Creating deletion strains to study the role of the argonaute and dicer genes in RNA interference in Cryptococcus neoformans

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Alan Dale Curtis II (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://www.wcu.edu/404.asp
Advisor
Indrani Bose

Abstract: Cryptococcus neoformans is a spherical, encapsulated, basidiomycetous yeast and the causative agent of cryptococcosis, a form of meningitis that affects the central nervous system of immunocompromised individuals (immunocompromised means patients with compromised immune systems). Since the 1980’s and the emergence of the AIDS epidemic, much study has been concentrated on this fungus because cryptococcosis is 100% fatal in untreated patients. Even with treatment, the condition does not always decrease in severity, and no major advancements in antifungal drugs have been made in a decade. Recently, Cryptococcus has been shown to possess the necessary machinery for RNA interference (RNAi). RNAi is a method of post-transcriptional gene silencing that may increase cryptococcal survival within mammalian hosts by controlling gene expression at various stages of the life cycle through heterochromatin and euchromatin rearrangement. RNAi was first described in Caenorhabditis elegans in 1998 by Andrew Fire and his colleagues, and is best studied in Drosophila melanogaster. The pathway includes argonaute and dicer proteins that aid in highly specific degradation of mRNA in vivo. To date, no organism has been shown to utilize RNAi as a factor for virulence, nor should it be thought of as such; however, this pathway may indeed play a role in the formation of certain virulence factors for Cryptococcus, including the transcription of heat stress response genes required for thermotolerance, oxidative stress response genes such as those required for melanin and mannitol biosynthesis, as well as the formation of the polysaccharide capsule. Two separate, yet equally effective methods were used in order to create deletion constructs of the AGO1 and AGO2 genes. Overlap PCR and traditional cloning methods were employed. While overlap PCR was optimized, molecular cloning techniques were also used to create the complementation constructs for the above genes. Through overlap PCR, this study resulted in the creation of deletion constructs for the AGO1 and AGO2 genes, which are thought to play a role in RNAi. This study also resulted in the creation of a complementation construct for the ago1 deletion. These constructs can be biolistically inserted into C. neoformans to produce the corresponding deletion strains. Once these strains are produced, future experiments will include many phenotypic studies between them and other known strains, as well as an eventual murine virulence study.

Additional Information

Publication
Thesis
Language: English
Date: 2009
Keywords
Argonaute, Cryptococcus neoformans, Dicer, RNA Interference, Virulence
Subjects
Cryptococcus neoformans
Cryptococcus
Genetic regulation