The Regulatory Pathway Of Aspergillus Fumigatus To Produce Glycerol Triphosphate And Its Effect On Stress Response And Virulence

Aspergillus fumigatus is a one of the most frequent human opportunistic fungus,and its virulence is regulated by a variety of factors.In the course of evolution,A.fumigatus formed a complex system to adapt to different host niches and avoid host defenses.Some metabolites of A.fumigatus play an important role in this system.Therefore,understanding the pathways of metabolic pathways of metabolites of A.fumigatus is essential for elucidating the pathogenicity of this pathogen.Glycerol kinase(GK)is an important enzyme in the glycerol metabolism pathway.GK,an important enzyme in the metabolic pathway of glycerol,exists widely in eukaryotes and is able to catalyze glycerol to glycerol-3-phosphate(G3P).In some pathogens,GK affects their pathogenicity.GK participates in the utilization of carbon sources in the most of cell biology,and is able to promote the carbon cycle in the environment.In addition,GK is also involved in industrial applications to participate in the production of a variety of industrial raw materials.Aspergillus is an important participant in the carbon cycle in nature,and many Aspergillus are industrial strains.However,the studies about GK in Aspergillus were few.The studying of GK in Aspergillus will make up for vacancies in the application,pathogenicity and carbon utilization in related fields,which is of great significance for the effective development of Aspergillus resources and prevention of aspergillosis.In this paper,the function of GK in A.fumigatus is studied in depth,revealing its multiple effects on A.fumigatus,and providing clues for preventing aspergillosis and related applications.Previous studies proved that the glc A gene might encode glycerol kinase in A.fumigatus,due to the growth defects of the glc A null mutant in glycerol medium but not in glucose medium.Next,another glycerol kinase gene glc B was obtained by bioinformatics analysis in A.fumigatus.The two genes were knocked out,and then cultured in different media with various carbon source.The result showed that the loss of glc A,not glc B,caused the defect of growth in the glycerol medium.The results of western blot and fluorescence microscopy showed that Glc A was overexpressed in glycerol medium.In addition,the GK enzyme activity and G3 P content of the null mutants were measured.The loss of glc A led to a decrease in the GK enzyme activity and G3 P content of A.fumigatus.Meanwhile,the expression of Glc B controlled by glc A promoter was able to rescue the defect of glc A null mutant in the glycerol medium.These findings indicate that the glc A promoter is able to be induced by glycerol,and thus start the expression of Glc A,which is essential for the utilization of glycerol in A.fumigatus.Glc A was response to oxidative stress by analyzing the growth in different stresses.Under oxidative stress,the expression of Glc A,the total enzyme activity of GK,and the G3 P content all showed an upward trend.In addition,measurement of mitochondrial membrane potential by flow cytometry revealed that the loss of glc A caused the decrease of mitochondrial membrane potential of the strain.The ROS content was found to be increased in glc A null mutant.These findings indicate that the antioxidant systems might be abnormal,which may be the reason why the glc A null mutant is sensitive to oxidative stress.In A.fumigatus,the gfd A gene,encoding glycerol-3-phosphate dehydrogenase(GPDH),is able to transform dihydroxyacetone phosphate(DHAP)to G3 P.The overexpressed of gfd A in wild-type strains and glc A null mutant was able to increase the G3 P content by analyzing of G3 P content in vivo and increase the tolerance of strains to oxidative stress by performing oxidative tolerance analysis.These results indicate that the defect caused by the loss of glc A might be due to reduction of G3 P.GK participates in the substance and energy metabolism of A.fumigatus,and is involved in responsing to oxidative stress.The capabilities of the carbon source utilization and tolerance to oxidative stress are necessary for A.fumigatus to invade the host.We examined glc A null mutant strains' virulence in a G.mellonella model.This results demonstrated that the virulence of the glc A null mutant is reduced in a model of simple innate immunity.This research elaborated that G3 P produced mainly by Glc A is involved in the oxidation resistance and virulence of A.fumigatus,which has deepened our understanding of A.fumigatus and has important significance for the prevention of aspergillosis and related treatments.