Blog 4

From the last post I mentioned I am glad to report I have substantial data which helps shed light on the entire purpose of my research.  First of all, I have completed my Opp  mutant growth trail and viability assay. From the results I have collected, I can conclude that glutathione (GSH) cannot enter within the cytoplasm of Opp mutants in great enough concentrations to cause any stress to the bacteria. This is seen as 0,4 and 8 mM GSH all had the same growth and viability. This confirms that Opp gene that encodes of the oligopeptide permease transporter is not only crucial but necessary for GSH stress.

I have also made a significant  discovery in my  assessment of GSH concentration in the cytoplasm of my model bacterium BCG. Using this fluorescent kit I was able to quantify approximately the amount of GSH that was entering the cytoplasm of the bacteria at 4 and 8 mM using 0 mM as a control. What I found was that the mount of GSH entering the cytoplasm at 4 mM was not statistically different than 0MM. However 8mM GSH we observed   5.73 fold increase in GSH content . These results help confirm the hypothesis that our bacteria, BCG is experiencing reductive stress from GSH and is unable to handle such stress when the concentration is 8mM and higher.

This project has greatly increased my understanding of the scientific process. After much failure and restarting experiments, I have learned that science does not always follow a straight path. And in order to become a successful scientist you need to have patience and respect for the scientific process.

Blog 3

It has been a difficult semester so far. Preparing for graduations and renovations in the labs have made performing experiments extremely difficult. At this point of my research project I have put a hold on my transposon mutagenesis project and have focused more on my Opp project. With this project I am trying to see if glutathione affects a BCG mutant that has a ABC transporter knocked out. I will be expressing this mutant to 0,4 and 8mM glutathione while measuring its optical density and viability over 4 days.

This experiment is somewhat difficult because Opp needs to be exposed to streptomycin and kanamycin antibiotics to ensure that the mutant plasmid stays within the bacteria. Giving it these antibiotics however retards the growth of the organism and makes it rather difficult to have mid-log growing bacteria at hand . After this project is completed and I solve my refrigerator problem then I will pick up back on the transposon mutagenesis work .

I also plan on using a thiol fluorescence’s kit to measure the amount of glutathione present in the cytoplasm’s of BCG at 0, 4 and 8mM. This test will also be conducted on persistent BCG using the Wayne model. The Wayne model comprises of using nunc tubes  to limit the oxygen available for the bacteria, while growing it for 100 days until it is in a environment of low nutrients and high hypoxia.

Hopefully I will be able to complete these three experiments before I graduate and obtain statistically significant data which will help  us to better understand the effects of glutathione.

Identifying and Characterizing Novel Genes Involved in Glutathione Sensitivity.

During the past several weeks we have collected peer reviewed articles and have hypothesized that fad genes might be a gene of interest responsible in GSH stress.  These genes are involved in fatty acid synthesis and degradation. It is hypothesized that these genes are also involved in the maintenance of the mycolic layer of the mycobacterium which protects the outer layers of the bacterium. This piece of information might prove useful in understanding the cellular interaction between Mycobacterium tuberculosis (M.tb) and glutathione (GSH).

We have since then infected a few Mycobacterium smegmatis with our phAE175 bacteriophage and we noticed that the titer count was rather low. This could be one of two reasons. The major reason can be due to the tween80 that is used in the media to grow the bacteria during infection, studies have shown that tween reduces the effectiveness of bacteriophages. Another reason is due to the fact that the phAE175 are over two years old and many of them are ineffective.

At this moment we are questioning the role that fad genes, the process of mycolic maintenance and GSH sensitivity all play a role in GSH stress. The ultimate goal of this research in the next coming months is to verify that fad as a novel gene involved in GSH stress and also find and characterize other genes to gain a better understanding of GSH toxicity to M.tb

 

Identifying and Characterizing Novel Genes Involved in Glutathione Sensitivity.

Identifying and Characterizing Novel Genes Involved in Glutathione Sensitivity.

