Growing Preference And Environmental Adaptation Of Aspergillus Fumigatus Revealed By Functional Proteome

Aspergillus fumigatus is one of the most wide-spread human pathogens in nature, and it comes mainly from biomass self-heating compost heaps. Studying dynamic changes of secretome has significance to analyze the biochemistry and physiology of fungi. It is ubiquitous in nature due to its saprophytic lifestyle. Aspergillus fumigatus Conidia can disperse in a long distance to the benefit of physical characteristics. Comparing with obligate parasite, it is more difficult to prevent Aspergillus fumigatus Conidia from distributing in a wide-range, therefore, Aspergillus fumigatus would bring huge potential hazards. So reducing the amount of Aspergillus fumigatus Conidia from source is crucial to relevant disease precaution, especially in environment where plant biomass or agricultural waste stacking. The investigation of microbial diversity revealed Aspergillus fumigatus distribution is significant different in varied environment, which suggested that environmental factors can affect the fungal growth to some extent. Therefore, it is of great significance to analysis growing preference and environmental adaptation of strain through extracellular functional proteome, which will provided a theoretical direction for the prevention and control of Aspergillus fumigatus.1. Isolation and identification of A.fumigatus from self-heating compostMassive metagenome analysis revealed that self-heating compost is the major environmental source of A.fumigatus, small-scale fermentor compost has been carried out using corn straw as substrate. Fermentor is a relatively closed container that affected only by a few factors. Compost was processed for 5 days under 34℃-50℃, the ratio of A.fumigatus contents to fungus was up to 30%. Thus a A.fumigatus was isolated from compost successfully, and evolution analysis revealed its close relative with A.niger, which also indicated that A.fumigatus has high performance in plant biomass degradation. The growing behavior monitored by colonial morphology at temperature from 20 ℃ to 60℃ suggested that A.fumigatus could be classified as a thermophilic fungus. All the features mentioned above comfirmed the dominant growth of A.fumigatus in compost.2. Proteome analysis of A.fumigatus cultured by substrates with different pH and moistureGenome analysis suggested that A.fumigatus has a large amount of glucoside hydrolase and protease encoding genes that were far more than that in A.niger, which suggested A.fumigatus has the broad adaptability in carbon and nitrogen source. A.fumigatus can secrete more proteins cultured by natural material than non-natural material. Besides, A.fumigatus grew well in a wide range of pH and moisture, and adjusted its protein secretion based on different culture condition. The results above suggested that it is difficult to control the fungal growth through changing pH and moisture of culture condition.3. Secretome analysis of A. fumigatus under different temperatureTemperature is the key factor to growth of A. fumigates. It secreted a large amount of extracellular biomass-degrading enzymes at 34℃ to 50 ℃, meanwhile, proteases and pathogenesis-related proteins also remain a high abundance, which suggested that A. fumigatus equips a strong ability to infect others within this temperature range, but more than 90% proteins cannot be detected when the culture temperature up to 55 ℃. The thermostability study of secretome by A. fumigatus suggested major polysaccharide degrading enzymes, such as endoglucanase and xylanase, can tolerate 2 hours with heat treatment of 60 ℃, and protease with 50 ℃, while beta-glucosidase has poor thermotolerance, indicating that high temperature can inhibit growth through weaken the amount and activity of extracellular functional proteins. All results suggested that temperature above 55 ℃ can lower fungal growth under high C/N ratio condition, therefore, this study can provide guidance on how to reduce the quantity of A. fumigatus from source. Moreover, there are many proteins in A.fumigatus secretome are of great importance in industry application, and their threats to health can be avoid by heterogeneous expression.4. Bioinformatics analysis and heterologous expression exploration of important enzymes secreted by A.fumigatusA.fumigatus can survive and grow in a wide range of environmental conditions, thus it was speculated that there always have some functional proteins play important role at different natural conditions. Endoglucanase eg25 (Q4WP32) and Endo-1,4-β-Xylanase xyn11A (V5R355) were identified by LC-MS/MS under all experimental conditions. Through bioinformatics analysis, finding that the structures of those two enzymes are similar with that of other members belonged to same GH families, and they both have β-jelly roll structures. However, those two enzymes have earlier evolutionary branching, which may suggested that they have more differences from other enzymes, hence, heterologous expression of those two proteins was carried out, and this work could provide a foundation for further study of enzymatic property.