Study On The Relationship Between The 3-phosphate Glycerol Dehydrogenase Encoded By Aspergillus Fumigatus GfdA And Related Pathway Members And HOG Pathway

Saprophytic filamentous fungi Aspergillus fumigatus is an important opportunistic pathogen that can cause immunodeficient patients to obtain invasive aspergillosis,which can even cause death in patients.In Aspergillus fumigatus,biosynthesis of glycerol is important for conidial production and coping with osmotic stress,but little is known about the genes involved in the regulation of biosynthesis.The gfdA gene,which encodes NAD+ dependent glycerol 3-phosphate dehydrogenase(G3PDH),has been studied to confirm that it does encode G3PDH enzyme and that the gfdA null mutant displays an osmoremdiable growth in the model organism Aspergillus nidulans.In view of this,this project uses Aspergillus fumigatus as experimental material to study the related biological function of gfdA.First,the gfdA gene was blasted with the Aspergillus fumigatus genome,and two homologous genes of AfgfdA and AfgfdB are found.We used CRISPR-Cas9 technology for gene editing,and had acquired AfgfdA and AfgfdB null mutants.Experiments show that:compared with the wild type,AfgfdA null mutant has a abnormal growth,its colony is smaller,mycelial extension and sporulation is blocked;however,AfgfdB null mutant has no difference growth to wild type;AgfdBAgfdA double mutant has a similar phenotype to AgfdA mutant,it all above also confirms that the gene encoding the functional G3PDH is predominantly gfdA rather than gfdB in Aspergillus fumigatus.In order to further confirm that the abnormal phenotype was indeed caused by the deletion of the gfdA gene,a rerecovery experiment was made,and the phenotype of retro-complementation strain was consistent with wild-type,which also proved that the construction of ?gfdA mutant was successful and right.Secondly,in order to further analyze the effect of gfdA on the glycerol metabolic pathway in Aspergillus fumigatus,?gfdA strain was cultured on medium with glycerol as the carbon source and found that its growth was the same as that of the wild type;whereas after cultured on the other carbon source(such as ethanol,fructose,glucose),the pathological phenotype did not ease,indicating that the gene was indeed involved in the process of glycerol metabolism.To further investigate how the abnormal phenotype was caused,the glcA gene was knocked out in Aspergillus fumigatus,which encodes glycerol kinase.The ?glcA was decreased sporulation quantity and the colony became smaller in the medium with glycerol as the carbon source.?glcA?gfdA double mutant strain was very ill,whether it was cultured on glycerol or glucose sugar as a carbon source of the medium can not grow normally.Since the two genes encode the proteases which catalyze to synthesize glycerol-3-phosphate,we consider that the ?gfdA strain is pathogenic due to the decrease containing of glycerol-3-phosphate in cell,which may be related to the cell wall integration pathway(CWI).The specific mechanism remains to be further explored.This result is consistent with Aspergillus nidulans.Thirdly,in order to understand more about the relationship between gfdA and other members of glycerol metabolic pathway in Aspergillus fumigatus,the gene gppA was knocked out which encodes 3-phosphoglycerolase,but the ?gppA showed a normal growth,?gppA?gfdA strain was consistent with the phenotype of ?gfdA,which indicated that the inactivation of 3-phosphoglycerate did not affect glycerol metabolism.Fourthly,in order to study the relationship between the gfdA and HOG pathway,1.2M polyols(Mannitol,Erythritol,Sorbitol)and 1M salts(NaCl and KC1)were added to the basal medium creating a high osmotic pressure to culture ?gfdA strain.It displayed that osmotic pressure would compensate for the defects of ?gfdA strain.It may be due to the increased osmotic pressure,the HOG pathway was activated,forced to produce some biocompatible substances to relieve osmotic pressure,making the phenotype to be restored.The?glcA?gfdA double mutant strain also restored to the phenotype consistent with the wild type under osmotic pressure,and it was clear that the phenotype was not dependent on the accumulation of glycerol-3-phosphate.Aspergillus fumigatus has four MAPKs,namely MpkA,MpkB,MpkC and SakA,while mpkC and sakA are homologous genes of Saccharomyces cerevisiae hog1,whereas SakA is the major regulator of hyperglycemic glycerol response pathway.Studies have shown that,after knocking out the sakA gene in Aspergillus fumigatus,it is found to be susceptible to salt stress.After we knocked out the gene gfdA in the ?sakA strain,and the phenotype did not recover when cultured under osmotic stress.It was found that colony of AsakA gfdA growth was more pathological than single gfdA null mutant.This shows that?gfdA strain to restore the phenotype is based on activated sakA gene under osmotic pressure.Fifthly,quantitative PCR showed that the expression of gfdA increased nearly 20 times under osmotic stress,but the expression of gfdB,gppA and glcA did not change much.However,when sakA was inactivated,the expression of gfdA increased 7 times,the rest of the gene changes were not great.This also indirectly indicates that osmotic stress may also be accompanied by activation of other pathways.In summary,the function of gfdA gene in Aspergillus fumigatus is consistent with gfdA gene in Aspergillus nidulans,we confirmed that polyol also regained normal growth of ?gfdA,and we also studied the role of other members of the glycerol metabolic pathway,as well as,we validated the HOG pathway indeed regulated gfdA,accompanied by activation of other pathways under osmotic stress.