Expression of Penicillium marneffei Chitin Synthase Genes in Response to Cell-Wall Stressors

Penicillium marneffei is a dimorphic fungus and an opportunistic pathogen responsible for the disease penicilliosis.Efforts to genetically characterize this organism have led to the development of a plasmid-dependent, mutant generating system known as Agrobacterium tumefaciens mediated transformation.This system produced one mutant, originally identified as strain I231, which was found to harbor a defective yakA gene.As determined by the work of Suwunnakorn et al. (2014), this defective gene resulted in an abnormal phenotype and unique stress responses when compared to a wild-type strain.The ΔyakA mutant possessed increased resistance to the anionic dye Congo Red (CR), known to inhibit glucan synthase activity, yet also yielded increased sensitivity to the similarly acting antifungal drug, caspofungin.These seemingly contradictory results, along with a increase in overall chitin content within the mutant, led to this study which seeks to determine overall chitin synthase gene expression in the ΔyakA mutant, the wildtype strain, and the complement strain, CY21, in terms of seven known chitin synthase genes.These strains were exposed to CR, the detergent sodium dodecyl sulfate (SDS), the chitin binding agent Calcofluor White (CW), as well as incubated under non-stressed control conditions.Subsequently, RNA from these cultures were isolated and chitin synthase gene expression levels were quantitated through qRT-PCR using the reference gene benA (encoding β-tubulin) for normalization.The results of this study indicated that the interrupted yakA gene produced no significant effect in terms of chitin synthase gene expression under stressed conditions, but the mutation produced significantly higher expression levels of the chitin synthases in the ΔyakA control conditions at both 25°C and iv 37°C.This response most likely represents a compensatory response to the weakened cell wall caused by an interrupted yakA gene.v vi Julio "Ed" Budde, I would like to thank Ed for training me in the art of qPCR and for all his help with ordering.He played a part in every aspect of the "bench work" and I couldn't have done it without him.Justin Waldern, I would like to thank Justin for his absolutely indispensable help with organizing and formatting my data for statistical analyses.His assistance was crucial to the success of my project.I wish him luck at Albany and know he and Kayla will do amazing work.Rachael D'auria, I would like to thank Rachael for her help with bettering the qPCR primer standards that back up all of my work.Despite only having five weeks of experience in a research lab, she did an amazing job and I wish her well in all her future endeavors at UCLA and beyond. Sarah Eisnaugle,Sarah is my lab mate and my best friend.We faced these past two years together and by sheer unbelievable luck managed to produce two wonderful, beautiful pieces of complementary research that, like us, are stronger together.