End of Year Report

Kate Becker

UGR Final Report

Mycobacterium bovis-BCG is the organism responsible for Tuberculosis (TB), an infection that can cause coughing, fever and chest pain (CDC, 2017). Tuberculosis can be characterized by two distinct states of infection, active and latent. In the active infection, the infected individual displays signs and symptoms of infection, while the latent infection hides from the host’s immune response and does not express any signs or symptoms. A latent infected individual can go long periods of time without knowing they are Tuberculosis, until the infection is reactivated. The infection will usually be reactivated when the host’s immune response is no longer strong enough to suppress the infection and when they have become immunocompromised (CDC, 2017).

While the infection is known as being latent, the organism behind the infection has entered a state called non replicative persistence (NRP). The NRP state is characterized by an active metabolism, without bacterial replication (K Patel et al., 2011). NRP allows the bacterium to live inside the body and successfully avoid any attempts the immune response makes to destroy the infection. With little information about NRP and its mechanisms, it is very difficult to treat latent Tuberculosis. The rise of antibiotic resistant strains of TB reinforces the difficulties of treating the infection.

According to the World Health Organization (WHO), one third of the world’s population is infected with Mtb. Of these infections, 50% are multidrug resistance and 10% are in the latent stage (WHO, 2017). As the population increases and the infection becomes harder and harder to treat, it is very important to find new ways to prevent and treat the infection. In order to do this, the mechanisms and metabolism behind NRP Mtb must be understood.

When TB is in the latent stage, Mtb has been sequestered in a granuloma, a cluster of cells that has absorbed the infection. The interior of the granuloma maintains a very harsh environment. Needing a carbon source to survive, the sequestered Mtb only has cholesterol available to it. Quigley and colleagues have shown that cholesterol is mandatory for the bacterium to enter NRP, leading to the latent state. It has also been shown by Pandey and colleagues that Mtb can completely break down cholesterol, using it for nutrients and the virulence factors that enable the bacteria’s ability to make a host sick.

Due to the fact that Mtb can use cholesterol as a sole carbon source for nutrients and persistence, it is necessary to understand Mtb’s cholesterol metabolism to create effective treatments and vaccinations for TB. The goal of this study is to determine the best cholesterol media that the bacterium can thrive on, allowing us to conduct energetics studies on the organism when they are only exposed to a minimal cholesterol media. Due to its near identical genome, and the deletion of its virulence factors, Mycobacterium bovis-BCG (BCG) will be used to determine the best cholesterol rich media for Mtb. BCG will be grown in two cholesterol medias, known as 7H12 ad 7H12T, where they will be tested for their ability to grow and if they remain viable after being exposed to the cholesterol. By determining the best media to grow these organisms, future metabolism studies will be able to be conducted.

When I first began this study, I did not foresee the cholesterol media being so difficult to make. Although I knew the fastidious nature of BCG, I did not understand how difficult it would actually be to culture. Before realizing this, my goal was conduct an NAD+ GLO assay by promega to understand how cholesterol was being metabolized within the organism and if this metabolic shift would enable active BCG to become resistant to glutathione induced reductive stress killing. After finding out how difficult cholesterol media can be, I shifted my main goal to just finding the best way I could get mycobacteria to grow in a nutrient depleted environment. After creating three different types of of media, 7H12, 7H12T1 and 7H12T2, I finally had success culturing the BCG. After conducting a growth trial, I found that my second attempt of making 7H12T media was actually working. Although the only difference between my attempts of making 7H12T was the way the cholesterol was dissolved, it seemed to make all the difference. By heating the cholesterol stock slightly above room temperature, I found that I was able to prevent the cholesterol from precipitating out of the base of the media, and therefore can remain in the media even after filter sterilization. I believe that in my first attempts, the inability to dissolve the cholesterol completely was causing the only available carbon source to be filtered out of the media.

Now that I have found a way to culture BCG in cholesterol, I can continue on with my experiment. In my final weeks of this semester I am planning on conducting a growth trial to understand how glutathione will effect BCG treated with and without cholesterol. I hypothesize that I will see decreased reductive stress killing and increased growth in the BCG treated with cholesterol, and decreased growth and viability in the active untreated BCG sample. Although I was not able to carry out my initial experiment, I think that this was a valuable lab experience. I think that this experience helped show me what working in a BSL2 laboratory is actually like, and that bacteria does not always grow like you want it to. Throughout my UGR experience I was able to learn about dealing with contamination, trial and error of making media and learning new techniques that I would not have come across if it wasn’t for this project.




