Proteomic analysis of Aspergillus nidulans during autophagy and the role of autophagy genes Anatg13 and Anatg8

Aspergilli represent an extremely important genus of microorganisms which can be both harmful pathogens, and beneficial pharmaceutical producers. In Aspergilli's interactions with man, suboptimal nutrient conditions are often present, and lead to a phenomenon known as autophagy. Autophagy is a cellular recycling mechanism that (in the case of macroautophagy) is augmented under nutrient-limited conditions to recycle cytoplasmic macromolecules and organelles for use in essential cell functions. Strategic manipulation of autophagy could ultimately lead to improved bioprocesses or anti-fungal treatments. Using the model filamentous fungus Aspergillus nidulans, a number of important questions about autophagy have been addressed.;Critical to the study of autophagy is the balance between self-degradation and self-preservation. Therefore, we adapted an XTT metabolic activity assay for use in filamentous fungi. The assay was first tested using a number of bioprocess-related stresses (e.g. temperature, shear), and found to be superior to DCW as an assessment of culture health. Next, the metabolic activity of fungal cultures was tested during autophagy-inducing conditions, demonstrating that the autophagy capable TN02A3 strain was more viable than an autophagy deficient DeltaAnatg13 strain in nutrient limiting conditions.;By analyzing the proteome of key autophagy mutants DeltaAnatg13 and DeltaAnatg8, an improved molecular understanding of autophagy in filamentous fungi was achieved. Using 2-dimensional electrophoresis, 44 unique proteins were found with significant expression changes caused either by addition of rapamycin (a chemical inducer of autophagy) or deletion of Anatg13. AnAtg13 dependent changes of multiple ribosomal and a key polyamine biosynthetic protein, spermidine synthase (AnSpdA), provides molecular evidence of AnAtg13 dependent lifespan extension in A. nidulans.;After establishing improved shotgun proteomic methods on the Thermo LTQ-XL, we generated a more thorough assessment of the A. nidulans response to autophagy by measuring protein expression as a function of time. It was found that autophagy induction caused a rapid and sustained increase in proteolysis, amino acid degradation, and lipid metabolism. Additionally, many proteins demonstrated a delayed change in expression. These included proteins involved in secretion, hydrolysis of alternative carbon sources, and secondary metabolite production; all of which are important to the bioprocess industry.