User:Punnania/sandbox

===Enzymatic interaction between TpsA and GsdA: two key enzymes in trehalose pathway and pentose phosphate pathway in Aspergillus fumigatus===

Aspergillus fumigatus is a saprophytic fungus that forms aireborne spores. Thus opportunistic human fungal pathogen can cause lethal invasive aspergillosis in immunocompromised hosts. It is the most common species recovered from patients among other molds and has a very high resistance to antifungal agents. A. fumigatus can produce trehalose, which has been primarily thought to serve as a storage carbohydrate and as an important stress protectant against a variety of stress conditions. The key enzymes at different key steps in the trehalose biosynthesis pathway are required for human and plant fungal pathogenesis. Thus, the examination of these enzymes and their interaction thereof could lead to a promising antifungal drug target. In my research project, we aimed to study the interaction between TpsA and GsdA, which encodes for G6PDH in pentose phosphate pathway. We further hypothesize that TpsA interacts and controls the enzymatic activity of GsdA in the aspect of the expression level under two different stressed conditions. We began by employing double-joint PCR to build the GsdA-S tag construction in order to insert the epitope tag ( S-tag ) into the genomic DNA. Then, we carried out Southern Blot Analysis to confirm the designated position in the genome and the copy of that fragment. After that, the Western Blot Analysis was utilized to confirm the S-tag expression. Finally, we performed Pulldown Assay and Co- Immunoprecipitation to study the interaction between the two proteins. Additionally, we studied the expression level in GsdA among trehalose mutants under the stressed condition. In the end, we discovered that there is a physical interaction between TpsA and GsdA confirmed by CO-IP, and there is an insignificant change in GsdA expression level among trehalose mutants.

The result from Co-immunoprecipitation indicates that there is an interaction between TpsA and GsdA, which are intermediates in trehalose pathway and pentose phosphate pathway respectively. However, using trehalose mutants (∆orlA, ∆tpsA, ∆tpsB, and ∆tpsA/B) to study the expression level of GsdA in a transcriptional level, it was found that there was not a significant decrease in GsdA level. This is a robust discovery suggesting that there is an alternative pathway that plays a role in compensating the lack of energy-rich compounds essential for survival and virulence in the absence of TpsA/B enzymes under stressed condition. “ALDA” is presumably thought to be the key player as a metabolic source of NADH that can substitute for the lack of the G6PDH activity.