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.

 

 

 

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