By: Raheem Lawrence

One third of the earth population is infected with Mycobacterium tuberculosis (M.tb) the causative agent for tuberculosis (TB). Of that one third, half are infected with the multi-drug resistant strain which kills more than 2 million people yearly. Tuberculosis is often viewed as disease of the poor, as majority of document cases of M.tb are in developing countries which makes advancing our knowledge of this disease of great importance. M.tb is an aerobic gram positive like bacterium that infects individuals through inhalation of aerosols, upon inhalation the bacteria resides in the linings of the lungs and rapidly proliferate in the oxygen rich environment. To prevent the continuous growth of the M.tb , macrophages which  function as part of the lung immune system quickly engulf the bacteria by phagocytosis. The invading bacteria are equipped with an innate mechanism to protect itself from the immune response by first lowering the local pH and then blocking phago-lysosomal fusion. This allows the bacteria to grow and persist within the macrophage and prevent its cell to be lysed. In response, the immune defense exposes the bacteria to high levels of oxidative radicals such as reactive oxygen and nitrogen intermediates. However these intermediates are ineffective against the bacteria as well as toxic to host cells, therefore the immune response produces glutathione (GSH) protect itself.

Glutathione is a   is a thiol based detoxification molecule that protects the host cell from oxidative radicals, recent studies conducted by our laboratory has observed that high concentrations of GSH are toxic in early in-vitro models used with Mycobacterium bovis (BCG) .BCG was chosen because of its availability to be used in a undergraduate facility and also shares 99% genetic similarity to M.tb and is used as a model organism in its study. From this study we have identified GSH as a possible compound to aid in the fight against M.tb, but the mechanisms behind GSH toxicity is still unknown and there is a need for further research to be conducted. The overarching goal of this research is to use transposon mutagenesis to create a panel of mutants to genetically classify the specific loci involved in GSH sensitivity. If successful this will lead us one step further in understanding the mechanism behind GSH stress and possibly help we to utilize GSH to make vaccines and anti-microbial to control the rampant spread of TB world wide.

Identifying and Characterizing Novel Genes Involved in Glutathione Sensitivity.

Identifying and Characterizing Novel Genes Involved in Glutathione Sensitivity.

By: Raheem Lawrence

One third of the earth’s population is infected with Mycobacterium tuberculosis (M.tb) the causative agent of tuberculosis. Of that one third, half are infected  with the multi-drug resistant strain, most of which are  found in developing countries which makes the study of this disease of great significance. M.tb is a Gram positive like bacterium which invades an individual through inhalation of the organism through aerosols. The bacteria infect alveolar tissues and are quickly engulfed by the host alveolar macrophages which functions as part of the lungs immune system. Within the macrophages the bacteria are exposed to nitrogen intermediates that are ineffective in retarding growth. These intermediates while ineffective against bacteria are toxic to host cells. Therefore host cells produce glutathione (GSH) which functions as a thiol based detoxification system to protect the host against radicals.

The data obtained by our lab has identified that GSH is toxic in early in-vitro Mycobacterium bovis (BCG) models, a close relative to M.tb sharing 99% genetic similarity. Using this knowledge GSH has been identified as a future tool to combat the rapid spread of multi-drug resistant M.tb.  However the mechanisms behind GSH sensitivity in BCG are not fully understood. The overarching goal of this research is to expose the mechanisms behind GSH toxicity. Like a good investigator I propose to start small, by first genetically characterizing loci that are involved in GSH sensitivity before expanding molecularly. Dr. Marcy Kelly and I have taken a pragmatic approach to elucidate the mysteries surrounding GSH sensitivity in BCG by using transposon mutagenesis with a mycobacteriophage phAE175. This method will create single and random mutations in the genes of BCG. These mutants will then be grown in toxic levels (8mM) GSH to select GSH resistant mutants. Mutants will then be sequenced to identify genes, involved in GSH sensitivity.

If successful we expect to acquire a panel of genes involved in GSH sensitivity, which is needed if GSH will ever be used as a tool to perturb the rapid spread of TB. GSH is naturally produced by human cells and has the potential to help develop highly potent antibacterial agents and vaccinations to treat and prevent the disease, respectively.