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’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.