Final Report

Kate Becker

UGR Grant Final Report

Summer 2017

In the beginning of the summer, I aimed to understand the intracellular response of Mycobacterium tuberculosis when attacked by the detoxification molecule, Glutathione. Using Mycobacterium bovis-BCG (BCG) as our model organism, our previous researched showed that non replicative persistent (NRP) BCG was resistant to glutathione induced reductive stress killing, while normal BCG succumbed to detoxification. This shows us that NRP BCG has an extra line of defense against the usual way the body would try to kill the bacteria. This data, combined with research from other laboratories stating that the presence of cholesterol in the granuloma can act as an electron sink helping to overcome reductive stress, gave way to my hypothesis.

I hypothesized that a cholesterol induced metabolism will protect M. bovis-BCG from GSH induced reductive stress killing similar to how NRP BCG is able to resist GSH. In order to test this, I planned to use an NAD/NADH-GloTM Assay in hopes of seeing BCG accumulating more NAD+/NADP when associated with cholesterol. These markers would indicate a metabolic shift towards an oxidative environment in the bacterial cytoplasm and would prevent glutathione induced reductive stress killing.

Before I could use the NAD/NADH-GloTM Assay, I had to create a media that contains enough cholesterol to induce a metabolic shift while also ensuring that the organism is not nutrient deprived. A recipe was created for 7H12 media using information from other labs who also worked with MTB and cholesterol. This recipe created our first obstacle because in order to fully dissolve the cholesterol, we had to heat 200 proof ethanol to 80C; which is highly dangerous. Ultimately, with much trial and error, we were able to create a media with the dissolved cholesterol without heating ethanol to 80C, called 7H12T. Additionally, a detergent to prevent the clumping of these naturally sticky organisms was needed. Originally, we attempted Tyloxapol, which was very hard to use due its thick consistency and a large amount was necessary to be effective. This made the media too bubbly when we had to filter sterilize it. Instead we found that we were able to use Tween80, a detergent that we use in our regular 7H9 media when we culture BCG without cholesterol.

Once we finished creating the media, we conducted growth trials to ensure that we would be able to culture BCG with it. When conducting growth trials, 1 mL of frozen BCG suspended in glycerol, then added to 4mL 7H9 media and was allowed to incubate for 24 hours. Then, 1 mL was added to 24mL of 7H12T media and incubated for three days. The growth was monitored each day by measuring optical density using a spectrophotometer. After the first day, we suspected contamination because of the doubling time. BCG usually takes three days to reach mid-log stage with an optical density of 0.6-0.8 nm. But, this culture reached 1.1 nm by day two. With other contamination issues being observed in the lab, by using a crystal violet stain we were able to observe three different types of contamination including yeast spores. The contamination was traced backed to our original frozen stock, forcing us to purchase fresh BCG.

The need for new BCG was a huge setback for the project, because we could not continue without it. Being a biosafety level 2 organism, it was difficult to obtain, and the shipping process took some time. After a few weeks, we finally received our new BCG and are currently working on culturing it to create new frozen stocks. Although I was not able to complete the goals that I had set, I still benefitted from this process. I was able to learn a lot about contamination; when to be suspicious, how to find it and how to get rid of it. A large part of the process included staining samples and observing them under microscopes, which was very interesting. Although the bacteria was damaging to my research it was fascinating to see all the other microbes living around us and how easily something could become infected. I also think that the many setbacks in creating the media was helpful to me as a researcher and scientist. With guidance from my advisor, I was able to learn how to go about making media that is suitable for a specific experiment and it really expanded my ability to comprehend scientific literature.

For the remainder of the summer and into the first semester, once the new BCG has been cultured; I will continue working on obtaining a successful growth curve which will lead to performing a NAD/NADH-GloTM Assay. Although scientific research does not always go as planned, I am incredibly grateful for the opportunity to work with BCG and further my knowledge in the field of microbiology.

Blog Post #3

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.

Blog Post #2

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.

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.

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.