure culture. Creating bacterial frozen stocks from a dry pellet takes about four weeks, putting my work slightly behind schedule, but I was able to start growth trials within the last week. Being in the first two days of the trial, I have not obtained any statistically significant data, but the bacteria appears to be growing in the harsh environment I have provided for it. I plan to continue this growth trial for about a week and determine my next steps from there. If the bacteria show a normal growth curve, I will treat new cultures with glutathione and hopefully observe their persistence. If they do not grow during this trial I will reassess my media recipe and attempt to create a more nutritious environment for the bacteria.
From the first half of my research I have learned that science does not always behave on a schedule! It is very difficult to try and force bacteria to grow, especially when you have deadlines to meet! I also learned a lot about controlling contamination and how to continue keeping the lab as pure as possible.
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.
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.
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.