| Aspergillus fumigatus is one of the most important airborne opportunistic pathogen and allergen worldwide.Several million people have invasive or chronic infections that lead to>600,000 deaths every year.Hence,a better understanding of the biology of this fungus may help to develop new or improve existing antifungal drugs in the future.Although classical gene deletion approaches by homologous integration have been used to explore the gene functions of fumigatus,they are technically complicated and time-intensive in inducing homology-directed repair(HDR)in wild-type strains.Based on the above,we have established a highly efficient CRISPR mutagenesis system with which we have dissected the functions of calcineurin in A.fumigatus.The conclusion we obtained as follows:1.In this thesis,we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient gene editing via very short(approximately 35-bp)homology arms in a process referred to as microhomology-mediated end joining(MMEJ),we referred to the system as MMEJ-CRISPR.Based on this system,we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion.Moreover,we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80,which is required for nonhomologous-end joining(NHEJ)DNA repair pathway,indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.2.As a potential drug target in Aspergillus,the heterodimer calcineurin is composed of catalytic subunit CnaA and regulatory subunit CnaB.It is beneficial to explore the functions of CnaA for dissecting the pathogenesis mechanisms of A.fumigatus.Based on MMEJ-CRISPR,we found the four serine residues of SPRR(the specific Serine Proline Rich Region of CnaA)are not necessary for the normal growth of Aspergillsu fumigatus.Meanwhile,the 374 tryptophan residues of CnaA is a sensitive site and it has a potential to be an antifungal drug target.3.The CnaA or CnaB deficiency was reported to lead to the growth defects and virulence attenuation of Aspergillus fumigatus.The calcineurin was also known as a central regulator of Ca2+homeostasis.However,it was still unclear that how calcineurin maintains the Ca2+homeostasis of Aspergillus fumigatus.In this thesis,we found that the dysfunction of calcineurin(?cnaA)increased the defects of hyphal polarity growth and induced cell autophagy,which were exacerbated by addition of calcium and alleviated in Ca2+starvation condition.These results suggest that the lack of calcineurin may induce the overload of intracellular Ca2+.In order to explore how calcineurin regulates the equilibrium of Ca2+inside the cell,the calcium reporter aequorin expression system was utilized to measure the dynamic free calcium.As a result,the dysfunction of calcineurin could decrease the calcium levels of cytosol and mitochondria,however,it promoted the influx of calcium into vacuole,suggesting that changes of subcellular Ca2+may be responsible for the polarity defect and autophagy of ?cnaA.Under the background of ?cnaA,deleting cchA(the putative voltage-gated Ca2+channels in plasma membrane and ER)not only alleviated the defects of hyphal polarity growth and cell autophagy but also rescued the dynamic distribution of Ca2+in cell.The above data suggest that the polarity growth defects and autophagy of ?cnaA are mainly induced by the calcium stress of endomembrane system.In conclusion,this work offered technical supports and theoretic basis for dissecting the pathogenesis mechanisms of A.fumigatus and for discovering the new antifungal drug target. |
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